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Find video protocols related to scientific articles indexed in Pubmed.
Effect of Mineralocorticoids on Acid-Base Balance.
Nephron Physiol
PUBLISHED: 11-06-2014
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Aldosterone is classically associated with the regulation of salt and potassium homeostasis but has also profound effects on acid-base balance. During acidosis, circulating aldosterone levels are increased and the hormone acts in concert with angiotensin II and other factors to stimulate renal acid excretion. Pharmacological blockade of aldosterone action as well as inherited or acquired syndromes of impaired aldosterone release or action impair the renal response to acid loading and cause hyperkalemic renal tubular acidosis. The mineralocorticoid receptor (MR) mediating the genomic effects of aldosterone is expressed in all cells of the distal nephron including all subtypes of intercalated cells. In acid-secretory type A intercalated cells, aldosterone stimulates proton secretion into urine, whereas in non-type A intercalated cells, aldosterone increases the activity of the luminal anion exchanger pendrin stimulating bicarbonate secretion and chloride reabsorption. Aldosterone has also stimulatory effects on proton secretion that may be mediated by a non-genomic pathway. In addition, aldosterone indirectly stimulates renal acid excretion by enhancing sodium reabsorption through the epithelial sodium channel ENaC. Increased sodium reabsorption enhances the lumen-negative transepithelial voltage that facilitates proton secretion by neighboring intercalated cells. This indirect coupling of sodium reabsorption and proton secretion is thought to underlie the fludrocortisone-furosemide test for maximal urinary acidification in patients with suspected distal renal tubular acidosis. In patients with CKD, acidosis-induced aldosterone may contribute to progression of kidney disease. In summary, aldosterone is a powerful regulator of renal acid excretion required for normal acid-base balance. © 2014 S. Karger AG, Basel.
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Proteinuria Increases Plasma Phosphate by Altering Its Tubular Handling.
J. Am. Soc. Nephrol.
PUBLISHED: 10-29-2014
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Proteinuria and hyperphosphatemia are cardiovascular risk factors independent of GFR. We hypothesized that proteinuria induces relative phosphate retention via increased proximal tubule phosphate reabsorption. To test the clinical relevance of this hypothesis, we studied phosphate handling in nephrotic children and patients with CKD. Plasma fibroblast growth factor 23 (FGF-23) concentration, plasma phosphate concentration, and tubular reabsorption of phosphate increased during the proteinuric phase compared with the remission phase in nephrotic children. Cross-sectional analysis of a cohort of 1738 patients with CKD showed that albuminuria?300 mg/24 hours is predictive of higher phosphate levels, independent of GFR and other confounding factors. Albuminuric patients also displayed higher plasma FGF-23 and parathyroid hormone levels. To understand the molecular mechanisms underlying these observations, we induced glomerular proteinuria in two animal models. Rats with puromycin-aminonucleoside-induced nephrotic proteinuria displayed higher renal protein expression of the sodium-phosphate co-transporter NaPi-IIa, lower renal Klotho protein expression, and decreased phosphorylation of FGF receptor substrate 2?, a major FGF-23 receptor substrate. These findings were confirmed in transgenic mice that develop nephrotic-range proteinuria resulting from podocyte depletion. In vitro, albumin did not directly alter phosphate uptake in cultured proximal tubule OK cells. In conclusion, we show that proteinuria increases plasma phosphate concentration independent of GFR. This effect relies on increased proximal tubule NaPi-IIa expression secondary to decreased FGF-23 biologic activity. Proteinuria induces elevation of both plasma phosphate and FGF-23 concentrations, potentially contributing to cardiovascular disease.
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The Murine Cl/HCO3(-) Exchanger Ae3 (Slc4a3) is Not Required for Acid-Base Balance but is Involved in Magnesium Handling by the Kidney.
Cell. Physiol. Biochem.
PUBLISHED: 09-01-2014
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Background: The Slc4 family of bicarbonate transporters consists of several members, many of which are highly expressed in the kidney and play an important role in acid-base homeostasis. Among them are Ae1 (Slc4a1) and Ae2 (Slc4a2). Another member, Ae3 (Slc4a3), is suggested to be expressed in the kidney, however, its localization and impact on renal function is still unknown. Ae3 has also been implicated in the central control of breathing. Aims: Here, we analyzed the expression of Ae3 transcripts in isolated nephron segments and investigated systemic and renal acid-base homeostasis and renal electrolyte handling in the absence of Ae3, using a knock out mouse model. Methods: qPCR was used to localize Ae3 transcripts in the murine nephron, metabolic studies and whole body plethysmography to assess the role of Ae3 in renal functions. Results: Two Ae3 transcripts, the brain variant bAe3 and the cardiac variant cAe3, are expressed at low levels in the murine kidney. Although differentially distributed, they localize mostly to the distal nephron and renal collecting duct system. At baseline and after an acid challenge, mice deficient for Ae3 did not show major disturbances in renal acid-base excretion. Respiratory responses in whole body plethysmography to acid loading and CO2 and O2 challenges were normal. No major differences in renal electrolyte handling were discovered except for small changes in magnesium, potassium and sodium excretion after 7 days of acid loading. We therefore challenged mice with diets with high and low magnesium diets and found no differences in renal magnesium excretion but elevated expression of the Trpm6 magnesium channel in Ae3 KO mice. In conclusion, Ae3 is expressed in murine kidney at very low levels. Conclusions: Ae3 plays no role in systemic acid-base homeostasis but may modify renal magnesium handling inducing a higher expression of Trpm6. © 2014 S. Karger AG, Basel.
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Genetic diseases of renal phosphate handling.
Nephrol. Dial. Transplant.
PUBLISHED: 08-29-2014
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Renal control of systemic phosphate homeostasis is critical as evident from inborn and acquired diseases causing renal phosphate wasting. At least three transport proteins are responsible for renal phosphate reabsorption: NAPI-IIa (SLC34A1), NAPI-IIc (SLC34A3) and PIT-2 (SLC20A2). These transporters are highly regulated by various cellular mechanisms and factors including acid-base status, electrolyte balance and hormones such as dopamine, glucocorticoids, growth factors, vitamin D3, parathyroid hormone and fibroblast growth factor 23 (FGF23). Whether renal phosphate wasting is caused by inactivating mutations in the NAPI-IIa transporter is controversial. Mutations in the NAPI-IIc transporter cause hereditary hypophosphatemic rickets with hypercalciuria. Besides the primary inherited defects, there are also inherited defects in major regulators of phosphate homeostasis that lead to alterations in phosphate handling. Autosomal dominant hypophosphatemic rickets is due to FGF23 mutations leading to resistance against its own degradation. Similarly, inactivating mutations in the PHEX gene, which causes FGF23 inactivation, cause X-linked hypophosphatemia due to renal phosphate losses. In contrast, mutations in galactosamine:polypeptide N-acetyl-galactosaminyltransferase, responsible for O-glycosylation of FGF23, or in klotho, a cofactor for FGF23 signalling result in hyperphosphatemia. Acquired syndromes of renal phosphate wasting, hypophosphatemia and osteomalacia (tumour-associated osteomalacia) can be due to the excessive synthesis or release of phosphaturic factors (FGF23, FGF-7, MEPE and sFRP4) from mesenchymal tumours.
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Acute metabolic acidosis in a GLUT2-deficient patient with Fanconi-Bickel syndrome: new pathophysiology insights.
Nephrol. Dial. Transplant.
PUBLISHED: 08-29-2014
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Fanconi-Bickel syndrome is a rare autosomal-recessive disorder caused by mutations in the SLC2A2 gene coding for the glucose transporter protein 2 (GLUT2). Major manifestations include hepatomegaly, glucose intolerance, post-prandial hypoglycaemia and renal disease that usually presents as proximal tubular acidosis associated with proximal tubule dysfunction (renal Fanconi syndrome). We report a patient harbouring a homozygous mutation of SLC2A2 who presented a dramatic exacerbation of metabolic acidosis in the context of a viral infection, owing to both ketosis and major urinary bicarbonate loss. The kidney biopsy revealed nuclear and cytoplasmic accumulation of glycogen in proximal tubule cells, a lack of expression of GLUT2, and major defects of key proteins of the proximal tubule such as megalin, cubilin and the B2 subunit of H(+)-ATPase. These profound alterations of the transport systems most likely contributed to proximal tubule alterations and profound bicarbonate loss.
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Dysregulation of the glutamine transporter Slc38a3 (SNAT3) and ammoniagenic enzymes in obese, glucose-intolerant mice.
Cell. Physiol. Biochem.
PUBLISHED: 08-11-2014
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Uric acid nephrolithiasis is prevalent among patients with type 2 diabetes and metabolic syndrome; it is correlated with an acidic urine and lower urinary ammonium excretion and is likely associated with insulin resistance. Insulin stimulates ammoniagenesis in renal cell lines via increased phosphate-dependent glutaminase (PDG) activity and glutamine metabolism. Ammonium excretion into the proximal tubule is mediated at least in part by the Na(+)/H(+)-exchanger NHE3 and in the collecting duct involving the Rhesus protein RhCG. Here we tested, whether obesity and insulin resistance in a diet-induced mouse model could contribute to deranged ammonium excretion.
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Mechanisms of Renal Control of Potassium Homeostasis in Complete Aldosterone Deficiency.
J. Am. Soc. Nephrol.
PUBLISHED: 07-30-2014
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Aldosterone-independent mechanisms may contribute to K(+) homeostasis. We studied aldosterone synthase knockout (AS(-/-)) mice to define renal control mechanisms of K(+) homeostasis in complete aldosterone deficiency. AS(-/-) mice were normokalemic and tolerated a physiologic dietary K(+) load (2% K(+), 2 days) without signs of illness, except some degree of polyuria. With supraphysiologic K(+) intake (5% K(+)), AS(-/-) mice decompensated and became hyperkalemic. High-K(+) diets induced upregulation of the renal outer medullary K(+) channel in AS(-/-) mice, whereas upregulation of the epithelial sodium channel (ENaC) sufficient to increase the electrochemical driving force for K(+) excretion was detected only with a 2% K(+) diet. Phosphorylation of the thiazide-sensitive NaCl cotransporter was consistently lower in AS(-/-) mice than in AS(+/+) mice and was downregulated in mice of both genotypes in response to increased K(+) intake. Inhibition of the angiotensin II type 1 receptor reduced renal creatinine clearance and apical ENaC localization, and caused severe hyperkalemia in AS(-/-) mice. In contrast with the kidney, the distal colon of AS(-/-) mice did not respond to dietary K(+) loading, as indicated by Ussing-type chamber experiments. Thus, renal adaptation to a physiologic, but not supraphysiologic, K(+) load can be achieved in aldosterone deficiency by aldosterone-independent activation of the renal outer medullary K(+) channel and ENaC, to which angiotensin II may contribute. Enhanced urinary flow and reduced activity of the thiazide-sensitive NaCl cotransporter may support renal adaptation by activation of flow-dependent K(+) secretion and increased intratubular availability of Na(+) that can be reabsorbed in exchange for K(+) secreted.
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The role of the renal ammonia transporter Rhcg in metabolic responses to dietary protein.
J. Am. Soc. Nephrol.
PUBLISHED: 03-20-2014
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High dietary protein imposes a metabolic acid load requiring excretion and buffering by the kidney. Impaired acid excretion in CKD, with potential metabolic acidosis, may contribute to the progression of CKD. Here, we investigated the renal adaptive response of acid excretory pathways in mice to high-protein diets containing normal or low amounts of acid-producing sulfur amino acids (SAA) and examined how this adaption requires the RhCG ammonia transporter. Diets rich in SAA stimulated expression of enzymes and transporters involved in mediating NH4 (+) reabsorption in the thick ascending limb of the loop of Henle. The SAA-rich diet increased diuresis paralleled by downregulation of aquaporin-2 (AQP2) water channels. The absence of Rhcg transiently reduced NH4 (+) excretion, stimulated the ammoniagenic pathway more strongly, and further enhanced diuresis by exacerbating the downregulation of the Na(+)/K(+)/2Cl(-) cotransporter (NKCC2) and AQP2, with less phosphorylation of AQP2 at serine 256. The high protein acid load affected bone turnover, as indicated by higher Ca(2+) and deoxypyridinoline excretion, phenomena exaggerated in the absence of Rhcg. In animals receiving a high-protein diet with low SAA content, the kidney excreted alkaline urine, with low levels of NH4 (+) and no change in bone metabolism. Thus, the acid load associated with high-protein diets causes a concerted response of various nephron segments to excrete acid, mostly in the form of NH4 (+), that requires Rhcg. Furthermore, bone metabolism is altered by a high-protein acidogenic diet, presumably to buffer the acid load.
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Influence of high-flux hemodialysis and hemodiafiltration on serum C-terminal agrin fragment levels in end-stage renal disease patients.
Transl Res
PUBLISHED: 03-06-2014
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C-terminal agrin fragment (CAF, 22 kDa) has been shown to be a promising new rapid biomarker for kidney function. This study evaluated the influence of hemodialysis (HD) and hemodiafiltration (HDF) treatment on serum CAF concentrations in patients with end-stage renal disease (ESRD). A total of 36 patients with ESRD undergoing chronic HD/HDF treatment were enrolled (21 high-flux-HD/Fx60 membrane, 7 high-flux-HD/Elisio19H membrane, and 8 HDF/Elisio19H membrane). On a midweek session, blood samples were obtained before, at halftime, and post-treatment. Dialysate samples were obtained 4 times during treatment. Serum and dialysate CAF, cystatin C, urea, and creatinine concentrations were measured. Reduction ratios (RRs), total solute removal, overall dialytic clearance, and instantaneous dialytic clearance at halftime were calculated and compared. Although HD/Elisio19H and HDF/Elisio19H treatments significantly reduced CAF concentrations (RR 46.6 ± 9.1% and 57.6 ± 11.7%, respectively, P = 0.018 and P = 0.001), HD/Fx60 treatment did not remove CAF from serum (RR 2.4 ± 15.4%, P = 0.25), there was no relevant CAF detection in dialysate. In the HD/Fx60 group, the RR of CAF was significantly lower compared with cystatin C, urea, and creatinine, in which significant removal was detected (37.9 ± 14.8%, 65.0 ± 10.7%, and 56.0 ± 9.8%, respectively, P < 0.001). CAF is a new biomarker for kidney function whose serum concentration is not influenced by conventional high-flux HD using Fx60 membrane. It might therefore represent a promising dialysis-independent biomarker for evaluation of kidney function, for example, in acute kidney failure.
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Renal-specific and inducible depletion of NaPi-IIc/Slc34a3, the cotransporter mutated in HHRH, does not affect phosphate or calcium homeostasis in mice.
Am. J. Physiol. Renal Physiol.
PUBLISHED: 02-19-2014
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The proximal renal epithelia express three different Na-dependent inorganic phosphate (Pi) cotransporters: NaPi-IIa/SLC34A1, NaPi-IIc/SLC34A3, and PiT2/SLC20A2. Constitutive mouse knockout models of NaPi-IIa and NaPi-IIc suggested that NaPi-IIa mediates the bulk of renal reabsorption of Pi whereas the contribution of NaPi-IIc to this process is minor and probably restricted to young mice. However, many reports indicate that mutations of NaPi-IIc in humans lead to hereditary hypophosphatemic rickets with hypercalciuria (HHRH). Here, we report the generation of a kidney-specific and inducible NaPi-IIc-deficient mouse model based on the loxP-Cre system. We found that the specific removal of the cotransporter from the kidneys of young mice does not impair the capacity of the renal epithelia to transport Pi. Moreover, the levels of Pi in plasma and urine as well as the circulating levels of parathyroid hormone, FGF-23, and vitamin D3 remained unchanged. These findings are in agreement with the data obtained with the constitutive knockout model and suggest that, under steady-state conditions of normal dietary Pi, NaPi-IIc is not an essential Na-Pi cotransporter in murine kidneys. However, and unlike the constitutive mutants, the kidney-specific depletion of NaPi-IIc does not result in alteration of the homeostasis of calcium. This suggests that the calcium-related phenotype observed in constitutive knockout mice may not be related to inactivation of the cotransporter in kidney.
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Renal expression of FGF23 and peripheral resistance to elevated FGF23 in rodent models of polycystic kidney disease.
Kidney Int.
PUBLISHED: 01-08-2014
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Fibroblast growth factor 23 (FGF23) regulates phosphate homeostasis and is linked to cardiovascular disease and all-cause mortality in chronic kidney disease. FGF23 rises in patients with CKD stages 2-3, but in patients with autosomal dominant polycystic kidney disease, the increase of FGF23 precedes the first measurable decline in renal function. The mechanisms governing FGF23 production and effects in kidney disease are largely unknown. Here we studied the relation between FGF23 and mineral homeostasis in two animal models of PKD. Plasma FGF23 levels were increased 10-fold in 4-week-old cy/+ Han:SPRD rats, whereas plasma urea and creatinine concentrations were similar to controls. Plasma calcium and phosphate levels as well as TmP/GFR were similar in PKD and control rats at all time points examined. Expression and activity of renal phosphate transporters, the vitamin D3-metabolizing enzymes, and the FGF23 co-ligand Klotho in the kidney were similar in PKD and control rats through 8 weeks of age, indicating resistance to FGF23, although phosphorylation of the FGF receptor substrate 2? protein was enhanced. In the kidneys of rats with PKD, FGF23 mRNA was highly expressed and FGF23 protein was detected in cells lining renal cysts. FGF23 expression in bone and spleen was similar in control rats and rats with PKD. Similarly, in an inducible Pkd1 knockout mouse model, plasma FGF23 levels were elevated, FGF23 was expressed in kidneys, but renal phosphate excretion was normal. Thus, the polycystic kidney produces FGF23 but is resistant to its action.
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The SLC34 family of sodium-dependent phosphate transporters.
Pflugers Arch.
PUBLISHED: 11-30-2013
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The SLC34 family of sodium-driven phosphate cotransporters comprises three members: NaPi-IIa (SLC34A1), NaPi-IIb (SLC34A2), and NaPi-IIc (SLC34A3). These transporters mediate the translocation of divalent inorganic phosphate (HPO4 (2-)) together with two (NaPi-IIc) or three sodium ions (NaPi-IIa and NaPi-IIb), respectively. Consequently, phosphate transport by NaPi-IIa and NaPi-IIb is electrogenic. NaPi-IIa and NaPi-IIc are predominantly expressed in the brush border membrane of the proximal tubule, whereas NaPi-IIb is found in many more organs including the small intestine, lung, liver, and testis. The abundance and activity of these transporters are mostly regulated by changes in their expression at the cell surface and are determined by interactions with proteins involved in scaffolding, trafficking, or intracellular signaling. All three transporters are highly regulated by factors including dietary phosphate status, hormones like parathyroid hormone, 1,25-OH2 vitamin D3 or FGF23, electrolyte, and acid-base status. The physiological relevance of the three members of the SLC34 family is underlined by rare Mendelian disorders causing phosphaturia, hypophosphatemia, or ectopic organ calcifications.
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The Proton-Activated Receptor GPR4 Modulates Glucose Homeostasis by Increasing Insulin Sensitivity.
Cell. Physiol. Biochem.
PUBLISHED: 10-21-2013
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Background: The proton-activated G protein-coupled receptor GPR4 is expressed in many tissues including white adipose tissue. GPR4 is activated by extracellular protons in the physiological pH range (i.e. pH 7.7 - 6.8) and is coupled to the production of cAMP. Methods: We examined mice lacking GPR4 and examined glucose tolerance and insulin sensitivity in young and aged mice as well as in mice fed with a high fat diet. Expression profiles of pro- and anti-inflammatory cytokines in white adipose tissue, liver and skeletal muscle was assessed. Results: Here we show that mice lacking GPR4 have an improved intraperitoneal glucose tolerance test and increased insulin sensitivity. Insulin levels were comparable but leptin levels were increased in GPR4 KO mice. Gpr4(-/-) showed altered expression of PPAR?, IL-6, IL-10, TNF?, and TGF-1? in skeletal muscle, white adipose tissue, and liver. High fat diet abolished the differences in glucose tolerance and insulin sensitivity between Gpr4(+/+) and Gpr4(-/-) mice. In contrast, in aged mice (12 months old), the positive effect of GPR4 deficiency on glucose tolerance and insulin sensitivity was maintained. Liver and adipose tissue showed no major differences in the mRNA expression of pro- and anti-inflammatory factors between aged mice of both genotypes. Conclusion: Thus, GPR4 deficiency improves glucose tolerance and insulin sensitivity. The effect may involve an altered balance between pro- and anti-inflammatory factors in insulin target tissues. © 2013 S. Karger AG, Basel.
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Differential cystine and dibasic amino acid handling after loss of function of the amino acid transporter b0,+AT (Slc7a9) in mice.
Am. J. Physiol. Renal Physiol.
PUBLISHED: 10-09-2013
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Cystinuria is an autosomal recessive disease caused by mutations in SLC3A1 (rBAT) and SLC7A9 (b(0,+)AT). Gene targeting of the catalytic subunit (Slc7a9) in mice leads to excessive excretion of cystine, lysine, arginine, and ornithine. Here, we studied this non-type I cystinuria mouse model using gene expression analysis, Western blotting, clearance, and brush-border membrane vesicle (BBMV) uptake experiments to further characterize the renal and intestinal consequences of losing Slc7a9 function. The electrogenic and BBMV flux studies in the intestine suggested that arginine and ornithine are transported via other routes apart from system b(0,+). No remarkable gene expression changes were observed in other amino acid transporters and the peptide transporters in the intestine and kidney. Furthermore, the glomerular filtration rate (GFR) was reduced by 30% in knockout animals compared with wild-type animals. The fractional excretion of arginine was increased as expected (?100%), but fractional excretions of lysine (?35%), ornithine (?16%), and cystine (?11%) were less affected. Loss of function of b(0,+)AT reduced transport of cystine and arginine in renal BBMVs and completely abolished the exchanger activity of dibasic amino acids with neutral amino acids. In conclusion, loss of Slc7a9 function decreases the GFR and increases the excretion of several amino acids to a lesser extent than expected with no clear regulation at the mRNA and protein level of alternative transporters and no increased renal epithelial uptake. These observations indicate that transporters located in distal segments of the kidney and/or metabolic pathways may partially compensate for Slc7a9 loss of function.
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C-Terminal Agrin Fragment - A New Fast Biomarker for Kidney Function in Renal Transplant Recipients.
Am. J. Nephrol.
PUBLISHED: 09-19-2013
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Background: The C-terminal agrin fragment (CAF) is a cleavage product of agrin, the major proteoglycan of the glomerular basement membrane. This article studies if CAF could serve as a biomarker for renal function in renal transplant recipients. Material and Methods: We measured serum CAF and creatinine concentrations and calculated estimated glomerular filtration rate (eGFR) (MDRD) in 96 healthy individuals and in 110 end-stage renal disease patients undergoing kidney transplantation before and after transplantation. Correlation between CAF and creatinine concentrations/eGFR was calculated as within-patient (cWP) and between-patient correlations (cBP). Moreover, we evaluated the association of CAF with delayed graft function (DGF). The diagnostic value of CAF for early detection of DGF compared to creatinine was evaluated by receiver operating characteristics (ROC) analysis. Results: CAF concentrations strongly correlated with creatinine (r = 0.86 (cWP), r = 0.74 (cBP)) and eGFR (MDRD) (r = 0.86 (cWP), r = 0.77 (cBP)). Pre-transplant (pre-Tx) CAF concentrations were 19-fold higher than in healthy individuals (1,115.0 (258.4-3,990.0) vs. 56.6 (20.0-109.5) pM). After transplantation, CAF decreased significantly faster than creatinine (postoperative days 1-3 (POD 1-3): 562.8 (101.6-2,113.0) pM; creatinine: pre-Tx 6.9 (3.1-15.7), POD 1-3: 6.4 (1.7-12.7) mg/dl, p < 0.001). Stable concentrations were reached 1-3 months after transplantation for CAF and creatinine (CAF 145.1 (6.7-851.0) pM; creatinine 1.6 (0.7-8.0) mg/dl). CAF concentrations at POD 1-3 were significantly associated with DGF and outperformed creatinine in early detection of DGF (area under the curve (AUC) CAF 80.7% (95% CI 72.3-89.1%) vs. AUC creatinine 71.3% (95% CI 61.8-81.1%), p = 0.061). Conclusion: CAF is a promising new and fast biomarker for kidney function and may serve as a new tool for the early detection of DGF. © 2013 S. Karger AG, Basel.
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Roux-en-Y gastric bypass surgery reduces bone mineral density and induces metabolic acidosis in rats.
Am. J. Physiol. Regul. Integr. Comp. Physiol.
PUBLISHED: 09-11-2013
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Roux-en-Y gastric bypass (RYGB) surgery leads to bone loss in humans, which may be caused by vitamin D and calcium malabsorption and subsequent secondary hyperparathyroidism. However, because these conditions occur frequently in obese people, it is unclear whether they are the primary causes of bone loss after RYGB. To determine the contribution of calcium and vitamin D malabsorption to bone loss in a rat RYGB model, adult male Wistar rats were randomized for RYGB surgery, sham-operation-ad libitum fed, or sham-operation-body weight-matched. Bone mineral density, calcium and phosphorus balance, acid-base status, and markers of bone turnover were assessed at different time points for 14 wk after surgery. Bone mineral density decreased for several weeks after RYGB. Intestinal calcium absorption was reduced early after surgery, but plasma calcium and parathyroid hormone levels were normal. 25-hydroxyvitamin D levels decreased, while levels of active 1,25-dihydroxyvitamin D increased after surgery. RYGB rats displayed metabolic acidosis due to increased plasma lactate levels and increased urinary calcium loss throughout the study. These results suggest that initial calcium malabsorption may play a key role in bone loss early after RYGB in rats, but other factors, including chronic metabolic acidosis, contribute to insufficient bone restoration after normalization of intestinal calcium absorption. Secondary hyperparathyroidism is not involved in postoperative bone loss. Upregulated vitamin D activation may compensate for any vitamin D malabsorption.
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The calcium-sensing receptor directly regulates proximal tubular functions.
Kidney Int.
PUBLISHED: 08-02-2013
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Capasso et al. show a role of the calcium-sensing receptor (CaSR) in enhancing proximal tubular fluid absorption and urinary acidification by stimulation of luminal Na(+)/H(+) exchanger (NHE) activity. NHE3 is required for sodium and fluid absorption, and its activity is coupled to passive reabsorption of a major fraction of calcium through the paracellular route. These data shed new light on the regulation of the kidney by the CaSR and whether it directly affects proximal tubular functions.
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Renal ?-intercalated cells maintain body fluid and electrolyte balance.
J. Clin. Invest.
PUBLISHED: 06-28-2013
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Inactivation of the B1 proton pump subunit (ATP6V1B1) in intercalated cells (ICs) leads to type I distal renal tubular acidosis (dRTA), a disease associated with salt- and potassium-losing nephropathy. Here we show that mice deficient in ATP6V1B1 (Atp6v1b1-/- mice) displayed renal loss of NaCl, K+, and water, causing hypovolemia, hypokalemia, and polyuria. We demonstrated that NaCl loss originated from the cortical collecting duct, where activity of both the epithelial sodium channel (ENaC) and the pendrin/Na(+)-driven chloride/bicarbonate exchanger (pendrin/NDCBE) transport system was impaired. ENaC was appropriately increased in the medullary collecting duct, suggesting a localized inhibition in the cortex. We detected high urinary prostaglandin E2 (PGE2) and ATP levels in Atp6v1b1-/- mice. Inhibition of PGE2 synthesis in vivo restored ENaC protein levels specifically in the cortex. It also normalized protein levels of the large conductance calcium-activated potassium channel and the water channel aquaporin 2, and improved polyuria and hypokalemia in mutant mice. Furthermore, pharmacological inactivation of the proton pump in ?-ICs induced release of PGE2 through activation of calcium-coupled purinergic receptors. In the present study, we identified ATP-triggered PGE2 paracrine signaling originating from ?-ICs as a mechanism in the development of the hydroelectrolytic imbalance associated with dRTA. Our data indicate that in addition to principal cells, ICs are also critical in maintaining sodium balance and, hence, normal vascular volume and blood pressure.
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Bile acids trigger cholemic nephropathy in common bile-duct-ligated mice.
Hepatology
PUBLISHED: 06-15-2013
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Tubular epithelial injury represents an underestimated but important cause of renal dysfunction in patients with cholestasis and advanced liver disease, but the underlying mechanisms are unclear. To address the hypothesis that accumulation and excessive alternative urinary elimination of potentially toxic bile acids (BAs) may contribute to kidney injury in cholestasis, we established a mouse model for detailed in vivo time course as well as treatment studies. Three-day common bile duct ligation (CBDL) induced renal tubular epithelial injury predominantly at the level of aquaporin 2-positive collecting ducts with tubular epithelial and basement membrane defects. This was followed by progressive interstitial nephritis and tubulointerstitial renal fibrosis in 3-, 6-, and 8-week CBDL mice. Farnesoid X receptor knockout mice (with a hydrophilic BA pool) were completely protected from CBDL-induced renal fibrosis. Prefeeding of hydrophilic norursodeoxycholic acid inhibited renal tubular epithelial injury in CBDL mice. In addition, we provide evidence for renal tubular injury in cholestatic patients with cholemic nephropathy. Conclusion: We characterized a novel in vivo model for cholemic nephropathy, which offers new perspectives to study the complex pathophysiology of this condition. Our findings suggest that urinary-excreted toxic BAs represent a pivotal trigger for renal tubular epithelial injury leading to cholemic nephropathy in CBDL mice. (Hepatology 2013; 58:2056-2069).
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Sulfatides are required for renal adaptation to chronic metabolic acidosis.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 05-28-2013
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Urinary ammonium excretion by the kidney is essential for renal excretion of sufficient amounts of protons and to maintain stable blood pH. Ammonium secretion by the collecting duct epithelia accounts for the majority of urinary ammonium; it is driven by an interstitium-to-lumen NH3 gradient due to the accumulation of ammonium in the medullary and papillary interstitium. Here, we demonstrate that sulfatides, highly charged anionic glycosphingolipids, are important for maintaining high papillary ammonium concentration and increased urinary acid elimination during metabolic acidosis. We disrupted sulfatide synthesis by a genetic approach along the entire renal tubule. Renal sulfatide-deficient mice had lower urinary pH accompanied by lower ammonium excretion. Upon acid diet, they showed impaired ammonuria, decreased ammonium accumulation in the papilla, and chronic hyperchloremic metabolic acidosis. Expression levels of ammoniagenic enzymes and Na(+)-K(+)/NH4(+)-2Cl(-) cotransporter 2 were higher, and transepithelial NH3 transport, examined by in vitro microperfusion of cortical and outer medullary collecting ducts, was unaffected in mutant mice. We therefore suggest that sulfatides act as counterions for interstitial ammonium facilitating its retention in the papilla. This study points to a seminal role of sulfatides in renal ammonium handling, urinary acidification, and acid-base homeostasis.
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(Pro)renin receptor is required for prorenin-dependent and -independent regulation of vacuolar H?-ATPase activity in MDCK.C11 collecting duct cells.
Am. J. Physiol. Renal Physiol.
PUBLISHED: 05-22-2013
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Prorenin binding to the prorenin receptor [(P)RR] results in nonproteolytic activation of prorenin but also directly (i.e., independent of angiotensin generation) activates signal transduction cascades that can lead to the upregulation of profibrotic factors. The (P)RR is an accessory protein of vacuolar-type H?-ATPase (V-ATPase) and is required for V-ATPase integrity. In addition, in collecting duct cells, prorenin-induced activation of Erk depends on V-ATPase activity. However, whether prorenin binding to the (P)RR directly regulates V-ATPase activity is as yet unknown. Here, we studied the effect of prorenin on plasma membrane V-ATPase activity in Madin-Darby canine kidney clone 11 (MDCK.C11) cells, which resemble intercalated cells of the collecting duct. Prorenin increased V-ATPase activity at low nanomolar concentrations, and the V-ATPase inhibitor bafilomycin A1, but not the angiotensin II type 1 and 2 receptor blockers irbesartan and PD-123319, prevented this. Increased, but not basal, V-ATPase activity was abolished by small interfering RNA depletion of the (P)RR. Unexpectedly, the putative peptidic (P)RR blocker handle region peptide also increased V-ATPase activity in a (P)RR-dependent manner. Finally, [Arg?]-vasopressin-stimulated V-ATPase activity and cAMP production were also abolished by (P)RR depletion. Our results show that in MDCK.C11 cells, the (P)RR is required for prorenin-dependent and -independent regulation of V-ATPase activity.
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The phosphate transporter NaPi-IIa determines the rapid renal adaptation to dietary phosphate intake in mouse irrespective of persistently high FGF23 levels.
Pflugers Arch.
PUBLISHED: 05-13-2013
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Renal reabsorption of inorganic phosphate (Pi) is mediated by the phosphate transporters NaPi-IIa, NaPi-IIc, and Pit-2 in the proximal tubule brush border membrane (BBM). Dietary Pi intake regulates these transporters; however, the contribution of the specific isoforms to the rapid and slow phase is not fully clarified. Moreover, the regulation of PTH and FGF23, two major phosphaturic hormones, during the adaptive phase has not been correlated. C57/BL6 and NaPi-IIa(-/-) mice received 5 days either 1.2 % (HPD) or 0.1 % (LPD) Pi-containing diets. Thereafter, some mice were acutely switched to LPD or HPD. Plasma Pi concentrations were similar under chronic diets, but lower when mice were acutely switched to LPD. Urinary Pi excretion was similar in C57/BL6 and NaPi-IIa(-/-) mice under HPD. During chronic LPD, NaPi-IIa(-/-) mice lost phosphate in urine compensated by higher intestinal Pi absorption. During the acute HPD-to-LPD switch, NaPi-IIa(-/-) mice exhibited a delayed decrease in urinary Pi excretion. PTH was acutely regulated by low dietary Pi intake. FGF23 did not respond to low Pi intake within 8 h whereas the phospho-adaptator protein FRS2? necessary for FGF-receptor cell signaling was downregulated. BBM Pi transport activity and NaPi-IIa but not NaPi-IIc and Pit-2 abundance acutely adapted to diets in C57/BL6 mice. In NaPi-IIa(-/-), Pi transport activity was low and did not adapt. Thus, NaPi-IIa mediates the fast adaptation to Pi intake and is upregulated during the adaptation to low Pi despite persistently high FGF23 levels. The sensitivity to FGF23 may be regulated by adapting FRS2? abundance and phosphorylation.
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?-Ketoglutarate regulates acid-base balance through an intrarenal paracrine mechanism.
J. Clin. Invest.
PUBLISHED: 04-11-2013
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Paracrine communication between different parts of the renal tubule is increasingly recognized as an important determinant of renal function. Previous studies have shown that changes in dietary acid-base load can reverse the direction of apical ?-ketoglutarate (?KG) transport in the proximal tubule and Henles loop from reabsorption (acid load) to secretion (base load). Here we show that the resulting changes in the luminal concentrations of ?KG are sensed by the ?KG receptor OXGR1 expressed in the type B and non-A-non-B intercalated cells of the connecting tubule (CNT) and the cortical collecting duct (CCD). The addition of 1 mM ?KG to the tubular lumen strongly stimulated Cl(-)-dependent HCO(3)(-) secretion and electroneutral transepithelial NaCl reabsorption in microperfused CCDs of wild-type mice but not Oxgr1(-/-) mice. Analysis of alkali-loaded mice revealed a significantly reduced ability of Oxgr1(-/-) mice to maintain acid-base balance. Collectively, these results demonstrate that OXGR1 is involved in the adaptive regulation of HCO(3)(-) secretion and NaCl reabsorption in the CNT/CCD under acid-base stress and establish ?KG as a paracrine mediator involved in the functional coordination of the proximal and the distal parts of the renal tubule.
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Multiday acute sodium bicarbonate intake improves endurance capacity and reduces acidosis in men.
J Int Soc Sports Nutr
PUBLISHED: 02-08-2013
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The purpose was to investigate the effects of one dose of NaHCO3 per day for five consecutive days on cycling time-to-exhaustion (Tlim) at Critical Power (CP) and acid-base parameters in endurance athletes.
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Regulation of two renal chloride transporters, AE1 and pendrin, by electrolytes and aldosterone.
PLoS ONE
PUBLISHED: 01-31-2013
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The renal handling of salt and protons and bicarbonate are intricately linked through shared transport mechanisms for sodium, chloride, protons, and bicarbonate. In the collecting duct, the regulated fine-tuning of salt and acid-base homeostasis is achieved by a series of transport proteins located in different cell types, intercalated and principal cells. Intercalated cells are considered to be of less importance for salt handling but recent evidence has suggested that the anion exchanger pendrin may participate in salt reabsorption and blood pressure regulation. Here, we examined the regulated expression of two functionally related but differentially expressed anion exchangers, AE1 and pendrin, by dietary electrolyte intake and aldosterone. Cortical expression of pendrin was regulated on mRNA and protein level. The combination of NaHCO? and DOCA enhanced pendrin mRNA and protein levels, whereas DOCA or NaHCO? alone had no effect. NaCl or KHCO? increased pendrin mRNA, KCl decreased its mRNA abundance. On protein level, NH?Cl, NaCl, and KCl reduced pendrin expression, the other treatments were without effect. In contrast, AE1 mRNA or protein expression in kidney cortex was regulated by none of these treatments. In kidney medulla, NaHCO?/DOCA or NaHCO? alone enhanced AE1 mRNA levels. AE1 protein abundance was increased by NH?Cl, NaHCO?/DOCA, and NaCl. Immunolocalization showed that during NH?Cl treatment the relative number of AE1 positive cells was increased and pendrin expressing cells reduced. Thus, pendrin and AE1 are differentially regulated with distinct mechanisms that separately affect mRNA and protein levels. Pendrin is regulated by acidosis and chloride intake, whereas AE1 is enhanced by acidosis, NaCl, and the combination of DOCA and NaHCO?.
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Haploinsufficiency of the ammonia transporter Rhcg predisposes to chronic acidosis: Rhcg is critical for apical and basolateral ammonia transport in the mouse collecting duct.
J. Biol. Chem.
PUBLISHED: 01-03-2013
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Ammonia secretion by the collecting duct (CD) is critical for acid-base homeostasis and, when defective, causes distal renal tubular acidosis (dRTA). The Rhesus protein RhCG mediates NH(3) transport as evident from cell-free and cellular models as well as from Rhcg-null mice. Here, we investigated in a Rhcg mouse model the metabolic effects of Rhcg haploinsufficiency, the role of Rhcg in basolateral NH(3) transport, and the mechanisms of adaptation to the lack of Rhcg. Both Rhcg(+/+) and Rhcg(+/-) mice were able to handle an acute acid load, whereas Rhcg(-/-) mice developed severe metabolic acidosis with reduced ammonuria and high mortality. However, chronic acid loading revealed that Rhcg(+/-) mice did not fully recover, showing lower blood HCO(3)(-) concentration and more alkaline urine. Microperfusion studies demonstrated that transepithelial NH(3) permeability was reduced by 80 and 40%, respectively, in CDs from Rhcg(-/-) and Rhcg(+/-) mice compared with controls. Basolateral membrane permeability to NH(3) was reduced in CDs from Rhcg(-/-) mice consistent with basolateral Rhcg localization. Rhcg(-/-) responded to acid loading with normal expression of enzymes and transporters involved in proximal tubular ammoniagenesis but reduced abundance of the NKCC2 transporter responsible for medullary accumulation of ammonium. Consequently, tissue ammonium content was decreased. These data demonstrate a role for apical and basolateral Rhcg in transepithelial NH(3) transport and uncover an incomplete dRTA phenotype in Rhcg(+/-) mice. Haploinsufficiency or reduced expression of RhCG may underlie human forms of (in)complete dRTA.
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Soluble klotho and autosomal dominant polycystic kidney disease.
Clin J Am Soc Nephrol
PUBLISHED: 12-22-2011
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Fibroblast growth factor 23 (FGF23) levels are elevated in patients with autosomal dominant polycystic kidney disease (ADPKD) and X-linked hypophosphatemia (XLH), but only the latter is characterized by a renal phosphate wasting phenotype. This study explored potential mechanisms underlying resistance to FGF23 in ADPKD.
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Impact of bicarbonate, ammonium chloride, and acetazolamide on hepatic and renal SLC26A4 expression.
Cell. Physiol. Biochem.
PUBLISHED: 10-11-2011
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SLC26A4 encodes pendrin, a transporter exchanging anions such as chloride, bicarbonate, and iodide. Loss of function mutations of SLC26A4 cause Pendred syndrome characterized by hearing loss and enlarged vestibular aqueducts as well as variable hypothyroidism and goiter. In the kidney, pendrin is expressed in the distal nephron and accomplishes HCO(3)(-) secretion and Cl(-) reabsorption. Renal pendrin expression is regulated by acid-base balance. The liver contributes to acid-base regulation by producing or consuming glutamine, which is utilized by the kidney for generation and excretion of NH(4)(+), paralleled by HCO(3)(-) formation. Little is known about the regulation of pendrin in liver. The present study thus examined the expression of Slc26a4 in liver and kidney of mice drinking tap water without or with NaHCO(3) (150 mM), NH(4)Cl (280 mM) or acetazolamide (3.6 mM) for seven days. As compared to Gapdh transcript levels, Slc26a4 transcript levels were moderately lower in liver than in renal tissue. Slc26a4 transcript levels were not significantly affected by NaHCO(3) in liver, but significantly increased by NaHCO(3) in kidney. Pendrin protein expression was significantly enhanced in kidney and reduced in liver by NaHCO(3). Slc26a4 transcript levels were significantly increased by NH(4)Cl and acetazolamide in liver, and significantly decreased by NH(4)Cl and by acetazolamide in kidney. NH(4)Cl and acetazolamide reduced pendrin protein expression significantly in kidney, but did not significantly modify pendrin protein expression in liver. The observations point to expression of pendrin in the liver and to opposite effects of acidosis on pendrin transcription in liver and kidney.
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Angiotensin II stimulates H?-ATPase activity in intercalated cells from isolated mouse connecting tubules and cortical collecting ducts.
Cell. Physiol. Biochem.
PUBLISHED: 09-21-2011
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Intercalated cells in the collecting duct system express V-type H(+)-ATPases which participate in acid extrusion, bicarbonate secretion, and chloride absorption depending on the specific subtype. The activity of H(+)-ATPases is regulated by acid-base status and several hormones, including angiotensin II and aldosterone. Angiotensin II stimulates chloride absorption mediated by pendrin in type B intercalated cells and this process is energized by the activity of H(+)-ATPases. Moreover, angiotensin II stimulates bicarbonate secretion by the connecting tubule (CNT) and early cortical collecting duct (CCD). In the present study we examined the effect of angiotensin II (10 nM) on H(+)-ATPase activity and localization in isolated mouse connecting tubules and cortical collecting ducts. Angiotensin II stimulated Na(+)-independent intracellular pH recovery about 2-3 fold, and this was abolished by the specific H(+)-ATPase inhibitor concanamycin. The effect of angiotensin II was mediated through type 1 angiotensin II receptors (AT(1)-receptors) because it could be blocked by saralasin. Stimulation of H(+)-ATPase activity required an intact microtubular network--it was completely inhibited by colchicine. Immunocytochemistry of isolated CNT/CCDs incubated in vitro with angiotensin II suggests enhanced membrane associated staining of H(+)-ATPases in pendrin expressing intercalated cells. In summary, angiotensin II stimulates H(+)-ATPases in CNT/CCD intercalated cells, and may contribute to the regulation of chloride absorption and bicarbonate secretion in this nephron segment.
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The anion exchanger pendrin (SLC26A4) and renal acid-base homeostasis.
Cell. Physiol. Biochem.
PUBLISHED: 08-22-2011
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The anion exchanger pendrin (Pds, SLC26A4) transports various anions including bicarbonate, chloride and iodide. In the kidney, pendrin is exclusively expressed on the luminal pole of bicarbonate-secretory type B intercalated cells. Genetic ablation of pendrin in mice abolishes luminal chloride-bicarbonate exchanger activity from type B intercalated cells suggesting that pendrin is the apical bicarbonate extruding pathway. The renal expression of pendrin is developmentally adapted and pendrin positive cells originate from both the uretric bud and mesenchyme. In adult kidney, pendrin expression and activity is regulated by systemic acid-base status, dietary electrolyte intake (mostly chloride), and hormones such as angiotensin II and aldosterone which can affect subcellular localization, the relative number of pendrin expressing cells, and the overall abundance consistent with a role of pendrin in maintaining normal acid-base homeostasis. This review summarizes recent findings on the role and regulation of pendrin in the context of the kidneys role in acid-base homeostasis in health and disease.
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Aldosterone stimulates vacuolar H(+)-ATPase activity in renal acid-secretory intercalated cells mainly via a protein kinase C-dependent pathway.
Am. J. Physiol., Cell Physiol.
PUBLISHED: 08-10-2011
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Urinary acidification in the collecting duct is mediated by the activity of H(+)-ATPases and is stimulated by various factors including angiotensin II and aldosterone. Classically, aldosterone effects are mediated via the mineralocorticoid receptor. Recently, we demonstrated a nongenomic stimulatory effect of aldosterone on H(+)-ATPase activity in acid-secretory intercalated cells of isolated mouse outer medullary collecting ducts (OMCD). Here we investigated the intracellular signaling cascade mediating this stimulatory effect. Aldosterone stimulated H(+)-ATPase activity in isolated mouse and human OMCDs. This effect was blocked by suramin, a general G protein inhibitor, and GP-2A, a specific G(?q) inhibitor, whereas pertussis toxin was without effect. Inhibition of phospholipase C with U-73122, chelation of intracellular Ca(2+) with BAPTA, and blockade of protein kinase C prevented the stimulation of H(+)-ATPases. Stimulation of PKC by DOG mimicked the effect of aldosterone on H(+)-ATPase activity. Similarly, aldosterone and DOG induced a rapid translocation of H(+)-ATPases to the luminal side of OMCD cells in vivo. In addition, PD098059, an inhibitor of ERK1/2 activation, blocked the aldosterone and DOG effects. Inhibition of PKA with H89 or KT2750 prevented and incubation with 8-bromoadenosine-cAMP mildly increased H(+)-ATPase activity. Thus, the nongenomic modulation of H(+)-ATPase activity in OMCD-intercalated cells by aldosterone involves several intracellular pathways and may be mediated by a G(?q) protein-coupled receptor and PKC. PKA and cAMP appear to have a modulatory effect. The rapid nongenomic action of aldosterone may participate in the regulation of H(+)-ATPase activity and contribute to final urinary acidification.
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Proliferation of acid-secretory cells in the kidney during adaptive remodelling of the collecting duct.
PLoS ONE
PUBLISHED: 06-30-2011
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The renal collecting duct adapts to changes in acid-base metabolism by remodelling and altering the relative number of acid or alkali secreting cells, a phenomenon termed plasticity. Acid secretory A intercalated cells (A-IC) express apical H(+)-ATPases and basolateral bicarbonate exchanger AE1 whereas bicarbonate secretory B intercalated cells (B-IC) express basolateral (and apical) H(+)-ATPases and the apical bicarbonate exchanger pendrin. Intercalated cells were thought to be terminally differentiated and unable to proliferate. However, a recent report in mouse kidney suggested that intercalated cells may proliferate and that this process is in part dependent on GDF-15. Here we extend these observations to rat kidney and provide a detailed analysis of regional differences and demonstrate that differentiated A-IC proliferate massively during adaptation to systemic acidosis. We used markers of proliferation (PCNA, Ki67, BrdU incorporation) and cell-specific markers for A-IC (AE1) and B-IC (pendrin). Induction of remodelling in rats with metabolic acidosis (with NH(4)Cl for 12 hrs, 4 and 7 days) or treatment with acetazolamide for 10 days resulted in a larger fraction of AE1 positive cells in the cortical collecting duct. A large number of AE1 expressing A-IC was labelled with proliferative markers in the cortical and outer medullary collecting duct whereas no labeling was found in B-IC. In addition, chronic acidosis also increased the rate of proliferation of principal collecting duct cells. The fact that both NH(4)Cl as well as acetazolamide stimulated proliferation suggests that systemic but not urinary pH triggers this response. Thus, during chronic acidosis proliferation of AE1 containing acid-secretory cells occurs and may contribute to the remodelling of the collecting duct or replace A-IC due to a shortened life span under these conditions.
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PKB/SGK-resistant GSK3 enhances phosphaturia and calciuria.
J. Am. Soc. Nephrol.
PUBLISHED: 04-14-2011
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Insulin and IGF1-dependent signaling activates protein kinase B and serum and glucocorticoid inducible kinase (PKB/SGK), which together phosphorylate and inactivate glycogen synthase kinase GSK3. Because insulin and IGF1 increase renal tubular calcium and phosphorus reabsorption, we examined GSK3 regulation of phosphate transporter activity and determined whether PKB/SGK inactivates GSK3 to enhance renal phosphate and calcium transport. Overexpression of GSK3 and the phosphate transporter NaPi-IIa in Xenopus oocytes decreased electrogenic phosphate transport compared with NaPi-IIa-expressing oocytes. PKB/SGK serine phosphorylation sites in GSK3 were mutated to alanine to create gsk3(KI) mice resistant to PKB/SGK inactivation. Compared with wildtype animals, gsk3(KI) animals exhibited greater urinary phosphate and calcium clearances with higher excretion rates and lower plasma concentrations. Isolated brush border membranes from gsk3(KI) mice showed less sodium-dependent phosphate transport and Na-phosphate co-transporter expression. Parathyroid hormone, 1,25-OH vitamin D levels, and bone mineral density were decreased in gsk3(KI) mice, suggesting a global dysregulation of bone mineral metabolism. Taken together, PKB/SGK phosphorylation of GSK3 increases phosphate transporter activity and reduces renal calcium and phosphate loss.
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Decreased bone density and increased phosphaturia in gene-targeted mice lacking functional serum- and glucocorticoid-inducible kinase 3.
Kidney Int.
PUBLISHED: 03-30-2011
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Insulin and growth factors activate the phosphatidylinositide-3-kinase pathway, leading to stimulation of several kinases including serum- and glucocorticoid-inducible kinase isoform SGK3, a transport regulating kinase. Here, we explored the contribution of SGK3 to the regulation of renal tubular phosphate transport. Coexpression of SGK3 and sodium-phosphate cotransporter IIa significantly enhanced the phosphate-induced current in Xenopus oocytes. In sgk3 knockout and wild-type mice on a standard diet, fluid intake, glomerular filtration and urine flow rates, and urinary calcium ion excretion were similar. However, fractional urinary phosphate excretion was slightly but significantly larger in the knockout than in wild-type mice. Plasma calcium ion, phosphate concentration, and plasma parathyroid hormone levels were not significantly different between the two genotypes, but plasma calcitriol and fibroblast growth factor 23 concentrations were significantly lower in the knockout than in wild-type mice. Moreover, bone density was significantly lower in the knockouts than in wild-type mice. Histological analysis of the femur did not show any differences in cortical bone but there was slightly less prominent trabecular bone in sgk3 knockout mice. Thus, SGK3 has a subtle but significant role in the regulation of renal tubular phosphate transport and bone density.
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CNNM2, encoding a basolateral protein required for renal Mg2+ handling, is mutated in dominant hypomagnesemia.
Am. J. Hum. Genet.
PUBLISHED: 02-14-2011
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Familial hypomagnesemia is a rare human disorder caused by renal or intestinal magnesium (Mg(2+)) wasting, which may lead to symptoms of Mg(2+) depletion such as tetany, seizures, and cardiac arrhythmias. Our knowledge of the physiology of Mg(2+) (re)absorption, particularly the luminal uptake of Mg(2+) along the nephron, has benefitted from positional cloning approaches in families with Mg(2+) reabsorption disorders; however, basolateral Mg(2+) transport and its regulation are still poorly understood. Here, by using a candidate screening approach, we identified CNNM2 as a gene involved in renal Mg(2+) handling in patients of two unrelated families with unexplained dominant hypomagnesemia. In the kidney, CNNM2 was predominantly found along the basolateral membrane of distal tubular segments involved in Mg(2+) reabsorption. The basolateral localization of endogenous and recombinant CNNM2 was confirmed in epithelial kidney cell lines. Electrophysiological analysis showed that CNNM2 mediated Mg(2+)-sensitive Na(+) currents that were significantly diminished in mutant protein and were blocked by increased extracellular Mg(2+) concentrations. Our data support the findings of a recent genome-wide association study showing the CNNM2 locus to be associated with serum Mg(2+) concentrations. The mutations found in CNNM2, its observed sensitivity to extracellular Mg(2+), and its basolateral localization signify a critical role for CNNM2 in epithelial Mg(2+) transport.
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Induction of metabolic acidosis with ammonium chloride (NH4Cl) in mice and rats--species differences and technical considerations.
Cell. Physiol. Biochem.
PUBLISHED: 11-04-2010
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Ammonium chloride addition to drinking water is often used to induce metabolic acidosis (MA) in rodents but may also cause mild dehydration. Previous microarray screening of acidotic mouse kidneys showed upregulation of genes involved in renal water handling. Thus, we compared two protocols to induce metabolic acidosis in mice and rats: standard 0.28M NH(4)Cl in drinking water or an equivalent amount of NH(4)Cl in food. Both treatments induced MA in mice and rats. In rats, NH (4)Cl in water caused signs of dehydration, reduced mRNA abundance of the vasopression receptor 2 (V2R), increased protein abundance of the aquaporin water channels AQP2 and AQP3 and stimulated phosphorylation of AQP2 at residues Ser256 and Ser261. In contrast, NH(4)Cl in food induced massive diuresis, decreased mRNA levels of V2R, AQP2, and AQP3, did not affect protein abundance of AQP2 and AQP3, and stimulated phosphorylation at Ser261 but not pSer256 of AQP2. In mice, NH(4)Cl in drinking water stimulated urine concentration, increased AQP2 and V2R mRNA levels, and enhanced AQP2 and AQP3 protein expression with higher levels of AQP2 pSer256 and pSer261. Addition of NH(4)Cl to food, stimulated diuresis, had no effect on mRNA levels of AQP2, AQP3, and V2R, and enhanced only AQP3 protein abundance whereas pSer256-AQP2 and pSer261-AQP2 remained unaltered. Similarly, AQP2 staining was more intense and luminal in kidney from mice with NH(4)Cl in water but not in food. Pendrin, SNAT3 and PEPCK mRNA expression in mouse kidney were not affected by the route of N(4)Cl application. Thus, addition of NH(4)Cl to water or food causes MA but has differential effects on diuresis and expression of mRNAs and proteins related to renal water handling. Moreover, mice and rats respond differently to NH(4)Cl loading, and increased water intake and diuresis may be a compensatory mechanism during MA. It may be necessary to consider these effects in planning and interpreting experiments of NH(4)Cl supplementation to animals.
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Toward quantitative proteomics of organ substructures: implications for renal physiology.
Semin. Nephrol.
PUBLISHED: 11-04-2010
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Organs are complex structures that consist of multiple tissues with different levels of gene expression. To achieve comprehensive coverage and accurate quantitation data, organs ideally should be separated into morphologic and/or functional substructures before gene or protein expression analysis. However, because of complex morphology and elaborate isolation protocols, to date this often has been difficult to achieve. Kidneys are organs in which functional and morphologic subdivision is especially important. Each subunit of the kidney, the nephron, consists of more than 10 subsegments with distinct morphologic and functional characteristics. For a full understanding of kidney physiology, global gene and protein expression analyses have to be performed at the level of the nephron subsegments; however, such studies have been extremely rare to date. Here we describe the latest approaches in quantitative high-accuracy mass spectrometry-based proteomics and their application to quantitative proteomics studies of the whole kidney and nephron subsegments, both in human beings and in animal models. We compare these studies with similar studies performed on other organ substructures. We argue that the newest technologies used for preparation, processing, and measurement of small amounts of starting material are finally enabling global and subsegment-specific quantitative measurement of protein levels in the kidney and other organs. These new technologies and approaches are making a decisive impact on our understanding of the (patho)physiological processes at the molecular level.
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Urinary pH and stone formation.
J. Nephrol.
PUBLISHED: 11-01-2010
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The formation of various types of kidney stones is strongly influenced by urinary pH. An alkaline pH favors the crystallization of calcium- and phosphate-containing stones, whereas and acidic urine pH promotes uric acid or cystine stones. The activity of many transport processes involved in calcium, citrate and phosphate handling are sensitive to changes in systemic or local pH as shown for several phosphate transporters, the citrate transporter NaDC1 and the TRPV5 calcium channel. Defects in urinary acidification (excretion of inappropriately alkaline or acidic urines, respectively) contribute to kidney stone disease. The low excretion of ammonium in patients with metabolic syndrome has been linked to more acidic urine and a higher incidence of uric acid stones. In this state, insulin resistance may reduce ammonium excretion by the proximal tubule. On the other hand, defensive mechanisms may protect from kidney stone formation in conditions such as hypercalciuria where high luminal calcium concentrations stimulate urinary acidification and reduce urinary concentration via a calcium-sensing receptor, resulting in the excretion of acidic and diluted urine. This review will discuss a few aspects that relate to the capacity of the kidney to regulate pH and its impact on the excretion of solutes that participate in the formation or prevention of stones.
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The rhesus protein RhCG: a new perspective in ammonium transport and distal urinary acidification.
Kidney Int.
PUBLISHED: 10-06-2010
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Urinary acidification is a complex process requiring the coordinated action of enzymes and transport proteins and resulting in the removal of acid and the regeneration of bicarbonate. Proton secretion is mediated by luminal H(+)-ATPases and requires the parallel movement of NH?, and its protonation to NH?(+), to provide sufficient buffering. It has been long assumed that ammonia secretion is a passive process occurring by means of simple diffusion driven by the urinary trapping of ammonium. However, new data indicate that mammalian cells possess specific membrane proteins from the family of rhesus proteins involved in ammonia/?m permeability. Rhesus proteins were first identified in yeast and later also in plants, algae, and mammals. In rodents, RhBG and RhCG are expressed in the collecting duct, whereas in humans only RhCG was detected. Their expression increases with maturation of the kidney and accelerates after birth in parallel with other acid-base transport proteins. Deletion of RhBG in mice had no effect on renal ammonium excretion, whereas RhCG deficiency reduces renal ammonium secretion strongly, causes metabolic acidosis in acid-challenged mice, and impairs restoration of normal acid-base status. Microperfusion experiments or functional reconstitution in liposomes demonstrates that ammonia is the most likely substrate of RhCG. Similarly, crystal structures of human RhCG and the homologous bacterial AmtB protein suggest that these proteins may form gas channels.
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Anion exchanger 1 interacts with nephrin in podocytes.
J. Am. Soc. Nephrol.
PUBLISHED: 06-24-2010
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The central role of the multifunctional protein nephrin within the macromolecular complex forming the glomerular slit diaphragm is well established, but the mechanisms linking the slit diaphragm to the cytoskeleton and to the signaling pathways involved in maintaining the integrity of the glomerular filter remain incompletely understood. Here, we report that nephrin interacts with the bicarbonate/chloride transporter kidney anion exchanger 1 (kAE1), detected by yeast two-hybrid assay and confirmed by immunoprecipitation and co-localization studies. We confirmed low-level glomerular expression of kAE1 in human and mouse kidneys by immunoblotting and immunofluorescence microscopy. We observed less kAE1 in human glomeruli homozygous for the NPHS1(FinMaj) nephrin mutation, whereas kAE1 expression remained unchanged in the collecting duct. We could not detect endogenous kAE1 expression in NPHS1(FinMaj) podocytes in primary culture, but heterologous re-introduction of wild-type nephrin into these podocytes rescued kAE1 expression. In kidneys of Ae1(-/-) mice, nephrin abundance was normal but its distribution was altered along with the reported kAE1-binding protein integrin-linked kinase (ILK). Ae1(-/-) mice had increased albuminuria with glomerular enlargement, mesangial expansion, mesangiosclerosis, and expansion of the glomerular basement membrane. Glomeruli with ILK-deficient podocytes also demonstrated altered AE1 and nephrin expression, further supporting the functional interdependence of these proteins. These data suggest that the podocyte protein kAE1 interacts with nephrin and ILK to maintain the structure and function of the glomerular basement membrane.
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Localization of members of MCT monocarboxylate transporter family Slc16 in the kidney and regulation during metabolic acidosis.
Am. J. Physiol. Renal Physiol.
PUBLISHED: 04-14-2010
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The monocarboxylate transporter family (MCT) comprises 14 members with distinct transport properties and tissue distribution. The kidney expresses several members of the MCT family, but only little is known about their exact distribution and function. Here, we investigated selected members of the MCT family in the mouse kidney. MCT1, MCT2, MCT7, and MCT8 localized to basolateral membranes of the epithelial cells lining the nephron. MCT1 and MCT8 were detected in proximal tubule cells whereas MCT7 and MCT2 were located in the thick ascending limb and the distal tubule. CD147, a beta-subunit of MCT1 and MCT4, showed partially overlapping expression with MCT1 and MCT2. However, CD147 was also found in intercalated cells. We also detected SMCT1 and SMCT2, two Na(+)-dependent monocarboxylate cotransporters, on the luminal membrane of type A intercalated cells. Moreover, mice were given an acid load for 2 and 7 days. Acidotic animals showed a marked but transient increase in urinary lactate excretion. During acidosis, a downregulation of MCT1, MCT8, and SMCT2 was observed at the mRNA level, whereas MCT7 and SMCT1 showed increased mRNA abundance. Only MCT7 showed lower protein abundance whereas all other transporters remained unchanged. In summary, we describe for the first time the localization of various MCT transporters in mammalian kidney and demonstrate that metabolic acidosis induces a transient increase in urinary lactate excretion paralleled by lower MCT7 protein expression.
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Acute parathyroid hormone differentially regulates renal brush border membrane phosphate cotransporters.
Pflugers Arch.
PUBLISHED: 02-12-2010
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Renal phosphate reabsorption across the brush border membrane (BBM) in the proximal tubule is mediated by at least three transporters, NaPi-IIa (SLC34A1), NaPi-IIc (SLC34A3), and Pit-2 (SLC20A2). Parathyroid hormone (PTH) is a potent phosphaturic factor exerting an acute and chronic reduction in proximal tubule phosphate reabsorption. PTH acutely induces NaPi-IIa internalization from the BBM and lysosomal degradation, but its effects on NaPi-IIc and Pit-2 are unknown. In rats adapted to low phosphate diet, acute (30 and 60 min) application of PTH decreased BBM phosphate transport rates both in the absence and the presence of phosphonoformic acid, an inhibitor of SLC34 but not SLC20 transporters. Immunohistochemistry showed NaPi-IIa expression in the S1 to the S3 segment of superficial and juxtamedullary nephrons; NaPi-IIc was only detectable in S1 segments and Pit-2 in S1 and weakly in S2 segments of superficial and juxtamedullary nephrons. PTH reduced NaPi-IIa staining in the BBM with increased intracellular and lysosomal appearance. NaPi-IIa internalization was most prominent in S1 segments of superficial nephrons. We did not detect changes in NaPi-IIc and Pit-2 staining over this time period. Blockade of lysosomal protein degradation with leupeptin revealed NaPi-IIa accumulation in lysosomes, but no lysosomal staining for NaPi-IIc or Pit-2 could be detected. Immunoblotting of BBM confirmed the reduction in NaPi-IIa abundance and the absence of any effect on NaPi-IIc expression. Pit-2 protein abundance was also significantly reduced by PTH. Thus, function and expression of BBM phosphate cotransporters are differentially regulated allowing for fine-tuning of renal phosphate reabsorption.
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New roles for renal potassium channels.
J. Nephrol.
PUBLISHED: 01-22-2010
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The role of the kidney in controlling and maintaining plasma potassium levels in the normal range requires the presence and activity of renal potassium channels, and their importance has been highlighted in patients with Bartter syndrome harboring mutations in the ROMK (Kir1.1, KCJN1) channel and hyperkalemia. However, the kidney expresses far more potassium channels than ROMK. Their functions are slowly emerging from studies in animal models and human rare inherited disorders that allow a better understanding of the plethora of functions that potassium channels fulfill in the kidney. Three recent studies shed light on the function of 2 members of the family of voltage-gated potassium channels. The group of René Bindels demonstrates that patients with isolated hypomagnesemia and inappropriately normal magnesuria carry mutations in the Kv1.1 (KCNA) potassium channel (Glaudemans B, et al. J Clin Invest. 2009;119:936-942). Two other studies elucidate a rather complex syndrome involving seizures, ataxia, deafness and renal salt loss, and show that mutations in the Kir4.1 (KCNJ10) potassium channel are responsible (Scholl UI, et al. Proc Natl Acad Sci U S A. 2009;106:5842-5847; Bockenhauer D, et al. N Engl J Med. 2009;360:1960-1970). This human disease is recapitulated by a mouse model deficient for the Kir4.1 channel presenting with similar symptoms. These studies together show that potassium channels in the kidney serve purposes far beyond controlling systemic potassium homeostasis, and are involved in various essential functions of the kidney. Moreover, defects of 2 different potassium channels expressed on opposing membrane domains of the same cells cause distinct symptoms.
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Downregulation of mouse intestinal Na(+)-coupled glucose transporter SGLT1 by gum arabic (Acacia Senegal).
Cell. Physiol. Biochem.
PUBLISHED: 01-12-2010
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Intestinal Na(+)-coupled glucose transporter SGLT1 determines the rate of glucose transport, which in turn influences glucose-induced insulin release and development of obesity. The present study explored effects of Gum Arabic (GA), a dietary polysaccharide from dried exudates of Acacia Senegal, on intestinal glucose transport and body weight in wild-type C57Bl/6 mice. Treatment with GA (100 g/l) in drinking water for four weeks did not affect intestinal SGLT1 transcript levels but decreased SGLT1 protein abundance in jejunal brush border membrane vesicles. Glucose-induced jejunal short-circuit currents revealed that GA treatment decreased electrogenic glucose transport. Drinking a 20% glucose solution for four weeks significantly increased body weight and fasting plasma glucose concentrations, effects significantly blunted by simultaneous treatment with GA. GA further significantly blunted the increase in body weight, fasting plasma glucose and fasting insulin concentrations during high fat diet. In conclusion, the present observations disclose a completely novel effect of gum arabic, i.e. its ability to decrease intestinal SGLT1 expression and activity and thus to counteract glucose-induced obesity.
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cAMP stimulates apical V-ATPase accumulation, microvillar elongation, and proton extrusion in kidney collecting duct A-intercalated cells.
Am. J. Physiol. Renal Physiol.
PUBLISHED: 01-06-2010
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Kidney proton-secreting A-intercalated cells (A-IC) respond to systemic acidosis by accumulating the vacuolar ATPase (V-ATPase) in their apical membrane and by increasing the length and number of apical microvilli. We show here that the cell-permeant cAMP analog CPT-cAMP, infused in vivo, results in an almost twofold increase in apical V-ATPase accumulation in AE1-positive A-IC within 15 min and that these cells develop an extensive array of apical microvilli compared with controls. In contrast, no significant change in V-ATPase distribution could be detected by immunocytochemistry in B-intercalated cells at the acute time point examined. To show a direct effect of cAMP on A-IC, we prepared cell suspensions from the medulla of transgenic mice expressing EGFP in IC (driven by the B1-subunit promoter of the V-ATPase) and exposed them to cAMP analogs in vitro. Three-dimensional reconstructions of confocal images revealed that cAMP induced a time-dependent growth of apical microvilli, starting within minutes after addition. This effect was blocked by the PKA inhibitor myristoylated PKI. These morphological changes were paralleled by increased cAMP-mediated proton extrusion (pHi recovery) by A-IC in outer medullary collecting ducts measured using the ratiometric probe BCECF. These results, and our prior data showing that the bicarbonate-stimulated soluble adenylyl cyclase (sAC) is highly expressed in kidney intercalated cells, support the idea that cAMP generated either by sAC, or by activation of other signaling pathways, is part of the signal transduction mechanism involved in acid-base sensing and V-ATPase membrane trafficking in kidney intercalated cells.
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The "antibodyome": or, how to find antibodies?
J. Nephrol.
PUBLISHED: 08-08-2009
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The importance of alloantibodies in acute and chronic allograft rejection has been highlighted over the past few decades. Besides human leukocyte antigen (HLA) antibodies, representing the most important group of antibodies in renal transplantation, non-HLA antigens such as MHC class I polypeptide-related sequence A (MICA) antibodies have also been implicated in renal allograft outcome. However, the identification of alloantibodies and autoantibodies and their antigens is technically difficult. Recently, Li and colleagues have performed an integrative genomic analysis of serological responses to non-HLA antigens in 18 pediatric renal transplant patients to identify new types of antibodies and their tissue specificity by cross-mapping kidney compartment-specific gene probes to protein targets on a protein array (1). Several non-HLA targets were detected against kidney compartment-specific antigens with the highest signal recognition for the renal pelvis. On the one hand, this integrative approach may provide a powerful and cost-efficient tool to detect autoantibodies involved in autoimmune diseases to investigate their specificity, relevance and pathogenetic role. On the other hand, the clinical relevance of these autoantibodies remains to be shown, and it should be explained how mainly intracellular proteins can provide relevant antigens.
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The calcium-sensing receptor promotes urinary acidification to prevent nephrolithiasis.
J. Am. Soc. Nephrol.
PUBLISHED: 05-21-2009
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Hypercalciuria increases the risk for urolithiasis, but renal adaptive mechanisms reduce this risk. For example, transient receptor potential vanilloid 5 knockout (TPRV5(-/-)) mice lack kidney stones despite urinary calcium (Ca(2+)) wasting and hyperphosphaturia, perhaps as a result of their significant polyuria and urinary acidification. Here, we investigated the mechanisms linking hypercalciuria with these adaptive mechanisms. Exposure of dissected mouse outer medullary collecting ducts to high (5.0 mM) extracellular Ca(2+) stimulated H(+)-ATPase activity. In TRPV5(-/-) mice, activation of the renal Ca(2+)-sensing receptor promoted H(+)-ATPase-mediated H(+) excretion and downregulation of aquaporin 2, leading to urinary acidification and polyuria, respectively. Gene ablation of the collecting duct-specific B1 subunit of H(+)-ATPase in TRPV5(-/-) mice abolished the enhanced urinary acidification, which resulted in severe tubular precipitations of Ca(2+)-phosphate in the renal medulla. In conclusion, activation of Ca(2+)-sensing receptor by increased luminal Ca(2+) leads to urinary acidification and polyuria. These beneficial adaptations facilitate the excretion of large amounts of soluble Ca(2+), which is crucial to prevent the formation of kidney stones.
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Potassium restriction, high protein intake, and metabolic acidosis increase expression of the glutamine transporter SNAT3 (Slc38a3) in mouse kidney.
Am. J. Physiol. Renal Physiol.
PUBLISHED: 05-20-2009
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Kidneys produce ammonium to buffer and excrete acids through metabolism of glutamine. Expression of the glutamine transporter Slc38a3 (SNAT3) increases in kidney during metabolic acidosis (MA), suggesting a role during ammoniagenesis. Potassium depletion and high dietary protein intake are known to elevate renal ammonium excretion. In this study, we examined SNAT3, phosphate-dependent glutaminase (PDG), and phosphoenolpyruvate carboxykinase (PEPCK) regulation during a control (0.36%) or low-K(+) (0.02%) diet for 7 or 14 days or a control (20%) or high-protein (50%) diet for 7 days. MA was induced in control and low-K(+) groups by addition of NH(4)Cl. Urinary ammonium excretion increased during MA, after 14-day K(+) restriction alone, and during high protein intake. SNAT3, PDG, and PEPCK mRNA abundance were elevated during MA and after 14-day K(+) restriction but not during high protein intake. SNAT3 protein abundance was enhanced during MA (both control and low K(+)), after 14-day low-K(+) treatment alone, and during high protein intake. Seven-day dietary K(+) depletion alone had no effect. Immunohistochemistry showed SNAT3 staining in earlier parts of the proximal tubule during 14-day K(+) restriction with and without NH(4)Cl treatment and during high protein intake. In summary, SNAT3, PDG, and PEPCK mRNA expression were congruent with urinary ammonium excretion during MA. Chronic dietary K(+) restriction, high protein intake, and MA enhance ammoniagenesis, an effect that may involve enhanced SNAT3 mRNA and protein expression. Our data suggest that SNAT3 plays an important role as the glutamine uptake mechanism in ammoniagenesis under these conditions.
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The calcineurin inhibitor FK506 (tacrolimus) is associated with transient metabolic acidosis and altered expression of renal acid-base transport proteins.
Am. J. Physiol. Renal Physiol.
PUBLISHED: 05-13-2009
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Calcineurin inhibitors like FK506 (tacrolimus) are routinely used for immunosuppression following transplantation. Its use is limited by many side effects, including renal tubular acidosis (RTA), mainly of the distal type. In this study, rats were treated with FK506 and at baseline (after 9 days) systemic acid-base status was similar to that in control animals. However, FK506-treated rats given NH(4)Cl in the drinking water for 2 days developed a more severe metabolic acidosis than control animals. Urine pH was more alkaline, but net acid excretion was normal. After 7 days of acid load, all differences related to acid-base homeostasis were equalized in both groups. Protein abundance of type IIa Na-P(i) cotransporter, type 3 Na(+)/H(+) exchanger, and electrogenic Na(+)-bicarbonate cotransporter, and both a4 and B2 subunits of the vacuolar H(+)-ATPase were reduced under baseline conditions, while induction of metabolic acidosis enhanced protein abundance of these transporters in FK506-treated animals. In parallel, protein expression of AE1 was reduced at baseline and increased together with pendrin during NH(4)Cl loading in FK506 rats. Protein abundance of the Na(+)-bicarbonate cotransporter NBCn1 was reduced under baseline conditions but remained downregulated during metabolic acidosis. Morphological analysis revealed an increase in the relative number of non-type A intercalated cells in the connecting tubule and cortical collecting duct at the expense of principal cells. Additionally, subcellular distribution of the a4 subunit of the vacuolar H(+)-ATPase was affected by FK506 with less luminal localization in the connecting tubule and outer medullary collecting duct. These data suggest that FK506 impacts on several major acid-base transport proteins in the kidney, and its use is associated with transient metabolic acidosis and altered expression of key renal acid-base transport proteins.
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Hydrogen sulfide: a new gaseous signal molecule and blood pressure regulator.
J. Nephrol.
PUBLISHED: 04-23-2009
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The gas hydrogen sulfide (H2S) is emerging as a novel regulator of important physiologic functions such as arterial diameter, blood flow and leukocyte adhesion. In addition, it may have antiinflammatory and antiapoptotic effects. H2S has recently attracted much interest as a potent vasorelaxative substance that may establish itself alongside another gaseous signal molecule, nitric oxide (NO). In contrast to NO, the major source of H2S in blood may be production by red blood cells or by vascular smooth muscle cells. H2S is produced from cysteine, involving the enzymes cystathionine beta-synthase and cystathionine gamma-lyase (CSE). The importance of CSE was recently demonstrated in a mouse lacking CSE which showed reduced H2S levels and developed hypertension and reduced endothelium-mediated vasorelaxation. These data establish H2S as a new and important biologic signal molecule and as a new regulator of vascular blood flow and blood pressure.
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Early inhibition of prostaglandin synthesis by n-3 fatty acids determinates histologic severity of necrotizing pancreatitis.
Pancreas
PUBLISHED: 03-20-2009
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Previously, we observed decreased histopathological severity of acute necrotizing pancreatitis (ANP) by parenteral nutrition with n-3 fatty acids. Thus, we now sequentially analyzed the impact of n-3 fatty acids on prostaglandin and leukotriene synthesis in ANP.
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Effects of receptor-mediated endocytosis and tubular protein composition on volume retention in experimental glomerulonephritis.
Am. J. Physiol. Renal Physiol.
PUBLISHED: 02-04-2009
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Human glomerulonephritis (GN) is characterized by sustained proteinuria, sodium retention, hypertension, and edema formation. Increasing quantities of filtered protein enter the renal tubule, where they may alter epithelial transport functions. Exaggerated endocytosis and consequent protein overload may affect proximal tubules, but intrinsic malfunction of distal epithelia has also been reported. A straightforward assignment to a particular tubule segment causing salt retention in GN is still controversial. We hypothesized that 1) trafficking and surface expression of major transporters and channels involved in volume regulation were altered in GN, and 2) proximal tubular endocytosis may influence locally as well as downstream expressed tubular transporters and channels. Effects of anti-glomerular basement membrane GN were studied in controls and megalin-deficient mice with blunted proximal endocytosis. Mice displayed salt retention and elevated systolic blood pressure when proteinuria had reached 10-15 mg/24 h. Surface expression of proximal Na(+)-coupled transporters and water channels was in part [Na(+)-P(i) cotransporter IIa (NaPi-IIa) and aquaporin-1 (AQP1)] increased by megalin deficiency alone, but unchanged (Na(+)/H(+) exchanger 3) or reduced (NaPi-IIa and AQP1) in GN irrespective of the endocytosis defect. In distal epithelia, significant increases in proteolytic cleavage products of alpha-epithelial Na(+) channel (ENaC) and gamma-ENaC were observed, suggesting enhanced tubular sodium reabsorption. The effects of glomerular proteinuria dominated over those of blunted proximal endocytosis in contributing to ENaC cleavage. Our data indicate that ENaC-mediated sodium entry may be the rate-limiting step in proteinuric sodium retention. Enhanced proteolytic cleavage of ENaC points to a novel mechanism of channel activation which may involve the action of filtered plasma proteases.
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Tissue-specific amino acid transporter partners ACE2 and collectrin differentially interact with hartnup mutations.
Gastroenterology
PUBLISHED: 02-03-2009
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Hartnup amino acid transporter B(0)AT1 (SLC6A19) is the major luminal sodium-dependent neutral amino acid transporter of small intestine and kidney proximal tubule. The expression of B(0)AT1 in kidney was recently shown to depend on its association with collectrin (Tmem27), a protein homologous to the membrane-anchoring domain of angiotensin-converting enzyme (ACE) 2.
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Regulated acid-base transport in the collecting duct.
Pflugers Arch.
PUBLISHED: 01-12-2009
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The renal collecting system serves the fine-tuning of renal acid-base secretion. Acid-secretory type-A intercalated cells secrete protons via a luminally expressed V-type H(+)-ATPase and generate new bicarbonate released by basolateral chloride/bicarbonate exchangers including the AE1 anion exchanger. Efficient proton secretion depends both on the presence of titratable acids (mainly phosphate) and the concomitant secretion of ammonia being titrated to ammonium. Collecting duct ammonium excretion requires the Rhesus protein RhCG as indicated by recent KO studies. Urinary acid secretion by type-A intercalated cells is strongly regulated by various factors among them acid-base status, angiotensin II and aldosterone, and the Calcium-sensing receptor. Moreover, urinary acidification by H(+)-ATPases is modulated indirectly by the activity of the epithelial sodium channel ENaC. Bicarbonate secretion is achieved by non-type-A intercalated cells characterized by the luminal expression of the chloride/bicarbonate exchanger pendrin. Pendrin activity is driven by H(+)-ATPases and may serve both bicarbonate excretion and chloride reabsorption. The activity and expression of pendrin is regulated by different factors including acid-base status, chloride delivery, and angiotensin II and may play a role in NaCl retention and blood pressure regulation. Finally, the relative abundance of type-A and non-type-A intercalated cells may be tightly regulated. Dysregulation of intercalated cell function or abundance causes various syndromes of distal renal tubular acidosis underlining the importance of these processes for acid-base homeostasis.
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Mechanisms of pH-gradient driven transport mediated by organic anion polypeptide transporters.
Am. J. Physiol., Cell Physiol.
PUBLISHED: 01-07-2009
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Organic anion transporting polypeptides (humans OATPs, rodents Oatps) are expressed in most mammalian tissues and mediate cellular uptake of a wide variety of amphipathic organic compounds such as bile salts, steroid conjugates, oligopeptides, and a large list of drugs, probably by acting as anion exchangers. In the present study we aimed to investigate the role of the extracellular pH on the transport activity of nine human and four rat OATPs/Oatps. Furthermore, we aimed to test the concept that OATP/Oatp transport activity is accompanied by extrusion of bicarbonate. By using amphibian Xenopus laevis oocytes expressing OATPs/Oatps and mammalian cell lines stably transfected with OATPs/Oatps, we could demonstrate that in all OATPs/Oatps investigated, with the exception of OATP1C1, a low extracellular pH stimulated transport activity. This stimulation was accompanied by an increased substrate affinity as evidenced by lower apparent Michaelis-Menten constant values. OATP1C1 is lacking a highly conserved histidine in the third transmembrane domain, which was shown by site-directed mutagenesis to be critically involved in the pH dependency of OATPs/Oatps. Using online intracellular pH measurements in OATP/Oatp-transfected Chinese Hamster Ovary (CHO)-K1 cells, we could demonstrate the presence of a 4,4-diisothiocyanatostilbene-2,2-disulfonic acid-sensitive chloride/bicarbonate exchanger in CHO-K1 cells and that OATP/Oatp-mediated substrate transport is paralleled by bicarbonate efflux. We conclude that the pH dependency of OATPs/Oatps may lead to a stimulation of substrate transport in an acidic microenvironment and that the OATP/Oatp-mediated substrate transport into cells is generally compensated or accompanied by bicarbonate efflux.
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Altered V-ATPase expression in renal intercalated cells isolated from B1 subunit-deficient mice by fluorescence-activated cell sorting.
Am. J. Physiol. Renal Physiol.
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Unlike human patients with mutations in the 56-kDa B1 subunit isoform of the vacuolar proton-pumping ATPase (V-ATPase), B1-deficient mice (Atp6v1b1(-/-)) do not develop metabolic acidosis under baseline conditions. This is due to the insertion of V-ATPases containing the alternative B2 subunit isoform into the apical membrane of renal medullary collecting duct intercalated cells (ICs). We previously reported that quantitative Western blots (WBs) from whole kidneys showed similar B2 protein levels in Atp6v1b1(-/-) and wild-type mice (P?unescu TG, Russo LM, Da Silva N, Kovacikova J, Mohebbi N, Van Hoek AN, McKee M, Wagner CA, Breton S, Brown D. Am J Physiol Renal Physiol 293: F1915-F1926, 2007). However, WBs from renal medulla (including outer and inner medulla) membrane and cytosol fractions reveal a decrease in the levels of the ubiquitous V-ATPase E1 subunit. To compare V-ATPase expression specifically in ICs from wild-type and Atp6v1b1(-/-) mice, we crossed mice in which EGFP expression is driven by the B1 subunit promoter (EGFP-B1(+/+) mice) with Atp6v1b1(-/-) mice to generate novel EGFP-B1(-/-) mice. We isolated pure IC populations by fluorescence-assisted cell sorting from EGFP-B1(+/+) and EGFP-B1(-/-) mice to compare their V-ATPase subunit protein levels. We report that V-ATPase A, E1, and H subunits are all significantly downregulated in EGFP-B1(-/-) mice, while the B2 protein level is considerably increased in these animals. We conclude that under baseline conditions B2 upregulation compensates for the lack of B1 and is sufficient to maintain basal acid-base homeostasis, even when other V-ATPase subunits are downregulated.
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DOCA sensitive pendrin expression in kidney, heart, lung and thyroid tissues.
Cell. Physiol. Biochem.
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Pendrin (SLC26A4), a transporter accomplishing anion exchange, is expressed in inner ear, thyroid gland, kidneys, lung, liver and heart. Loss or reduction of function mutations of SLC26A4 underlie Pendred syndrome, a disorder invariably leading to hearing loss with enlarged vestibular aqueducts and in some patients to hypothyroidism and goiter. Renal pendrin expression is up-regulated by mineralocorticoids such as aldosterone or deoxycorticosterone (DOCA). Little is known about the impact of mineralocorticoids on pendrin expression in extrarenal tissues.
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Programmed death 1 protects from fatal circulatory failure during systemic virus infection of mice.
J. Exp. Med.
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The inhibitory programmed death 1 (PD-1)-programmed death ligand 1 (PD-L1) pathway contributes to the functional down-regulation of T cell responses during persistent systemic and local virus infections. The blockade of PD-1-PD-L1-mediated inhibition is considered as a therapeutic approach to reinvigorate antiviral T cell responses. Yet previous studies reported that PD-L1-deficient mice develop fatal pathology during early systemic lymphocytic choriomeningitis virus (LCMV) infection, suggesting a host protective role of T cell down-regulation. As the exact mechanisms of pathology development remained unclear, we set out to delineate in detail the underlying pathogenesis. Mice deficient in PD-1-PD-L1 signaling or lacking PD-1 signaling in CD8 T cells succumbed to fatal CD8 T cell-mediated immunopathology early after systemic LCMV infection. In the absence of regulation via PD-1, CD8 T cells killed infected vascular endothelial cells via perforin-mediated cytolysis, thereby severely compromising vascular integrity. This resulted in systemic vascular leakage and a consequential collapse of the circulatory system. Our results indicate that the PD-1-PD-L1 pathway protects the vascular system from severe CD8 T cell-mediated damage during early systemic LCMV infection, highlighting a pivotal physiological role of T cell down-regulation and suggesting the potential development of immunopathological side effects when interfering with the PD-1-PD-L1 pathway during systemic virus infections.
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Secreted Klotho and FGF23 in chronic kidney disease Stage 1 to 5: a sequence suggested from a cross-sectional study.
Nephrol. Dial. Transplant.
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Klotho and fibroblast growth factor 23 (FGF23) are key regulators of mineral metabolism in renal insufficiency. FGF23 levels have been shown to increase early in chronic kidney disease (CKD); however, the corresponding soluble Klotho levels at the different CKD stages are not known.
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OSR1-sensitive renal tubular phosphate reabsorption.
Kidney Blood Press. Res.
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The oxidative stress-responsive kinase 1 (OSR1) participates in the WNK-(with no K) kinase dependent regulation of renal salt excretion and blood pressure. Little is known, however, about the role of OSR1 in the regulation of further renal transport systems. The present study analyzed the effect of OSR1 on NaPiIIa, the major renal tubular phosphate transporter.
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Aldosterone deficiency adversely affects pregnancy outcome in mice.
Pflugers Arch.
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Circulating aldosterone levels are increased in human pregnancy. Inadequately low aldosterone levels as present in preeclampsia, a life-threatening disease for both mother and child, are discussed to be involved in its pathogenesis or severity. Moreover, inactivating polymorphisms in the aldosterone synthase gene have been detected in preeclamptic women. Here, we used aldosterone synthase-deficient (AS(-/-)) mice to test whether the absence of aldosterone is sufficient to impair pregnancy or even to cause preeclampsia. AS(-/-) and AS(+/+) females were mated with AS(+/+) and AS(-/-) males, respectively, always generating AS(+/-) offspring. With maternal aldosterone deficiency in AS(-/-) mice, systolic blood pressure was low before and further reduced during pregnancy with no increase in proteinuria. Yet, AS(-/-) had smaller litters due to loss of fetuses as indicated by a high number of necrotic placentas with massive lymphocyte infiltrations at gestational day 18. Surviving fetuses and their placentas from AS(-/-) females were smaller. High-salt diet before and during pregnancy increased systolic blood pressure only before pregnancy in both genotypes and abolished the difference in blood pressure during late pregnancy. Litter size from AS(-/-) was slightly improved and the differences in placental and fetal weights between AS(+/+) and AS(-/-) mothers disappeared. Overall, an increased placental efficiency was observed in both groups paralleled by a normalization of elevated HIF1? levels in the AS(-/-) placentas. Our results demonstrate that aldosterone deficiency has profound adverse effects on placental function. High dietary salt intake improved placental function. In this animal model, aldosterone deficiency did not cause preeclampsia.
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Sirolimus induced phosphaturia is not caused by inhibition of renal apical sodium phosphate cotransporters.
PLoS ONE
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The vast majority of glomerular filtrated phosphate is reabsorbed in the proximal tubule. Posttransplant phosphaturia is common and aggravated by sirolimus immunosuppression. The cause of sirolimus induced phosphaturia however remains elusive. Male Wistar rats received sirolimus or vehicle for 2 or 7 days (1.5mg/kg). The urine phosphate/creatinine ratio was higher and serum phosphate was lower in sirolimus treated rats, fractional excretion of phosphate was elevated and renal tubular phosphate reabsorption was reduced suggesting a renal cause for hypophosphatemia. PTH was lower in sirolimus treated rats. FGF 23 levels were unchanged at day 2 but lower in sirolimus treated rats after 7 days. Brush border membrane vesicle phosphate uptake was not altered in sirolimus treated groups or by direct incubation with sirolimus. mRNA, protein abundance, and subcellular transporter distribution of NaPi-IIa, Pit-2 and NHE3 were not different between groups but NaPi-IIc mRNA expression was lower at day 7. Transcriptome analyses revealed candidate genes that could be involved in the phosphaturic response. Sirolimus caused a selective renal phosphate leakage, which was not mediated by NaPi-IIa or NaPi-IIc regulation or localization. We hypothesize that another mechanism such as a basolateral phosphate transporter may be responsible for the sirolimus induced phosphaturia.
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The proton-activated G protein coupled receptor OGR1 acutely regulates the activity of epithelial proton transport proteins.
Cell. Physiol. Biochem.
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The Ovarian cancer G protein-coupled Receptor 1 (OGR1; GPR68) is proton-sensitive in the pH range of 6.8 - 7.8. However, its physiological function is not defined to date. OGR1 signals via inositol trisphosphate and intracellular calcium, albeit downstream events are unclear. To elucidate OGR1 function further, we transfected HEK293 cells with active OGR1 receptor or a mutant lacking 5 histidine residues (H5Phe-OGR1). An acute switch of extracellular pH from 8 to 7.1 (10 nmol/l vs 90 nmol/l protons) stimulated NHE and H(+)-ATPase activity in OGR1-transfected cells, but not in H5Phe-OGR1-transfected cells. ZnCl(2) and CuCl(2) that both inhibit OGR1 reduced the stimulatory effect. The activity was blocked by chelerythrine, whereas the ERK1/2 inhibitor PD 098059 had no inhibitory effect. OGR1 activation increased intracellular calcium in transfected HEK293 cells. We next isolated proximal tubules from kidneys of wild-type and OGR1-deficient mice and measured the effect of extracellular pH on NHE activity in vitro. Deletion of OGR1 affected the pH-dependent proton extrusion, however, in the opposite direction as expected from cell culture experiments. Upregulated expression of the pH-sensitive kinase Pyk2 in OGR1 KO mouse proximal tubule cells may compensate for the loss of OGR1. Thus, we present the first evidence that OGR1 modulates the activity of two major plasma membrane proton transport systems. OGR1 may be involved in the regulation of plasma membrane transport proteins and intra- and/or extracellular pH.
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Uriniferous tubule: structural and functional organization.
Compr Physiol
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The uriniferous tubule is divided into the proximal tubule, the intermediate (thin) tubule, the distal tubule and the collecting duct. The present chapter is based on the chapters by Maunsbach and Christensen on the proximal tubule, and by Kaissling and Kriz on the distal tubule and collecting duct in the 1992 edition of the Handbook of Physiology, Renal Physiology. It describes the fine structure (light and electron microscopy) of the entire mammalian uriniferous tubule, mainly in rats, mice, and rabbits. The structural data are complemented by recent data on the location of the major transport- and transport-regulating proteins, revealed by morphological means(immunohistochemistry, immunofluorescence, and/or mRNA in situ hybridization). The structural differences along the uriniferous tubule strictly coincide with the distribution of the major luminal and basolateral transport proteins and receptors and both together provide the basis for the subdivision of the uriniferous tubule into functional subunits. Data on structural adaptation to defined functional changes in vivo and to genetical alterations of specified proteins involved in transepithelial transport importantly deepen our comprehension of the correlation of structure and function in the kidney, of the role of each segment or cell type in the overall renal function,and our understanding of renal pathophysiology.
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Molecular mechanisms of acid-base sensing by the kidney.
J. Am. Soc. Nephrol.
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A major function of the kidney is to collaborate with the respiratory system to maintain systemic acid-base status within limits compatible with normal cell and organ function. It achieves this by regulating the excretion and recovery of bicarbonate (mainly in the proximal tubule) and the secretion of buffered protons (mainly in the distal tubule and collecting duct). How proximal tubular cells and distal professional proton transporting (intercalated) cells sense and respond to changes in pH, bicarbonate, and CO(2) status is a question that has intrigued many generations of renal physiologists. Over the past few years, however, some candidate molecular pH sensors have been identified, including acid/alkali-sensing receptors (GPR4, InsR-RR), kinases (Pyk2, ErbB1/2), pH-sensitive ion channels (ASICs, TASK, ROMK), and the bicarbonate-stimulated adenylyl cyclase (sAC). Some acid-sensing mechanisms in other tissues, such as CAII-PDK2L1 in taste buds, might also have similar roles to play in the kidney. Finally, the function of a variety of additional membrane channels and transporters is altered by pH variations both within and outside the cell, and the expression of several metabolic enzymes are altered by acid-base status in parts of the nephron. Thus, it is possible that a master pH sensor will never be identified. Rather, the kidney seems equipped with a battery of molecules that scan the epithelial cell environment to mount a coordinated physiologic response that maintains acid-base homeostasis. This review collates current knowledge on renal acid-base sensing in the context of a whole organ sensing and response process.
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