Inhibiting calpain 1 and 2 in cyclin G associated kinase–knockout mice mitigates podocyte injury

Abstract Background and Aims The G-protein-coupled receptor 43 (GPR43) is a post-transcriptional regulator involved in cholesterol metabolism. Our previous studies demonstrated that intracellular cholesterol accumulation contributed to podocyte injury in diabetic nephropathy (DN). This study aimed to investigate possible roles of GPR43 activation in lipid nephrotoxicity in DN and to explore its potential mechanisms. Method The experiments were conducted by using diabetic GPR43 knockout mice and cell culture model of podocytes. Renal pathological changes were checked by periodic acid schiff staining, immunohistochemical staining and transmission electron microscopy (TEM). The lipid deposition and free cholesterol contents in kidney tissues were detected by Oil Red O staining, BODIPY staining, and cholesterol quantitative assay. The protein expressions of GPR43, LC3II, p62 and related molecules of LDLR pathway in kidney tissues and podocytes were detected by real-time PCR, immunofluorescent staining, and Western blotting. Results There were decreased plasma level of LDL-cholesterol and cholesterol accumulation in kidney tissues of diabetic GPR43 knockout mice. In vitro study demonstrated that acetate, a stimulator of GPR43, increased LDLR-mediated cholesterol uptake in podocytes, accompanied with reduced cholesterol autophagic degeneration, characterized by inhibited LC3 maturation, p62 degradation, and autophagosome formation. These may evoke synergistic effects attributing to cellular cholesterol accumulation in podocytes. While genetic knockdown or pharmacological inhibition of GPR43 inhibited this effect in podocytes. Furthermore, the activation of GPR43 led to the activation of ERK1/2/EGR1 pathway in podocytes, whereas blocking ERK1/2 activity or reducing EGR1 expression reversed cholesterol influx and the inhibition of autophagy mediated by GPR43 activation in podocytes. Meanwhile, deletion of the GPR43 improved autophagy and inhibited the ERK1/2-EGR1 pathway of podocyte in diabetic mice in vivo. Conclusion Activation of the GPR43 mediated lipid nephrotoxicity contributes to podocyte injury in DN, which was mainly through the activation of ERK/EGR1 pathways. These findings suggested that the GPR43 receptor could be a potential therapeutic target for the prevention of DN progression.

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SIRT1 Alleviates Aldosterone-Induced Podocyte Injury by Suppressing Mitochondrial Dysfunction and NLRP3 Inflammasome Activation

Kidney Diseases ◽

10.1159/000513884 ◽

2021 ◽

pp. 1-13

Author(s):

Mingzhu Jiang ◽

Min Zhao ◽

Mi Bai ◽

Juan Lei ◽

Yanggang Yuan ◽

...

Keyword(s):

Mitochondrial Dysfunction ◽

Knockout Mice ◽

Nlrp3 Inflammasome ◽

Inflammasome Activation ◽

Glomerular Injury ◽

Glomerular Diseases ◽

Podocyte Injury ◽

Pyrin Domain ◽

Nlrp3 Inflammasome Activation

Background: Podocyte injury contributes to progressive glomerulosclerosis. Previously, we demonstrated the important role of the NLR family pyrin domain containing 3 (NLRP3) inflammasome in mediating the podocyte injury induced by aldosterone. Silent mating type information regulation 2 homolog 1 (SIRT1) is an NAD+-dependent deacetylase that is associated with the regulation of cellular inflammation. However, whether the activation of the NLRP3 inflammasome in podocytes is regulated by SIRT1, and the mechanism involved, remains unknown. Methods: In this study, we detected SIRT1 expression in patients with podocyte disease and performed an aldosterone infusion model in podocyte-specific Sirt1 knockout mice. In cultured podocytes, we used plasmids to overexpress SIRT1 and treated the podocyte with aldosterone. Results: SIRT1 was significantly decreased in the glomeruli of patients with podocyte disease. Sirt1-deficient mice showed significant urinary albumin excretion after aldosterone infusion, and the severity of the glomerular injury was significantly greater in podocyte-specific Sirt1 knockout mice than in the wild-type mice. Moreover, podocyte conditional Sirt1 knockout aggravated NLRP3 inflammasome activation and mitochondrial dysfunction (MtD). In vitro, overexpression of SIRT1 inhibited NLRP3 activation, protected against MtD and podocyte injury. Conclusion: Taken together, these findings revealed a novel regulatory mechanism of the NLRP3 inflammasome by SIRT1 by promoting mitochondrial function, which provides some potential targets for the treatment of glomerular diseases.

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Loss of CLDN5 in podocytes deregulates WIF1 to activate WNT signaling and contributes to kidney disease

10.21203/rs.3.rs-285550/v1 ◽

2021 ◽

Author(s):

Jie Yan ◽

Hui Li ◽

Hui Sun ◽

Haotian Guo ◽

Jieying Liu ◽

...

Keyword(s):

Wnt Signaling ◽

Knockout Mice ◽

Targeted Delivery ◽

Nuclear Translocation ◽

Wnt Signaling Pathway ◽

Integral Membrane Protein ◽

Tubular Epithelial Cells ◽

Podocyte Injury ◽

Kidney Fibrosis ◽

Glomerular Ultrastructure

Abstract Although mature podocytes lack tight junctions (TJs) and form slit diaphragms between opposing foot processes, TJ integral membrane protein CLDN5 is predominantly expressed throughout the plasma membrane of podocytes under normal conditions. Here using podocyte specific Cldn5 knockout mice as a model, we identify CLDN5 as a crucial regulator of podocyte function and reveal Cldn5 deletion exacerbates podocyte injury and proteinuria in diabetic nephropathy (DN) mouse model. Mechanistically, CLDN5 absence reduces ZO1 expression and induces the nuclear translocation of ZONAB, followed by transcriptional downregulation of WIF1, which leads to activation of WNT signaling pathway. Knockout Wif1 in podocytes result in the development of proteinuria and typical glomerular ultrastructure change occurring in Cldn5 knockout mice, while targeted delivery of Wif1 to podocytes prevents the development of glomerular nephropathy in Cldn5 knockout diabetic mice. Podocyte-derived WIF1 also plays a paracrine role on tubular epithelial cells, evidenced by animals with podocyte deletion of Cldn5 or Wif1 have worse kidney fibrosis after unilateral ureteral obstruction when compared with littermate controls with intact podocyte WIF1 expression. These findings establish a novel function of podocyte CLDN5 in restricting WNT activity in the kidney.

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Nephrin-Binding Ephrin-B1 at the Slit Diaphragm Controls Podocyte Function through the JNK Pathway

Journal of the American Society of Nephrology ◽

10.1681/asn.2017090993 ◽

2018 ◽

Vol 29 (5) ◽

pp. 1462-1474 ◽

Cited By ~ 8

Author(s):

Yoshiyasu Fukusumi ◽

Ying Zhang ◽

Ryohei Yamagishi ◽

Kanako Oda ◽

Toru Watanabe ◽

...

Keyword(s):

Knockout Mice ◽

Kinase Inhibitor ◽

Conditional Knockout ◽

Slit Diaphragm ◽

Hek293 Cells ◽

Podocyte Injury ◽

Jnk Pathway ◽

Conditional Knockout Mice ◽

Membrane Bound ◽

In Cells

Background B-type ephrins are membrane-bound proteins that maintain tissue function in several organs. We previously reported that ephrin-B1 is localized at the slit diaphragm of glomerular podocytes. However, the function of ephrin-B1 at this location is unclear.Methods We analyzed the phenotype of podocyte-specific ephrin-B1 knockout mice and assessed the molecular association of ephrin-B1 and nephrin, a key molecule of the slit diaphragm, in HEK293 cells and rats with anti-nephrin antibody-induced nephropathy.Results Compared with controls, ephrin-B1 conditional knockout mice displayed altered podocyte morphology, disarrangement of the slit diaphragm molecules, and proteinuria. Ephrin-B1 expressed in HEK293 cells immunoprecipitated with nephrin, which required the basal regions of the extracellular domains of both proteins. Treatment of cells with an anti-nephrin antibody promoted the phosphorylation of nephrin and ephrin-B1. However, phosphorylation of ephrin-B1 did not occur in cells expressing a mutant nephrin lacking the ephrin-B1 binding site or in cells treated with an Src kinase inhibitor. The phosphorylation of ephrin-B1 enhanced the phosphorylation of nephrin and promoted the phosphorylation of c-Jun N-terminal kinase (JNK), which was required for ephrin-B1–promoted cell motility in wound-healing assays. Notably, phosphorylated JNK was detected in the glomeruli of control mice but not ephrin-B1 conditional knockout mice. In rats, the phosphorylation of ephrin-B1, JNK, and nephrin occurred in the early phase (24 hours) of anti-nephrin antibody-induced nephropathy.Conclusions Through interactions with nephrin, ephrin-B1 maintains the structure and barrier function of the slit diaphragm. Moreover, phosphorylation of ephrin-B1 and, consequently, JNK are involved in the development of podocyte injury.

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