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.
Sclerostin is secreted by osteocytes. As a circulating inhibitor of the Wnt-signaling pathway it inhibits bone formation and contributes to the development of osteoporosis. Sclerostin levels are elevated in patients with chronic kidney disease and end-stage renal disease. Since data for patients after kidney transplantation are scarce, we have prospectively measured sclerostin levels before and during the first year after renal transplantation and have examined the association of sclerostin with parameters of bone mineral metabolism and with bone mineral density.
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.
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.
Renal tubular epithelial cell proliferation and transepithelial cyst fluid secretion are key features in the progression of polycystic kidney disease (PKD). As the role of the apical renal sodium-glucose cotransporters in these processes is not known, we tested whether phlorizin inhibits cyst growth and delays renal disease progression in a rat model of PKD. Glycosuria was induced by subcutaneous injection of phlorizin in male heterozygous (Cy/+) and wild-type Han:SPRD rats. Phlorizin induced immediate and sustained glycosuria and osmotic diuresis in these rats. Cy/+ rats treated with phlorizin for 5 weeks showed a significant increase in creatinine clearance, a lower 2-kidneys/body weight ratio, a lower renal cyst index, and reduced urinary albumin excretion as compared with vehicle-treated Cy/+ rats. Measurement of Ki67 staining found significantly lower cell proliferation in dilated tubules and cysts of Cy/+ rats treated with phlorizin, as well as a marked inhibition of the activated MAP kinase pathway. In contrast, the mTOR pathway remained unaltered. Phlorizin dose dependently inhibited MAP kinase in cultured tubular epithelial cells from Cy/+ rats. Thus, long-term treatment with phlorizin significantly inhibits cystic disease progression in a rat model of PKD. Hence, induction of glycosuria and osmotic diuresis (glycuresis) by renal sodium-glucose cotransporters inhibition could have a therapeutic effect in polycystic kidney disease.
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?.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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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