Diabetic Podocytopathy Research Articles

A human stem cell-derived model reveals pathologic extracellular matrix remodeling in diabetic podocyte injury

Diabetic nephropathy results from chronic (or uncontrolled) hyperglycemia and is the leading cause of kidney failure. The kidney’s glomerular podocytes are highly susceptible to diabetic injury and subsequent non-reversible degeneration. We generated a human induced pluripotent stem (iPS) cell-derived model of diabetic podocytopathy to investigate disease pathogenesis and progression. The model recapitulated hallmarks of podocytopathy that precede proteinuria including retraction of foot processes and podocytopenia (detachment from the extracellular matrix (ECM)). Moreover, hyperglycemia-induced injury to podocytes exacerbated remodeling of the ECM. Specifically, mature podocytes aberrantly increased expression and excessively deposited collagen (IV)α1α1α2 that is normally abundant in the embryonic glomerulus. This collagen (IV) imbalance coincided with dysregulation of lineage-specific proteins, structural abnormalities of the ECM, and podocytopenia – a mechanism not shared with endothelium and is distinct from drug-induced injury. Intriguingly, repopulation of hyperglycemia-injured podocytes on decellularized ECM scaffolds isolated from healthy podocytes attenuated the loss of synaptopodin (a mechanosensitive protein associated with podocyte health). These results demonstrate that human iPS cell-derived podocytes can facilitate in vitro studies to uncover the mechanisms of chronic hyperglycemia and ECM remodeling and guide disease target identification.

RIPK3 causes mitochondrial dysfunction and albuminuria in diabetic podocytopathy through PGAM5-Drp1 signaling

BackgroundReceptor-interacting protein kinase (RIPK)3 is an essential molecule for necroptosis and its role in kidney fibrosis has been investigated using various kidney injury models. However, the relevance and the underlying mechanisms of RIPK3 to podocyte injury in albuminuric diabetic kidney disease (DKD) remain unclear. Here, we investigated the role of RIPK3 in glomerular injury of DKD. MethodsWe analyzed RIPK3 expression levels in the kidneys of patients with biopsy-proven DKD and animal models of DKD. Additionally, to confirm the clinical significance of circulating RIPK3, RIPK3 was measured by ELISA in plasma obtained from a prospective observational cohort of patients with type 2 diabetes, and estimated glomerular filtration rate (eGFR) and urine albumin-to-creatinine ratio (UACR), which are indicators of renal function, were followed up during the observation period. To investigate the role of RIPK3 in glomerular damage in DKD, we induced a DKD model using a high-fat diet in Ripk3 knockout and wild-type mice. To assess whether mitochondrial dysfunction and albuminuria in DKD take a Ripk3-dependent pathway, we used single-cell RNA sequencing of kidney cortex and immortalized podocytes treated with high glucose or overexpressing RIPK3. ResultsRIPK3 expression was increased in podocytes of diabetic glomeruli with increased albuminuria and decreased podocyte numbers. Plasma RIPK3 levels were significantly elevated in albuminuric diabetic patients than in non-diabetic controls (p = 0.002) and non-albuminuric diabetic patients (p = 0.046). The participants in the highest tertile of plasma RIPK3 had a higher incidence of renal progression (hazard ratio [HR] 2.29 [1.05–4.98]) and incident chronic kidney disease (HR 4.08 [1.10–15.13]). Ripk3 knockout improved albuminuria, podocyte loss, and renal ultrastructure in DKD mice. Increased mitochondrial fragmentation, upregulated mitochondrial fission-related proteins such as phosphoglycerate mutase family member 5 (PGAM5) and dynamin-related protein 1 (Drp1), and mitochondrial ROS were decreased in podocytes of Ripk3 knockout DKD mice. In cultured podocytes, RIPK3 inhibition attenuated mitochondrial fission and mitochondrial dysfunction by decreasing p-mixed lineage kinase domain-like protein (MLKL), PGAM5, and p-Drp1 S616 and mitochondrial translocation of Drp1. ConclusionsThe study demonstrates that RIPK3 reflects deterioration of renal function of DKD. In addition, RIPK3 induces diabetic podocytopathy by regulating mitochondrial fission via PGAM5-Drp1 signaling through MLKL. Inhibition of RIPK3 might be a promising therapeutic option for treating DKD.

The redox-sensitive GSK3β is a key regulator of glomerular podocyte injury in type 2 diabetic kidney disease

Emerging evidence suggests that GSK3β, a redox-sensitive transducer downstream of insulin signaling, acts as a convergent point for myriad pathways implicated in kidney injury, repair, and regeneration. However, its role in diabetic kidney disease remains controversial. In cultured glomerular podocytes, exposure to a milieu of type 2 diabetes elicited prominent signs of podocyte injury and degeneration, marked by loss of homeostatic marker proteins like synaptopodin, actin cytoskeleton disruption, oxidative stress, apoptosis, and stress-induced premature senescence, as shown by increased staining for senescence-associated β-galactosidase activity, amplified formation of γH2AX foci, and elevated expression of mediators of senescence signaling, like p21 and p16INK4A. These degenerative changes coincided with GSK3β hyperactivity, as evidenced by GSK3β overexpression and reduced inhibitory phosphorylation of GSK3β, and were averted by tideglusib, a highly-selective small molecule inhibitor of GSK3β. In agreement, post-hoc analysis of a publicly-available glomerular transcriptomics dataset from patients with type 2 diabetic nephropathy revealed that the curated diabetic nephropathy-related gene set was enriched in high GSK3β expression group. Mechanistically, GSK3β-modulated nuclear factor Nrf2 signaling is involved in diabetic podocytopathy, because GSK3β knockdown reinforced Nrf2 antioxidant response and suppressed oxidative stress, resulting in an improvement in podocyte injury and senescence. Conversely, ectopic expression of the constitutively active mutant of GSK3β impaired Nrf2 antioxidant response and augmented oxidative stress, culminating in an exacerbated diabetic podocyte injury and senescence. Moreover, IRS-1 was found to be a cognate substrate of GSK3β for phosphorylation at IRS-1S332, which negatively regulates IRS-1 activity. GSK3β hyperactivity promoted IRS-1 phosphorylation, denoting a desensitized insulin signaling. Consistently, in vivo in db/db mice with diabetic nephropathy, GSK3β was hyperactive in glomerular podocytes, associated with IRS-1 hyperphosphorylation, impaired Nrf2 response and premature senescence. Our finding suggests that GSK3β is likely a novel therapeutic target for treating type 2 diabetic glomerular injury.

Sulforaphane ameliorated podocyte injury according to regulation of the Nrf2/PINK1 pathway for mitophagy in diabetic kidney disease

Mitophagy, a mechanism of self-protection against oxidative stress, plays a critical role in podocyte injury caused by diabetic kidney disease (DKD). Sulforaphane (SFN), an isothiocyanate compound, is a potent antioxidant that affords protection against diabetes mellitus-mediated podocyte injury. However, its role and underlying mechanism in DKD especially in diabetic podocytopathy is not clearly defined. In the current study, we demonstrated SFN remarkably activated mitophagy in podocytes, restored urine albumin to creatinine ration, and prevented the glomerular hypertrophy and extensive foot process fusion in diabetic mice. Simultaneously, nephroprotective effects of SFN on kidney injury were abolished in podocyte-specific Nuclear factor erythroid 2-related factor 2 (Nrf2) conditional knockout mouse (cKO), indicating that SFN alleviating DM-induced podocyte injury dependent on Nrf2. In vitro study, supplement with SFN augmented the expression of PTEN induced kinase 1(PINK1) and mediated the activation of mitophagy in podocytes treated with high glucose. Further study revealed that SFN treatment enabled Nrf2 translocate into nuclear and bind to the specific site of PINK1 promoter, ultimately reinforcing the transcription of PINK1. Moreover, SFN failed to confer protection to podocytes treated with high glucose in presence of PINK1 knockdown. On the contrary, exogenous overexpression of PINK1 reversed mitochondrial abnormalities in Nrf2 cKO diabetic mice. In conclusion, SFN alleviated podocyte injury in DKD through activating Nrf2/PINK1 signaling pathway and balancing mitophagy, thus maintaining the mitochondrial homeostasis.

Cellular mechanism of action of forsythiaside for the treatment of diabetic kidney disease.

Background: Diabetic kidney disease (DKD) becomes the leading cause of death for end-stage renal disease, whereas the potential mechanism is unclear and effective therapy is still rare. Our study was designed to investigate the cellular mechanism of Forsythiaside against DKD. Materials and Methods: The targets of Forsythiaside and the DKD-related targets were obtained from databases. The overlapping targets in these two sets were regarded as potential targets for alleviation of DKD by Forsythiaside. The targets of diabetic podocytopathy and tubulopathy were also detected to clarify the mechanism of Forsythiaside ameliorating DKD from the cellular level. Results: Our results explored that PRKCA and RHOA were regarded as key therapeutic targets of Forsythiaside with excellent binding affinity for treating DKD podocytopathy. Enrichment analysis suggested the underlying mechanism was mainly focused on the oxidative stress and mTOR signaling pathway. The alleviated effects of Forsythiaside on the reactive oxidative species accumulation and PRKCA and RHOA proteins upregulation in podocytes were also confirmed. Conclusion: The present study elucidates that Forsythiaside exerts potential treatment against DKD which may act directly RHOA and PRKCA target by suppressing the oxidative stress pathway in podocytes. And Forsythiaside could be regarded as one of the candidate drugs dealing with DKD in future experimental or clinical researches.

Dynein-Mediated Trafficking: A New Mechanism of Diabetic Podocytopathy.

Key Points The expression of dynein is increased in human and rodent models of diabetic nephropathy (DN), eliciting a new dynein-driven pathogenesis.Uncontrolled dynein impairs the molecular sieve of kidney by remodeling the postendocytic triage and homeostasis of nephrin.The delineation of the dynein-driven pathogenesis promises a broad spectrum of new therapeutic targets for human DN. Background Diabetic nephropathy (DN) is characterized by increased endocytosis and degradation of nephrin, a protein that comprises the molecular sieve of the glomerular filtration barrier. While nephrin internalization has been found activated in diabetes-stressed podocytes, the postinternalization trafficking steps that lead to the eventual depletion of nephrin and the development of DN are unclear. Our work on an inherited podocytopathy uncovered that dysregulated dynein could compromise nephrin trafficking, leading us to test whether and how dynein mediates the pathogenesis of DN. Methods We analyzed the transcription of dynein components in public DN databases, using the Nephroseq platform. We verified altered dynein transcription in diabetic podocytopathy by quantitative PCR. Dynein-mediated trafficking and degradation of nephrin was investigated using an in vitro nephrin trafficking model and was demonstrated in a mouse model with streptozotocin (STZ)-induced DN and in human kidney biopsy sections. Results Our transcription analysis revealed increased expression of dynein in human DN and diabetic mouse kidney, correlated significantly with the severity of hyperglycemia and DN. In diabetic podocytopathy, we observed that dynein-mediated postendocytic sorting of nephrin was upregulated, resulting in accelerated nephrin degradation and disrupted nephrin recycling. In hyperglycemia-stressed podocytes, Dynll1, one of the most upregulated dynein components, is required for the recruitment of dynein complex that mediates the postendocytic sorting of nephrin. This was corroborated by observing enhanced Dynll1-nephrin colocalization in podocytes of diabetic patients, as well as dynein-mediated trafficking and degradation of nephrin in STZ-induced diabetic mice with hyperglycemia. Knockdown of Dynll1 attenuated lysosomal degradation of nephrin and promoted its recycling, suggesting the essential role of Dynll1 in dynein-mediated mistrafficking. Conclusions Our studies show that hyperglycemia stimulates dynein-mediated trafficking of nephrin to lysosomes by inducing its expression. The decoding of dynein-driven pathogenesis of diabetic podocytopathy offers a spectrum of new dynein-related therapeutic targets for DN.

Urolithins Modulate the Viability, Autophagy, Apoptosis, and Nephrin Turnover in Podocytes Exposed to High Glucose.

Urolithins are bioactive compounds generated in human and animal intestines because of the bacterial metabolism of dietary ellagitannins (and their constituent, ellagic acid). Due to their multidirectional effects, including anti-inflammatory, antioxidant, anti-cancer, neuroprotective, and antiglycative properties, urolithins are potential novel therapeutic agents. In this study, while considering the future possibility of using urolithins to improve podocyte function in diabetes, we assessed the results of exposing mouse podocytes cultured in normal (NG, 5.5 mM) and high (HG, 25 mM) glucose concentrations to urolithin A (UA) and urolithin B (UB). Podocytes metabolized UA to form glucuronides in a time-dependent manner; however, in HG conditions, the metabolism was lower than in NG conditions. In HG milieu, UA improved podocyte viability more efficiently than UB and reduced the reactive oxygen species level. Both types of urolithins showed cytotoxic activity at high (100 µM) concentration. The UA upregulated total and surface nephrin expression, which was paralleled by enhanced nephrin internalization. Regulation of nephrin turnover was independent of ambient glucose concentration. We conclude that UA affects podocytes in different metabolic and functional aspects. With respect to its pro-survival effects in HG-induced toxicity, UA could be considered as a potent therapeutic candidate against diabetic podocytopathy.

ROCK2-induced metabolic rewiring in diabetic podocytopathy

Loss of podocytes is a common feature of diabetic renal injury and a key contributor to the development of albuminuria. We found that podocyte Rho associated coiled-coil containing protein kinase 2 (ROCK2) is activated in rodent models and patients with diabetes. Mice that lacked ROCK2 only in podocytes (PR2KO) were resistant to albuminuria, glomerular fibrosis, and podocyte loss in multiple animal models of diabetes (i.e., streptozotocin injection, db/db, and high-fat diet feeding). RNA-sequencing of ROCK2-null podocytes provided initial evidence suggesting ROCK2 as a regulator of cellular metabolism. In particular, ROCK2 serves as a suppressor of peroxisome proliferator-activated receptors α (PPARα), which rewires cellular programs to negatively control the transcription of genes involved in fatty acid oxidation and consequently induce podocyte apoptosis. These data establish ROCK2 as a nodal regulator of podocyte energy homeostasis and suggest this signaling pathway as a promising target for the treatment of diabetic podocytopathy.

Dynein-Driven Pathogenesis of Diabetic Podocytopathy

Dynein-Driven Pathogenesis of Diabetic Podocytopathy

392-P: ROCK2-Mediated Podocyte Dysfunction in Diabetic Nephropathy

The small GTPase RhoA and its downstream effector, Rho-kinase (ROCK), are implicated in a variety of cellular functions (e.g., cell adhesion, migration) via effect on cytoskeletal reorganization. We and others have illuminated ROCK as an important pathogenic regulator of diabetic kidney disease. Renal ROCK activity is elevated in both type 1 and type 2 diabetic murine models, as well as in diabetic patients. ROCK has two isoforms, ROCK1 and ROCK2. ROCK1 has been shown to regulate mitochondrial fission and epithelial-to-mesenchymal transition in the setting of diabetic kidney disease: however, the role of renal ROCK2 remain unclear. In the present study, we have demonstrated the upregulation of ROCK2 in glomerular podocytes obtained from 3 established models of diabetic kidney disease, (i.e., streptozotocin injection, db/db, and high-fat diet treatment), and diabetic patients. In an attempt to elucidate the roles of podocyte ROCK2, we generated podocyte-specific ROCK2 knockout mice (PR2KO) by breeding ROCK2 flox mice with Podocin-Cre mice. We found that podocyte-restricted ROCK2 deletion did not lead to any defects in the kidney development and functions. Of note, PR2KO were protected against albuminuria and glomerular sclerosis in streptozotocin-induced diabetic mice. In addition, renoprotective actions of podocyte ROCK2 deletion were also observed in type 2 diabetic db/db mice and high-fat diet-fed animals. Mechanistically, RNA-Seq analysis in ROCK2-null podocytes revealed the recovery of fatty acid utilization. When considered alongside our previous observations, the current work highlights the importance of ROCK2 in diabetic podocytopathy. Disclosure K. Matoba: Research Support; Self; Eli Lilly Japan K. K. Y. Takeda: None. T. Akamine: None. Y. Nagai: None. R. Ukichi: None. Y. Kanazawa: None. K. Utsunomiya: None. R. Nishimura: Speaker’s Bureau; Self; Abbott Japan Co., Ltd., Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly Japan K. K., Kissei Pharmaceutical Co., Ltd., Medtronic, MSD Corporation, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. Funding Japan Society for the Promotion of Science (20K08645, 18K15985); Suzuken Memorial Foundation; Takeda Science Foundation; Yokoyama Foundation for Clinical Pharmacology; MSD Life Science Foundation; Ichiro Kanehara Foundation; Japan Diabetes Foundation; Ueh

481-P: Renal Distribution and Expression Analysis of ROCK Isoforms in Diabetic Kidney Disease

The small GTPase Rho and its effector, Rho-kinase (ROCK), are implicated in the fundamental cellular processes (e.g., cell adhesion, migration). Studies over the past decade have illuminated ROCK as an important regulator of diabetic kidney disease. We previously demonstrated elevated renal ROCK activity in both type 1 and type 2 diabetic murine models. In an attempt to elucidate the roles of the activated ROCK, we provided evidence that ROCK inhibition attenuates an increase of urinary albumin excretion, glomerular sclerosis, and podocyte loss in rodents. Mechanistically, ROCK regulates inflammatory events as well as hypoxia-induced fibrotic reactions. However, specific roles of ROCK isoforms, ROCK1 and ROCK2, in diabetic kidneys remain unclear. In the present study, we first investigated the renal distribution of ROCK1 and ROCK2 using wild type mice and human kidneys obtained at necropsy. The immunohistochemical analysis detected both isoforms in glomeruli, tubules, and vessel walls. Whereas ROCK1 was mainly expressed in the cytoplasm, ROCK2 was accumulated in the nucleus of these cells. Double staining with anti-nephrin antibody showed co-localization of ROCK2 with nephrin, indicating distinctive roles of ROCK2 in the glomerular podocyte. In order to elucidate the role of podocyte ROCK2, we next evaluated gene expression levels of ROCK2 in high-fat diet-fed mice and patients with diabetic kidney disease. In both mice and patients, ROCK2 was increased in the setting of diabetes compared to healthy subjects. Of note, glomerular ROCK2 mRNA levels were positively correlated with the increase of urinary albumin excretion in mice. When considered alongside our previous observations, the current work highlights the importance of ROCK2 in diabetic podocytopathy. Disclosure K. Matoba: Research Support; Self; Sanofi K.K., Takeda Pharmaceutical Company Limited. Y. Takeda: None. Y. Nagai: None. T. Akamine: None. Y. Kanazawa: None. D. Kawanami: None. K. Utsunomiya: None. R. Nishimura: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc. Speaker’s Bureau; Self; Abbott, AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly Japan K.K., Medtronic, Merck & Co., Inc., Novo Nordisk Inc., Sanofi K.K., Takeda Pharmaceutical Company Limited. Funding Japan Society for the Promotion of Science (18K15985)

RNA-binding proteins tristetraprolin and human antigen R are novel modulators of podocyte injury in diabetic kidney disease

Diabetic kidney disease (DKD) is one of the most common complications of diabetes, and the most common cause of end-stage renal disease, for which no effective therapies are yet available. RNA-binding proteins (RBPs) play a pivotal role in epigenetic regulation; tristetraprolin (TTP) and human antigen R (HuR) competitively bind cytokine mRNAs, exert contrasting effects on RNA stability, and drive inflammation. However, RBPs’ roles in diabetes-related glomerulopathy are poorly understood. Herein, we investigated whether TTP and HuR are involved in post-transcriptional regulation of podocytopathic molecules and inflammatory cytokines in DKD. In DKD patients and db/db mice, TTP expression was significantly decreased and HuR expression was increased in glomerular podocytes, concurrent with podocyte injury, histological signs of DKD, and augmented glomerular expression of interleukin (IL)-17 and claudin-1, which are targets of TTP and HuR, as evidenced by RNA immunoprecipitation. In cultured podocytes, exposure to high ambient glucose amplified HuR expression and repressed TTP expression, upregulated IL-17 and claudin-1, and promoted podocyte injury. Thus, TTP hypoactivity or HuR hyperactivity is sufficient and essential to diabetic podocytopathy. Moreover, in silico analysis revealed that several kinases govern phosphorylation and activation of TTP and HuR, and glycogen synthase kinase (GSK)-3β activated both TTP and HuR, which harbor putative GSK-3β consensus phosphorylation motifs. Treatment of db/db mice with a small molecule inhibitor of GSK-3β abrogated the changes in TTP and HuR in glomeruli and mitigated the overexpression of their target genes (IL-17, claudin-1, B7-1, and MCP-1) thus also mitigating proteinuria and DKD pathology. Our study indicates that TTP and HuR are dysregulated in DKD via a GSK-3β-mediated mechanism and play crucial roles in podocyte injury through post-transcriptional regulation of diverse genes. It also provides novel insights into DKD’s pathophysiology and identifies potential therapeutic targets.

RNA-Binding Proteins Tristetraprolin and Human Antigen R Are Novel Modulators of Podocyte Injury and Inflammation in Diabetic Kidney Disease

Diabetic kidney disease (DKD) is one of the most common complications of diabetes, and the most common cause of end-stage renal disease, for which no effective therapies are yet available. RNA-binding proteins (RBPs) play a pivotal role in epigenetic regulation; tristetraprolin (TTP) and human antigen R (HuR) competitively bind cytokine mRNAs, exert contrasting effects on RNA stability, and drive inflammation. However, RBPs’ roles in diabetes-related glomerulopathy are poorly understood. Herein, we investigated whether TTP and HuR are involved in post-transcriptional regulation of podocytopathic molecules and inflammatory cytokines in DKD. In DKD patients and db/db mice, TTP expression was significantly decreased and HuR expression was increased in glomerular podocytes, concurrent with podocyte injury, histological signs of DKD, and augmented glomerular expression of interleukin (IL)-17 and claudin-1, which are targets of TTP and HuR, as evidenced by RNA immunoprecipitation. In cultured podocytes, exposure to high ambient glucose amplified HuR expression and repressed TTP expression, upregulated IL-17 and claudin-1, and promoted podocyte injury. Thus, TTP hypoactivity or HuR hyperactivity is sufficient and essential to diabetic podocytopathy. Moreover, in silico analysis revealed that several kinases govern phosphorylation and activation of TTP and HuR, and glycogen synthase kinase (GSK)-3β activated both TTP and HuR, which harbor putative GSK-3β consensus phosphorylation motifs. Treatment of db/db mice with a small molecule inhibitor of GSK-3β abrogated the changes in TTP and HuR in glomeruli and mitigated the overexpression of their target genes (IL-17 , claudin-1 , B7-1 ,and MCP-1) thus also mitigating proteinuria and DKD pathology. Our study indicate that TTP and HuR are dysregulated in DKD via a GSK-3β-mediated mechanism and play crucial roles in podocyte injury through post-transcriptional regulation of diverse genes. It also provides novel insights into DKD’s pathophysiology and identifies potential therapeutic targets. Funding Statement: This work was partially supported by the Natural Science Foundation of China (grant numbers U1604284 and 81670663). Declaration of Interests: The authors declare that they have no conflicts of interest. Ethics Approval Statement: The Ethical Committee of the First Affiliated Hospital of Zhengzhou University approved this study. All participants waived informed consent. The animal study was approved by the Institutional Animal Care and Use Committee of the University of Toledo.

501-P: ROCK2 Activation Accelerates Podocyte Injury in Diabetic Kidney Disease

Podocyte apoptosis is a key pathological finding in the progression of diabetic kidney disease. Here we demonstrate that Rho-kinase (ROCK) mediates transforming growth factor β (TGF-β)-induced podocyte apoptosis. Mechanistically, ROCK regulates Notch ligand induction via the extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) but not Smad pathways. Consistently, the ROCK inhibitor fasudil suppressed albuminuria and the urinary excretion of nephrin in type 2 diabetic db/db mice and attenuated podocyte apoptosis. The expression of Notch ligand and apoptosis markers such as Bax and cyclin-dependent kinase inhibitor 1A (CDKN1A) was decreased in podocytes derived from fasudil-treated db/db mice. In order to elucidate the isoform-specific roles of ROCK, we knocked down ROCK1 and ROCK2 separately using siRNA duplexes. Importantly, Notch ligand expression was specifically regulated by ROCK2 but not ROCK1. In renal tissue, ROCK2 was expressed mainly in nucleus of glomerular cells, tubules, and vascular walls in wild type mice. Podocyte colocalization was revealed by immunofluorescence of ROCK2 and nephrin, indicating the involvement of ROCK2 in diabetic podocytopathy. The present study provides evidence that ROCK plays a key role in podocyte apoptosis. ROCK2 may be an attractive therapeutic target for diabetic kidney disease. Disclosure K. Matoba: Research Support; Self; Daiichi Sankyo Company, Limited, Novo Nordisk Inc., Sanofi, Shionogi & Co., Ltd., Takeda Pharmaceutical Company Limited. D. Kawanami: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc. Y. Takeda: None. Y. Nagai: None. K. Utsunomiya: None. Funding Japan Society for the Promotion of Science; Uehara Memorial Foundation; Japan Diabetes Society; Ichiro Kanehara Foundation; MSD Life Science Foundation; Yokoyama Foundation

MIF/CD74 axis is a target for metformin therapy in diabetic podocytopathy - real world evidence.

202 uncontrolled type 2 diabetics, who were previously prescribed sulfonylurea monotherapy(n=100) or sulfonylurea in combination with metformin (n=102) were enrolled in the study. The amount of macrophage migration inhibitory factor(MIF) and CD74 in serum, urinary MIF to creatine ratio(UMCR), urinary CD74 to creatine ratio(UCCR), urinary albumin to creatine ratio(UACR) and urinary podocalyxin to creatine ratio (UPCR) were determined. Metabolic parameters including fasting blood glucose, postprandial 2 hours blood glucose, hemoglobin A1c, MIF and CD74 in serum were comparable between the two groups. Moreover, metformin add-on therapy showed significantly better efficacy in reducing UMCR, UCCR, UPCR and UACR in comparison with those in sulfonylurea monotherapy group, respectively. UPCR had positive correlation with UACR, UMCR and UCCR (r＝0.73, r=0.69, r=0.62, P < 0.01), respectively. Metformin could present its podocyte-protective capacity in type 2 diabetics and the underlying mechanisms may be partly attributed to its effects in suppressing MIF-CD74 axis mediated inflammatory cascade response. < p > < /p >.

Modulation of apolipoprotein L1-microRNA-193a axis prevents podocyte dedifferentiation in high-glucose milieu.

The loss of podocyte (PD) molecular phenotype is an important feature of diabetic podocytopathy. We hypothesized that high glucose (HG) induces dedifferentiation in differentiated podocytes (DPDs) through alterations in the apolipoprotein (APO) L1-microRNA (miR) 193a axis. HG-induced DPD dedifferentiation manifested in the form of downregulation of Wilms' tumor 1 (WT1) and upregulation of paired box 2 (PAX2) expression. WT1-silenced DPDs displayed enhanced expression of PAX2. Immunoprecipitation of DPD cellular lysates with anti-WT1 antibody revealed formation of WT1 repressor complexes containing Polycomb group proteins, enhancer of zeste homolog 2, menin, and DNA methyltransferase (DNMT1), whereas silencing of either WT1 or DNMT1 disrupted this complex with enhanced expression of PAX2. HG-induced DPD dedifferentiation was associated with a higher expression of miR193a, whereas inhibition of miR193a prevented DPD dedifferentiation in HG milieu. HG downregulated DPD expression of APOL1. miR193a-overexpressing DPDs displayed downregulation of APOL1 and enhanced expression of dedifferentiating markers; conversely, silencing of miR193a enhanced the expression of APOL1 and preserved DPD phenotype. Moreover, stably APOL1G0-overexpressing DPDs displayed the enhanced expression of WT1 but attenuated expression of miR193a; nonetheless, silencing of APOL1 reversed these effects. Since silencing of APOL1 enhanced miR193a expression as well as dedifferentiation in DPDs, it appears that downregulation of APOL1 contributed to dedifferentiation of DPDs through enhanced miR193a expression in HG milieu. Vitamin D receptor agonist downregulated miR193a, upregulated APOL1 expression, and prevented dedifferentiation of DPDs in HG milieu. These findings suggest that modulation of the APOL1-miR193a axis carries a potential to preserve DPD molecular phenotype in HG milieu.

Renin-angiotensin system within the diabetic podocyte.

Diabetic kidney disease is the leading cause of end-stage renal disease. Podocytes are differentiated cells necessary for the development and maintenance of the glomerular basement membrane and the capillary tufts, as well as the function of the glomerular filtration barrier. The epithelial glomerular cells express a local renin-angiotensin system (RAS) that varies in different pathological situations such as hyperglycemia or mechanical stress. RAS components have been shown to be altered in diabetic podocytopathy, and their modulation may modify diabetic nephropathy progression. Podocytes are a direct target for angiotensin II-mediated injury by altered expression and distribution of podocyte proteins. Furthermore, angiotensin II promotes podocyte injury indirectly by inducing cellular hypertrophy, increased apoptosis, and changes in the anionic charge of the glomerular basement membrane, among other effects. RAS blockade has been shown to decrease the level of proteinuria and delay the progression of chronic kidney disease. This review summarizes the local intraglomerular RAS and its imbalance in diabetic podocytopathy. A better understanding of the intrapodocyte RAS might provide a new approach for diabetic kidney disease treatment.

Insulin stimulates glucose transport via protein kinase G type I alpha-dependent pathway in podocytes

Podocyte resistance to the actions of insulin on glucose transport could contribute to the pathogenesis of diabetic podocytopathy (DP) via disturbances in cyclic-dependent protein kinase signaling. To determine whether cGMP-dependent protein kinase (PKG) is involved in the insulin regulation of glucose transport, we measured insulin-dependent glucose uptake into cultured rat podocytes under conditions of modified PKG activity using pharmacological (PKG activator or inhibitor) and biochemical (siRNA PKGIα, siRNA insulin receptor β) means. Our findings indicate the participation of PKG in insulin-stimulated transport and provide new insights into how PKG may trigger the resistance of glucose transport to insulin in DP.

MicroRNA-29a Promotion of Nephrin Acetylation Ameliorates Hyperglycemia-Induced Podocyte Dysfunction

Podocyte dysfunction is a detrimental feature in diabetic nephropathy, with loss of nephrin integrity contributing to diabetic podocytopathy. MicroRNAs (miRs) reportedly modulate the hyperglycemia-induced perturbation of renal tissue homeostasis. This study investigated whether regulation of histone deacetylase (HDAC) actions and nephrin acetylation by miR-29 contributes to podocyte homeostasis and renal function in diabetic kidneys. Hyperglycemia accelerated podocyte injury and reduced nephrin, acetylated nephrin, and miR-29a levels in primary renal glomeruli from streptozotocin-induced diabetic mice. Diabetic miR-29a transgenic mice had better nephrin levels, podocyte viability, and renal function and less glomerular fibrosis and inflammation reaction compared with diabetic wild-type mice. Overexpression of miR-29a attenuated the promotion of HDAC4 signaling, nephrin ubiquitination, and urinary nephrin excretion associated with diabetes and restored nephrin acetylation. Knockdown of miR-29a by antisense oligonucleotides promoted HDAC4 action, nephrin loss, podocyte apoptosis, and proteinuria in nondiabetic mice. In vitro, interruption of HDAC4 signaling alleviated the high glucose-induced apoptosis and inhibition of nephrin acetylation in podocyte cultures. Furthermore, HDAC4 interference increased the acetylation status of histone H3 at lysine 9 (H3K9Ac), the enrichment of H3K9Ac in miR-29a proximal promoter, and miR-29a transcription in high glucose-stressed podocytes. In conclusion, hyperglycemia impairs miR-29a signaling to intensify HDAC4 actions that contribute to podocyte protein deacetylation and degradation as well as renal dysfunction. HDAC4, via epigenetic H3K9 hypoacetylation, reduces miR-29a transcription. The renoprotective effects of miR-29a in diabetes-induced loss of podocyte integrity and renal homeostasis highlights the importance of post-translational acetylation reactions in podocyte microenvironments. Increasing miR-29a action may protect against diabetic podocytopathy.

Podocyte-specific overexpression of human angiotensin-converting enzyme 2 attenuates diabetic nephropathy in mice

Angiotensin-converting enzyme 2 (ACE2) degrades angiotensin II to angiotensin-(1–7) and is expressed in podocytes. Here we overexpressed ACE2 in podocytes in experimental diabetic nephropathy using transgenic methods where a nephrin promoter drove the expression of human ACE2. Glomeruli from these mice had significantly increased mRNA, protein, and activity of ACE2 compared to wild-type mice. Male mice were treated with streptozotocin to induce diabetes. After 16 weeks, there was no significant difference in plasma glucose levels between wild-type and transgenic diabetic mice. Urinary albumin was significantly increased in wild-type diabetic mice at 4 weeks, whereas albuminuria in transgenic diabetic mice did not differ from wild-type nondiabetic mice. However, this effect was transient and by 16 weeks both transgenic and nontransgenic diabetic mice had similar rates of proteinuria. Compared to wild-type diabetic mice, transgenic diabetic mice had an attenuated increase in mesangial area, decreased glomerular area, and a blunted decrease in nephrin expression. Podocyte numbers decreased in wild-type diabetic mice at 16 weeks, but were unaffected in transgenic diabetic mice. At 8 weeks, kidney cortical expression of transforming growth factor-β1 was significantly inhibited in transgenic diabetic mice as compared to wild-type diabetic mice. Thus, the podocyte-specific overexpression of human ACE2 transiently attenuates the development of diabetic nephropathy.