unilateral-ureteral-obstruction-model-for-investigating-kidney

Unilateral Ureteral Obstruction Model for Investigating Kidney Interstitial Fibrosis

Kidney fibrosis is the final pathological outcome of progressive chronic kidney disease (CKD). The unilateral ureteral obstruction (UUO) model is widely ...

Nanjing Medical University

The mammalian target of rapamycin (mTOR) was recently identified in two structurally distinct multiprotein complexes: mTORC1 and mTORC2. Previously, we found that Rictor/mTORC2 protects against cisplatin-induced acute kidney injury, but the role and mechanisms for Rictor/mTORC2 in TGFβ1-induced fibroblast activation and kidney fibrosis remains unknown. To study this, we initially treated NRK-49F cells with TGFβ1 and found that TGFβ1 could activate Rictor/mTORC2 signaling in cultured cells. Blocking Rictor/mTORC2 signaling with Rictor or Akt1 small interfering RNAs markedly inhibited TGFβ1-induced fibronection and α-smooth muscle actin expression. Ensuing western blotting or immunostaining results showed that Rictor/mTORC2 signaling was activated in kidney interstitial myofibroblasts from mice with unilateral ureteral obstruction. Next, a mouse model with fibroblast-specific deletion of Rictor was generated. These knockout mice were normal at birth and had no obvious kidney dysfunction or kidney morphological abnormality within 2 months of birth. Compared with control littermates, the kidneys of Rictor knockout mice developed less interstitial extracellular matrix deposition and inflammatory cell infiltration at 1 or 2 weeks after ureteral obstruction. Thus our study suggests that Rictor/mTORC2 signaling activation mediates TGFβ1-induced fibroblast activation and contributes to the development of kidney fibrosis. This may provide a therapeutic target for chronic kidney diseases.

Rictor/mTORC2 signaling mediates TGFβ1-induced fibroblast activation and kidney fibrosis.

Quercetin, a flavonoid found in a wide variety of plants and presented in human diet, displays promising potential in preventing kidney fibroblast activation. However, whether quercetin can ameliorate kidney fibrosis in mice with obstructive nephropathy and the underlying mechanisms remain to be further elucidated. In this study, we found that administration of quercetin could largely ameliorate kidney interstitial fibrosis and macrophage accumulation in the kidneys with obstructive nephropathy. MTORC1, mTORC2, β-catenin as well as Smad signaling were activated in the obstructive kidneys, whereas quercetin could markedly reduce their abundance except Smad3 phosphorylation. In cultured NRK-49F cells, quercetin could inhibit α-SMA and fibronectin (FN) expression induced by TGFβ1 treatment. MTORC1, mTORC2, β-catenin and Smad signaling pathways were stimulated by TGFβ1 at a time dependent manner. Similar to those findings in the obstructive kidneys, mTORC1, mTORC2 and β-catenin, but not Smad signaling pathways were remarkably blocked by quercetin treatment. Together, these results suggest that quercetin inhibits fibroblast activation and kidney fibrosis involving a combined inhibition of mTOR and β-catenin signaling transduction, which may act as a therapeutic candidate for patients with chronic kidney diseases.

Quercetin Inhibits Fibroblast Activation and Kidney Fibrosis Involving the Suppression of Mammalian Target of Rapamycin and β-catenin Signaling.

Metformin, one of the most common prescriptions for patients with type 2 diabetes, is reported to protect the kidney from gentamicin-induced nephrotoxicity. However, the role and mechanisms for metformin in preventing cisplatin-induced nephrotoxicity remains largely unknown. In this study, a single intraperitoneal injection of cisplatin was employed to induce acute kidney injury (AKI) in CD1 mice. The mice exhibited severe kidney dysfunction and histological damage at day 2 after cisplatin injection. Pretreatment of metformin could markedly attenuate cisplatin-induced acute kidney injury, tubular cell apoptosis and inflammatory cell accumulation in the kidneys. Additionally, pretreatment of metformin could enhance both AMPKα phosphorylation and autophagy induction in the kidneys after cisplatin injection. In cultured NRK-52E cells, a rat kidney tubular cell line, metformin could stimulate AMPKα phosphorylation, induce autophagy and inhibit cisplatin-induced cell apoptosis. Blockade of either AMPKα activation or autophagy induction could largely abolish the protective effect of metformin in cisplatin-induced cell death. Together, this study demonstrated that metformin may protect against cisplatin-induced tubular cell apoptosis and AKI through stimulating AMPKα activation and autophagy induction in the tubular cells.

Metformin Protects Against Cisplatin-Induced Tubular Cell Apoptosis and Acute Kidney Injury via AMPKα-regulated Autophagy Induction.

Epidemiologic studies showed the correlation between the deficiency of omega-3 polyunsaturated fatty acids (n-3 PUFAs) and the progression of chronic kidney diseases (CKD), however, the role and mechanisms for n-3 PUFAs in protecting against kidney fibrosis remain obscure. In this study, NRK-49F cells, a rat kidney interstitial fibroblast cell line, were stimulated with TGFβ1. A Caenorhabditis elegans fat-1 transgenic mouse model in which n-3 PUFAs are endogenously produced from n-6 PUFAs owing to the expression of n-3 fatty acid desaturase were deployed. Docosahexaenoic acid (DHA), one member of n-3 PUFAs family, could suppress TGFβ1-induced fibroblast activation at a dose and time dependent manner. Additionally, DHA could largely inhibit TGFβ1-stimulated Akt but not S6 or Smad3 phosphorylation at a time dependent manner. To decipher the role for n-3 PUFAs in protecting against kidney fibrosis, fat-1 transgenic mice were operated with unilateral ureter obstruction (UUO). Compared to the wild types, fat-1 transgenics developed much less kidney fibrosis and inflammatory cell accumulation accompanied by less p-Akt (Ser473), p-Akt (Thr308), p-S6 and p-Smad3 in kidney tissues at day 7 after UUO. Thus, n-3 PUFAs can attenuate fibroblast activation and kidney fibrosis, which may be associated with the inhibition of mTORC2 signaling.

Omega-3 Polyunsaturated Fatty Acids Attenuate Fibroblast Activation and Kidney Fibrosis Involving MTORC2 Signaling Suppression.

Mammalian target of rapamycin (mTOR) signalling controls many essential cellular functions. However, the role of Rictor/mTOR complex 2 (mTORC2) in regulating macrophage activation and kidney fibrosis remains largely unknown. We report here that Rictor/mTORC2 was activated in macrophages from the fibrotic kidneys of mice. Ablation of Rictor in macrophages reduced kidney fibrosis, inflammatory cell accumulation, macrophage proliferation and polarization after unilateral ureter obstruction or ischaemia/reperfusion injury. In bone marrow-derived macrophages (BMMs), deletion of Rictor or blockade of protein kinase Cα inhibited cell migration. Additionally, deletion of Rictor or blockade of Akt abolished interleukin-4-stimulated or transforming growth factor (TGF)-β1-stimulated macrophage M2 polarization. Furthermore, deletion of Rictor downregulated TGF-β1-stimulated upregulation of multiple profibrotic cytokines, including platelet-derived growth factor, vascular endothelial growth factor and connective tissue growth factor, in BMMs. Conditioned medium from TGF-β1-pretreated Rictor macrophages stimulated fibroblast activation less efficiently than that from TGF-β1-pretreated Rictor macrophages. These results demonstrate that Rictor/mTORC2 signalling can promote macrophage activation and kidney fibrosis. Targeting this signalling pathway in macrophages may shine light on ways to protect against kidney fibrosis in patients with chronic kidney diseases. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Rictor/mammalian target of rapamycin complex 2 promotes macrophage activation and kidney fibrosis.

The Wnt/-catenin pathway is crucial in normal development and throughout life, but aberrant activation of this pathway has been linked to kidney fibrosis, although the mechanisms involved remain incompletely determined. Here, we investigated the role of Wnt/-catenin in regulating macrophage activation and the contribution thereof to kidney fibrosis. Treatment of macrophages with Wnt3a exacerbated IL-4- or TGF1-induced macrophage alternative (M2) polarization and the phosphorylation and nuclear translocation of STAT3  Conversely, inhibition of Wnt/-catenin signaling prevented these IL-4- or TGF1-induced processes. In a mouse model, induced deletion of -catenin in macrophages attenuated the fibrosis, macrophage accumulation, and M2 polarization observed in the kidneys of wild-type littermates after unilateral ureter obstruction. This study shows that activation of Wnt/-catenin signaling promotes kidney fibrosis by stimulating macrophage M2 polarization.

Wnt/-Catenin-Promoted Macrophage Alternative Activation Contributes to Kidney Fibrosis.

Kidney fibrosis is a histological hallmark of chronic kidney disease and arises in large part through extracellular matrix deposition by activated fibroblasts. The signaling protein complex mTOR complex 2 (mTORC2) plays a critical role in fibroblast activation and kidney fibrosis. Protein kinase Cα (PKCα) is one of the major sub-pathways of mTORC2, but its role in fibroblast activation and kidney fibrosis remains to be determined. Here, we found that transforming growth factor β1 (TGFβ1) activates PKCα signaling in cultured NRK-49F cells in a time-dependent manner. Blocking PKCα signaling with the chemical inhibitor Go6976 or by transfection with PKCα siRNA largely reduced expression of the autophagy-associated protein lysosomal-associated membrane protein 2 (LAMP2) and also inhibited autophagosome-lysosome fusion and autophagic flux in the cells. Similarly to chloroquine, Go6976 treatment and PKCα siRNA transfection also markedly inhibited TGFβ1-induced fibroblast activation. In murine fibrotic kidneys with unilateral ureteral obstruction (UUO) nephropathy, PKCα signaling is activated in the interstitial myofibroblasts. Go6976 administration largely blocked autophagic flux in fibroblasts in the fibrotic kidneys and attenuated the UUO nephropathy. Together, our findings suggest that blocking PKCα activity may retard autophagic flux and thereby prevent fibroblast activation and kidney fibrosis.

Protein kinase Cα drives fibroblast activation and kidney fibrosis by stimulating autophagic flux.

Wnt/β-catenin, an evolutionary conserved signaling pathway, plays an essential role in modulating kidney injury and repair. Our previous studies demonstrated that Wnt/β-catenin signaling could stimulate macrophage M2 polarization and contribute to kidney fibrosis. However, whether canonical Wnt signaling activation leads to macrophage proliferation during kidney fibrosis remains to be determined.

Canonical Wnt Signaling Promotes Macrophage Proliferation during Kidney Fibrosis.

Sphingosine kinases (Sphks) are the rate-limiting enzymes in the conversion of sphingosine to biologically active sphingosine-1-phosphate. The present study aimed to determine the role of Sphk2 and its downstream targets in renal fibroblast activation and interstitial fibrosis. In the kidney interstitium of patients with renal fibrosis, Sphk2-expressing cells (mainly interstitial fibroblasts) were significantly elevated and highly correlated with disease progression in patients. In a murine model of renal interstitial fibrosis, Sphk2 was upregulated in the kidney of wild-type mice in response to disease progression. Importantly, Sphk2-knockout (KO) mice exhibited significantly lower levels of extracellular matrix (ECM) production and a suppressed inflammatory response in the kidney tissues, compared to those in their wild-type counterparts, whereas the expression of TGF-β1 was unaffected. TGF-β1 effectively upregulated Sphk2 expression in the renal interstitial fibroblast line, NRK-49F, independent of canonical Smad signaling activation. Furthermore, siRNA-mediated Sphk2 knockdown or suppression of Sphk2 activity by ABC294640 exposure effectively attenuated AKT and STAT3 activation and ECM production, but had no effects on Smad2 and Smad3 activation. Sphk2 phosphorylated Fyn to activate downstream STAT3 and AKT, thereby promoting ECM synthesis. Therefore, our findings indicate that targeting Sphk2-Fyn-STAT3/AKT signaling pathway may be a novel therapeutic approach for renal fibrosis.

Sphingosine kinase 2 cooperating with Fyn promotes kidney fibroblast activation and fibrosis via STAT3 and AKT.

Upon occurrence of kidney injury, tubular cells arrested in G2/M stage may promote interstitial fibroblast activation and kidney fibrosis through producing large amounts of pro-fibrotic cytokines. MTORC1 signaling is essential for controlling cell growth, however, the role and mechanisms for mTORC1 in regulating tubular cell cycle progression during kidney fibrosis are not clear. Here we reported that p-S6 abundance was increased at 15 min, reached peak at 1 h and declined from 3 h to 24 h, while the abundance of p-4E-BP1 and p-Histone H3 was increased from 15 min to 24 h in tubular epithelial cells at the similar pattern after serum stimulation. The phosphorylation of 4E-BP1 was prohibited in NRK-52E cells by the transfection of 4E-BP1 plasmid with four phospho-sites mutation (4E-BP1A4). 4E-BP1A4 transfection led to less G2/M cell arrest as well as the production of pro-fibrotic cytokine and extracellular matrix in NRK-52E cells. In addition, aristolochic acid (AA)-induced tubular cell G2/M arrest induced by treatment was also largely attenuated in NRK-52E cells transfected with 4E-BP1A4. In mouse kidneys with UUO nephropathy, p-4E-BP1 abundance was markedly elevated in the mitotic tubular cells. Therefore, these data indicates that suppressing 4E-BP1 phosphorylation may inhibit tubular cell G2/M-arrest and kidney fibrosis.

Inhibition of 4E-BP1 phosphorylation promotes tubular cell escaping from G2/M arrest and ameliorates kidney fibrosis.

The transcription factor Twist1 regulates several processes that could impact kidney disease progression, including epithelial cell differentiation and inflammatory cytokine induction. Podocytes are specialized epithelia that exhibit features of immune cells and could therefore mediate unique effects of Twist1 on glomerular disease. To study Twist1 functions in podocytes during proteinuric kidney disease, we employed a conditional mutant mouse in which Twist1 was selectively ablated in podocytes (Twist1-PKO). Deletion of Twist1 in podocytes augmented proteinuria, podocyte injury, and foot process effacement in glomerular injury models. Twist1 in podocytes constrained renal accumulation of monocytes/macrophages and glomerular expression of CCL2 and the macrophage cytokine TNF-α after injury. Deletion of TNF-α selectively from podocytes had no impact on the progression of proteinuric nephropathy. By contrast, the inhibition of CCL2 abrogated the exaggeration in proteinuria and podocyte injury accruing from podocyte Twist1 deletion. Collectively, Twist1 in podocytes mitigated urine albumin excretion and podocyte injury in proteinuric kidney diseases by limiting CCL2 induction that drove monocyte/macrophage infiltration into injured glomeruli. Myeloid cells, rather than podocytes, further promoted podocyte injury and glomerular disease by secreting TNF-α. These data highlight the capacity of Twist1 in the podocyte to mitigate glomerular injury by curtailing the local myeloid immune response.

Twist1 in podocytes ameliorates podocyte injury and proteinuria by limiting CCL2-dependent macrophage infiltration.

Interleukin (IL)-1 receptor type 1 (IL-1R1) activation triggers a proinflammatory signaling cascade that can exacerbate kidney injury. However, the functions of podocyte IL-1R1 in glomerular disease remain unclear. To study the role of IL-1R1 signaling in podocytes, we selectively ablated podocyte IL-1R1 in mice (PKO mice). We then subjected PKO mice and wild-type controls to two glomerular injury models: nephrotoxic serum (NTS)- and adriamycin-induced nephropathy. Surprisingly, we found that IL-1R1 activation in podocytes limited albuminuria and podocyte injury during NTS- and adriamycin-induced nephropathy. Moreover, deletion of IL-1R1 in podocytes drove podocyte apoptosis and glomerular injury through diminishing Akt activation. Activation of Akt signaling abrogated the differences in albuminuria and podocyte injury between wild-type and PKO mice during NTS. Thus, IL-1R1 signaling in podocytes limits susceptibility to glomerular injury via an Akt-dependent signaling pathway. These data identify an unexpected protective role for IL-1R1 signaling in podocytes in the pathogenesis of glomerular disease. The present study establishes that activation of the receptor for interleukin-1 limits susceptibility to damage to the kidney glomerulus in preclinical mouse models by stimulating Akt signaling cascades inside the podocyte.

IL-1 receptor signaling in podocytes limits susceptibility to glomerular damage.

Activation of the type 1 IL-1 receptor (IL-1R1) triggers a critical innate immune signaling cascade that contributes to the pathogenesis of AKI. However, blockade of IL-1 signaling in AKI has not consistently demonstrated kidney protection. The current murine experiments show that IL-1R1 activation in the proximal tubule exacerbates toxin-induced AKI and cell death through local suppression of apolipoprotein M. By contrast, IL-1R1 activation in endothelial cells ameliorates AKI by restoring VEGFA-dependent endothelial cell viability. Using this information, future delivery strategies can maximize the protective effects of blocking IL-1R1 while mitigating unwanted actions of IL-1R1 manipulation.

Divergent Actions of Renal Tubular and Endothelial Type 1 IL-1 Receptor Signaling in Toxin-Induced AKI.

The transcription factor Twist1 plays a vital role in normal development in many tissue systems and continues to be important throughout life. However, inappropriate Twist1 activity has been associated with kidney injury and fibrosis, though the underlying mechanisms involved remain incomplete. Here, we explored the role of Twist1 in regulating fibroblast behaviors and the development kidney fibrosis. Initially Twist1 protein and activity was found to be markedly increased within interstitial myofibroblasts in fibrotic kidneys in both humans and rodents. Treatment of rat kidney interstitial fibroblasts with transforming growth factor-β1 (a profibrotic factor) also induced Twist1 expression in vitro. Gain- and loss-of-function experiments supported that Twist1 signaling was responsible for transforming growth factor-β1-induced fibroblast activation and fetal bovine serum-induced fibroblast proliferation. Mechanistically, Twist1 protein promoted kidney fibroblast activation by driving the expression of downstream signaling proteins, Prrx1 and Tnc. Twist1 directly enhanced binding to the promoter of Prrx1 but not TNC, whereas the promoter of TNC was directly bound by Prrx1. Finally, mice with fibroblast-specific deletion of Twist1 exhibited less Prrx1 and TNC protein abundance, interstitial extracellular matrix deposition and kidney inflammation in both the unilateral ureteral obstruction and ischemic-reperfusion injury-induced-kidney fibrotic models. Inhibition of Twist1 signaling with Harmine, a β-carboline alkaloid, improved extracellular matrix deposition in both injury models. Thus, our results suggest that Twist1 signaling promotes the activation and proliferation of kidney fibroblasts, contributing to the development of interstitial fibrosis, offering a potential therapeutic target for chronic kidney disease.

Transcription factor Twist1 drives fibroblast activation to promote kidney fibrosis via signaling proteins Prrx1/TNC.

 Chronic kidney disease (CKD) is a global public health issue, with vascular calcification (VC) being a common and deadly complication. Despite its prevalence, the underlying mechanisms of VC remain unclear. In this study, we aimed to investigate whether and how Otubain-2 (OTUB2) contributes to VC.  The relationship between OTUB2 and VC was examined via immunohistochemical and immunofluorescence staining of discarded calcified radial arteries from uremic patients who underwent arteriovenous fistula operations. Additionally, mice were fed a 0.2% adenine diet supplemented with 1.2% phosphorus to establish a model of CKD-related VC. Vascular smooth muscle cell (VSMC)-specific OTUB2 knockout and overexpression were performed  via the delivery of adeno-associated virus 9 vectors to manipulate the expression of OTUB2. Additionally, a calcified VSMC model was established to explore the roles of OTUB2 in VC by evaluating changes in osteogenic marker expression and calcium deposition.  Our results revealed a significant upregulation of OTUB2 expression during VC progression. OTUB2 overexpression upregulated the expression of osteogenic markers and exacerbated VSMC calcification, as verified by Von Kossa and Alizarin red staining. Conversely, VSMC-specific OTUB2 deficiency significantly mitigated adenine diet-induced VC in CKD mice. OTUB2 knockdown or inhibition decreased Yes-associated protein (YAP) abundance. Mechanistically, OTUB2 bound to YAP, decreasing its K48-linked polyubiquitination and inhibiting its subsequent degradation. Knockdown or inhibition of YAP abolished the effect of OTUB2 overexpression on VSMC calcification, indicating a YAP-mediated mechanism. Furthermore, the YAP/TEAD1 complex bound to the promoter of PFKFB3, increasing its transcriptional activity, as determined by CUT&RUN-qPCR. The knockdown or inhibition of PFKFB3 alleviated the procalcific effects of OTUB2.  Our findings indicate that OTUB2 promotes VC at least partially by activating the YAP-PFKFB3 signaling pathway. Targeting OTUB2 may be an appealing therapeutic strategy for VC.

Article	Year
Rictor/mTORC2 signaling mediates TGFβ1-induced fibroblast activation and kidney fibrosis. Kidney international\| PubMed ID: 25970154	2015
Quercetin Inhibits Fibroblast Activation and Kidney Fibrosis Involving the Suppression of Mammalian Target of Rapamycin and β-catenin Signaling. Scientific reports\| PubMed ID: 27052477	2016
Metformin Protects Against Cisplatin-Induced Tubular Cell Apoptosis and Acute Kidney Injury via AMPKα-regulated Autophagy Induction. Scientific reports\| PubMed ID: 27052588	2016
Omega-3 Polyunsaturated Fatty Acids Attenuate Fibroblast Activation and Kidney Fibrosis Involving MTORC2 Signaling Suppression. Scientific reports\| PubMed ID: 28393852	2017
Rictor/mammalian target of rapamycin complex 2 promotes macrophage activation and kidney fibrosis. The Journal of pathology\| PubMed ID: 28585302	2017
Wnt/-Catenin-Promoted Macrophage Alternative Activation Contributes to Kidney Fibrosis. Journal of the American Society of Nephrology : JASN\| PubMed ID: 29021383	2018
Protein kinase Cα drives fibroblast activation and kidney fibrosis by stimulating autophagic flux. The Journal of biological chemistry\| PubMed ID: 29794026	2018
Canonical Wnt Signaling Promotes Macrophage Proliferation during Kidney Fibrosis. Kidney diseases (Basel, Switzerland)\| PubMed ID: 29998124	2018
Sphingosine kinase 2 cooperating with Fyn promotes kidney fibroblast activation and fibrosis via STAT3 and AKT. Biochimica et biophysica acta. Molecular basis of disease\| PubMed ID: 30251698	2018
Inhibition of 4E-BP1 phosphorylation promotes tubular cell escaping from G2/M arrest and ameliorates kidney fibrosis. Cellular signalling\| PubMed ID: 31154001	2019
Twist1 in podocytes ameliorates podocyte injury and proteinuria by limiting CCL2-dependent macrophage infiltration. JCI insight\| PubMed ID: 34369383	2021
IL-1 receptor signaling in podocytes limits susceptibility to glomerular damage. American journal of physiology. Renal physiology\| PubMed ID: 34894725	2022
Divergent Actions of Renal Tubular and Endothelial Type 1 IL-1 Receptor Signaling in Toxin-Induced AKI. Journal of the American Society of Nephrology : JASN\| PubMed ID: 37545036	2023
Transcription factor Twist1 drives fibroblast activation to promote kidney fibrosis via signaling proteins Prrx1/TNC. Kidney international\| PubMed ID: 39181396	2024
OTUB2 contributes to vascular calcification in chronic kidney disease via the YAP-mediated transcription of PFKFB3. Theranostics\| PubMed ID: 39776804	2025

Jiafa Ren

Biography

JoVE Journal Publications

Other Publications