Therapeutic Target For Diabetic Nephropathy Research Articles

Transforming Growth Factor Beta 1 Drives a Switch in Connexin Mediated Cell-to-Cell Communication in Tubular Cells of the Diabetic Kidney

Background/Aims: Changes in cell-to-cell communication have been linked to several secondary complications of diabetes, but the mechanism by which connexins affect disease progression in the kidney is poorly understood. This study examines a role for glucose-evoked changes in the beta1 isoform of transforming growth factor (TGFβ1), on connexin expression, gap-junction mediated intercellular communication (GJIC) and hemi-channel ATP release from tubular epithelial cells of the proximal renal nephron. Methods: Biopsy material from patients with and without diabetic nephropathy was stained for connexin-26 (CX26) and connexin-43 (CX43). Changes in expression were corroborated by immunoblot analysis in human primary proximal tubule epithelial cells (hPTECs) and model epithelial cells from human renal proximal tubules (HK2) cultured in either low glucose (5mmol/L) ± TGFβ1 (2-10ng/ml) or high glucose (25mmol/L) for 48h or 7days. Secretion of the cytokine was determined by ELISA. Paired whole cell patch clamp recordings were used to measure junctional conductance in control versus TGFβ1 treated (10ng/ml) HK2 cells, with carboxyfluorescein uptake and ATP-biosensing assessing hemi-channel function. A downstream role for ATP in mediating the effects of TGF-β1 on connexin mediated cell communication was assessed by incubating cells with ATPγS (1-100µM) or TGF-β1 +/- apyrase (5 Units/ml). Implications of ATP release were measured through immunoblot analysis of interleukin 6 (IL-6) and fibronectin expression. Results: Biopsy material from patients with diabetic nephropathy exhibited increased tubular expression of CX26 and CX43 (P<0.01, n=10), data corroborated in HK2 and hPTEC cells cultured in TGFβ1 (10ng/ml) for 7days (P<0.001, n=3). High glucose significantly increased TGFβ1 secretion from tubular epithelial cells (P<0.001, n=3). The cytokine (10ng/ml) reduced junctional conductance between HK2 cells from 4.5±1.3nS in control to 1.15±0.9nS following 48h TGFβ1 and to 0.42±0.2nS after 7days TGFβ1 incubation (P<0.05, n=5). Acute (48h) and chronic (7day) challenge with TGFβ1 produced a carbenoxolone (200µM)-sensitive increase in carboxyfluorescein loading, matched by an increase in ATP release from 0.29±0.06μM in control to 1.99±0.47μM after 48hr incubation with TGFβ1 (10ng/ml; P<0.05, n=3). TGF-β1 (2-10ng/ml) and ATPγs (1-100µM) increased expression of IL-6 (P<0.001 n=3) and fibronectin (P<0.01 n=3). The effect of TGF-β1 on IL-6 and fibronectin expression was partially blunted when preincubated with apyrase (n=3). Conclusion: These data suggest that chronic exposure to glucose-evoked TGFβ1 induce an increase in CX26 and CX43 expression, consistent with changes observed in tubular epithelia from patients with diabetic nephropathy. Despite increased connexin expression, direct GJIC communication decreases, whilst hemichannel expression/function and paracrine release of ATP increases, changes that trigger increased levels of expression of interleukin 6 and fibronectin. Linked to inflammation and fibrosis, local increases in purinergic signals may exacerbate disease progression and highlight connexin mediated cell communication as a future therapeutic target for diabetic nephropathy.

High glucose augments angiotensinogen in human renal proximal tubular cells through hepatocyte nuclear factor-5.

High glucose has been demonstrated to induce angiotensinogen (AGT) synthesis in the renal proximal tubular cells (RPTCs) of rats, which may further activate the intrarenal renin-angiotensin system (RAS) and contribute to diabetic nephropathy. This study aimed to investigate the effects of high glucose on AGT in the RPTCs of human origin and identify the glucose-responsive transcriptional factor(s) that bind(s) to the DNA sequences of AGT promoter in human RPTCs. Human kidney (HK)-2 cells were treated with normal glucose (5.5 mM) and high glucose (15.0 mM), respectively. Levels of AGT mRNA and AGT secretion of HK-2 cells were measured by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Consecutive 5’-end deletion mutant constructs and different site-directed mutagenesis products of human AGT promoter sequences were respectively transfected into HK-2 cells, followed by AGT promoter activity measurement through dual luciferase assay. High glucose significantly augmented the levels of AGT mRNA and AGT secretion of HK-2 cells, compared with normal glucose treatment. High glucose also significantly augmented AGT promoter activity in HK-2 cells transfected with the constructs of human AGT promoter sequences, compared with normal glucose treatment. Hepatocyte nuclear factor (HNF)-5 was found to be one of the glucose-responsive transcriptional factors of AGT in human RPTCs, since the mutation of its binding sites within AGT promoter sequences abolished the above effects of high glucose on AGT promoter activity as well as levels of AGT mRNA and its secretion. The present study has demonstrated, for the first time, that high glucose augments AGT in human RPTCs through HNF-5, which provides a potential therapeutic target for diabetic nephropathy.

Identification of a protective proteomic signature and a potential therapeutic target in diabetic nephropathy

In a recent paper in Nature Medicine, Qi etal. set out to identify protective pathways in patients with longstanding diabetes who do not develop diabetic nephropathy. Unbiased proteomic analysis of kidney tissue identified a unique protein signature in unaffected individuals. Studies invitro and in murine models identified the highly upregulated glycolytic enzyme pyruvate kinase M2 as a potential therapeutic target to prevent or treat diabetic nephropathy.

Rho-Kinase Blockade Attenuates Podocyte Apoptosis by Inhibiting the Notch Signaling Pathway in Diabetic Nephropathy.

Podocyte apoptosis is a key process in the onset of diabetic nephropathy. A significant body of evidence shows that the Notch signaling pathway plays a central role in this process. We found that Rho-kinase mediates transforming growth factor β (TGF-β)-induced Notch ligand Jag1 expression. Importantly, TGF-β-mediated podocyte apoptosis was attenuated by Rho-kinase inhibition. Mechanistically, Rho-kinase regulated Jag1 induction via the extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) but not Smad pathways. Consistently, the Rho-kinase inhibitor fasudil prevented albuminuria and the urinary excretion of nephrin in db/db mice and reduced the prevalence of podocyte apoptosis and Jag1 expression. Finally, the expression of Jag1 and apoptosis markers such as Bax and cyclin-dependent kinase inhibitor 1A (CDKN1A) was decreased in podocytes derived from db/db mice treated with fasudil. The present study provides evidence that Rho-kinase plays a key role in podocyte apoptosis. Rho-kinase is an attractive therapeutic target for diabetic nephropathy.

Regulating Autophagy as a Therapeutic Target for Diabetic Nephropathy.

Autophagy promotes cellular health in response to various cellular stresses and to changes in nutrient conditions. In this review, we focus on the role of autophagy in the pathogenesis of diabetic nephropathy and discuss the regulation of autophagy as a new therapeutic target for the suppression of diabetic nephropathy. Previous studies have indicated that autophagy deficiency or insufficiency in renal cells, including podocytes, mesangial cells, endothelial cells and tubular cells, contributes to the pathogenesis of diabetic nephropathy. Alterations in the nutrient-sensing pathways, including mammalian target of rapamycin complex1 (mTORC1), AMP-activated kinase (AMPK) and Sirt1, due to excess nutrition in diabetes are implicated in the impairment of autophagy. Maintaining both basal and adaptive autophagy against cellular stress may protect the kidney from diabetes-induced cellular stresses. Therefore, the activation of autophagy through the modulation of nutrient-sensing pathways may be a new therapeutic option for the suppression of diabetic nephropathy.

Podocan Is Expressed in Blood and Adipose Tissue and Correlates Negatively With the Induction of Diabetic Nephropathy.

Podocan, a member of the small leucine-rich repeat proteoglycans (SLRPs), is expressed in vascular endothelial cells with high levels of expression in the sclerotic glomerular lesions of experimental HIV-associated nephropathy. It is also found in vascular smooth muscle cells and is involved in atherosclerosis. Decorin, a protein similar to podocan, also belongs to the SLRP family and is highly expressed in adipose tissues. It is a secreted protein associated with obesity, type 2 diabetes, and diabetic nephropathy. Based on the similarity of podocan to decorin and its functions reported in the renal and cardiovascular systems, we hypothesized that podocan levels might correlate with the occurrence of metabolic syndromes such as obesity, diabetes, and diabetic nephropathy. We found that podocan was highly expressed in the adipose tissue of mice and humans and its expression was regulated by tumor necrosis factor-α in mouse 3T3-L1 adipocytes. In addition, podocan was detected in the plasma, and its levels tended to increase in diet-induced obese C57BL/6J mice and decrease in obese-diabetic KKAy and db/db mice. Podocan messenger RNA (mRNA) levels in the renal cortex correlated negatively with the urinary albumin-to-creatinine ratio, a surrogate marker of glomerular injury in uninephrectomized db/db mice used as a model of diabetic nephropathy. Our results suggest that podocan is involved in kidney function and could be a unique therapeutic target for diabetic nephropathy.

Short-Chain Fatty Acids Inhibit Oxidative Stress and Inflammation in Mesangial Cells Induced by High Glucose and Lipopolysaccharide.

Recently, an connection between Short-chain fatty acids (SCFAs) produced by intestinal microbiota and kidney has been revealed. The aim of this study was to explore whether SCFAs or their specific G protein-coupled receptors 43 (GPR43) agonist inhibit oxidative stress and inflammatory response in glomerular mesangial cells (GMCs) induced by high glucose and lipopolysaccharide (LPS). Our research showed that treatment with SCFAs, especially acetate and butyrate, or GPR43 agonist significantly inhibited GMCs proliferation induced by high glucose and LPS, and then reversed the production of reactive oxygen species (ROS) and malondialdehyde (MDA) but increased levels of antioxidant enzyme superoxide dismutase (SOD). Furthermore, SCFAs or GPR43 agonist obviously increased the protein expression of GPR43 induced by high glucose and LPS, but diminished the expression of adhesion molecule intercellular adhesion molecule-1 (ICAM-1), and then decreased the proinflammatory cytokine monocyte chemoattractant protein (MCP-1) and interleukin-1β (IL-1β) release from GMCs stimulated by the high glucose and LPS. These combined results support the hypothesis that SCFAs or GPR43 agonist can inhibit oxidative stress and inflammation of GMCs induced by high glucose and LPS, suggesting that SCFAs induced signaling pathway may act as new therapeutic targets of diabetic nephropathy (DN).

KCa3.1 as a novel therapeutic target for diabetic nephropathy

KCa3.1 as a novel therapeutic target for diabetic nephropathy

NMDA Receptors as Potential Therapeutic Targets in Diabetic Nephropathy: Increased Renal NMDA Receptor Subunit Expression in Akita Mice and Reduced Nephropathy Following Sustained Treatment With Memantine or MK-801.

N-methyl-d-aspartate (NMDA) receptors are expressed throughout the kidney, and the abundance of these receptors and some of their endogenous agonists are increased in diabetes. Moreover, sustained activation of podocyte NMDA receptors induces Ca(2+) influx, oxidative stress, loss of slit diaphragm proteins, and apoptosis. We observed that NMDA receptor subunits and their transcripts are increased in podocytes and mesangial cells cultured in elevated glucose compared with controls. A similar increase in NMDA subunits, especially NR1, NR2A, and NR2C, was observed in glomeruli and tubules of Akita mice. Sustained continuous treatment with the strong NMDA receptor antagonist dizocilpine (MK-801) for 28 days starting at 8 weeks of age reduced 24-h albumin excretion and mesangial matrix expansion and improved glomerular ultrastructure in Akita mice. MK-801 did not alleviate reduced Akita mouse body weight and had no effect on kidney histology or ultrastructure in DBA/2J controls. The structurally dissimilar NMDA antagonist memantine also reduced diabetic nephropathy, although it was less effective than MK-801. Inhibition of NMDA receptors may represent a valid therapeutic approach to reduce renal complications of diabetes, and it is possible to develop well-tolerated agents with minimal central nervous system effects. Two such agents, memantine and dextromethorphan, are already in widespread clinical use.

Coagulation Factor Xa and Protease-Activated Receptor 2 as Novel Therapeutic Targets for Diabetic Nephropathy.

The role of hypercoagulability in the pathogenesis of diabetic nephropathy (DN) remains elusive. We recently reported the increased infiltration of macrophages expressing tissue factor in diabetic kidney glomeruli; tissue factor activates coagulation factor X (FX) to FXa, which in turn stimulates protease-activated receptor 2 (PAR2) and causes inflammation. Here, we demonstrated that diabetes mellitus increased renal FX mRNA, urinary FXa activity, and FX expression in glomerular macrophages. Administration of an oral FXa inhibitor, edoxaban, ameliorated DN with concomitant reductions in the expression of PARs (Par1 and Par2) and of proinflammatory and profibrotic genes. Diabetes mellitus induced PAR2, and lack of Par2 ameliorated DN. FXa or PAR2 agonist increased inflammatory cytokines in endothelial cells and podocytes in vitro. We conclude that enhanced FXa and PAR2 exacerbate DN and that both are promising targets for preventing DN. Alleviating inflammation is probably more important than inhibiting coagulation per se when treating kidney diseases using anticoagulants.

Autophagy protects human podocytes from high glucose-induced injury by preventing insulin resistance

ObjectiveInsulin resistance is correlated with the progress of albuminuria in diabetic patients, and podocytes are crucial for maintaining the normal function of the glomerular filtration barrier. In the present study, we aimed to investigate the high glucose-induced insulin resistance and cell injury in human podocytes and the putative role of autophagy in this process. MethodsHuman podocytes were cultured in high glucose-supplemented medium and low glucose and high osmotic conditions were used for the controls. Autophagy in the podocytes was regulated using rapamycin or 3-methyladenine stimulation. Next, autophagy markers including LC3B, Beclin-1, and p62 were investigated using western blot and qPCR, and the insulin responsiveness was analyzed based on glucose uptake and by using the phosphorylation of the insulin receptor with Nephrin as a podocyte injury marker. ResultsThe basal autophagy level decreased under the high glucose conditions, which was accompanied by a decrease in the glucose uptake and phosphorylation of the insulin receptor in the human podocytes. More interestingly, the glucose uptake and the phosphorylation of the insulin receptor were decreased by 3-MA stimulation and increased by rapamycin, illustrating that the responsiveness of insulin was regulated by autophagy. The activation of autophagy by rapamycin also ameliorated cell injury in the human podocytes. ConclusionsThe presence or activation of autophagy was found to play a protective role in human podocytes against high glucose-induced insulin resistance and cell injury, which indicates a novel cellular mechanism and provides a potential therapeutic target for diabetic nephropathy (DN).

Mir-217 promotes inflammation and fibrosis in high glucose cultured rat glomerular mesangial cells via Sirt1/HIF-1α signaling pathway.

Silent information regulator 1 (Sirt1) plays a protective role in kidney. Sirt1 suppresses activation of hypoxia-inducible factor-1 alpha (HIF-1α), with MircroRNA-217 (Mir-217) being closely related to Sirt1. The relationship of Sirt1, HIF-1α and Mir-217, however, has never been reported in high glucose cultured rat glomerular mesangial cells (RMCs). Thus, we explored the role of Mir-217 on inflammation and fibrosis in RMCs cultured with high glucose in vitro through Sirt1/HIF-1α signaling pathway. Rat glomerular mesangial cells were pre-incubated with Sirt1 activator Resveratrol prior to high glucose treatment. Furthermore the cells were transiently transfected with Sirt1 small interfering RNA (siRNA), HIF-1α siRNA and Mir-217 inhibitor using Lipofectamine 2000. Real-time PCR was used to analyse the expression of Mir-217, Sirt1 mRNA and HIF-1α mRNA; Western Blot was used to observe protein expression of Sirt1, HIF-1α, connective tissue growth factor, endothelin-1 and fibronectin; enzyme-linked immunosorbent assay was used to detect protein expression of transforming growth factor-β1 and vascular endothelial growth factor. High glucose increased Mir-217 expression. High glucose decreased Sirt1 expression, accompanied by the increased HIF-1α expression and then promoted inflammation and fibrosis. In addition, Mir-217 gene silencing or Resveratrol could suppress the expression of HIF-1α, which in turn restrained inflammation and fibrosis in rat glomerular mesangial cells cultured with high glucose. This study clarified the role of Mir-217 in high glucose cultured rat glomerular mesangial cells through Sirt1/HIF-1α signaling pathway and provided new therapeutic targets for diabetic nephropathy. Copyright © 2016 John Wiley & Sons, Ltd.

Long non-coding RNA ENSMUST00000147869 protects mesangial cells from proliferation and fibrosis induced by diabetic nephropathy.

Diabetic nephropathy as the primary cause of end-stage renal disease reveals an increased incidence in patients with kidney disease as the continuous rising of type 2 diabetes. Long non-coding RNAs (lncRNAs) are involved in the development of many diseases including diabetes; however, the role of lncRNAs in diabetic nephropathy is still unclear. In the present study, lncRNA microarray analysis was used to identify abnormally expressed lncRNAs and nearby mRNAs in renal cortical tissues dissected from kidney of db/db and db/m mice. After verifying the data from microarray analysis by quantitative RT-PCR, downregulated ENSMUST00000147869 associated with Cyp4a12a was selected for overexpression in mouse mesangial cells among differentially expressed lncRNAs. Cell Counting Kit-8, Western blotting, and quantitative RT-PCR showed that proliferation and fibrosis indexes were reversed in mesangial cells with ENSMUST00000147869 overexpression. Our data suggested the potential role of ENSMUST00000147869 in proliferation and fibrosis of mesangial cells, which provided a molecular biomarker and therapeutic target for diabetic nephropathy.

Autophagy downregulation contributes to insulin resistance mediated injury in insulin receptor knockout podocytes in vitro.

It is unknown whether autophagy activity is altered in insulin resistant podocytes and whether autophagy could be a therapeutic target for diabetic nephropathy (DN). Here we used shRNA transfection to knockdown the insulin receptor (IR) gene in cultured human immortalized podocytes as an in vitro insulin resistant model. Autophagy related proteins LC3, Beclin, and p62 as well as nephrin, a podocyte injury marker, were assessed using western blot and immunofluorescence staining. Our results show that autophagy is suppressed when podocytes lose insulin sensitivity and that treatment of rapamycin, an mTOR specific inhibitor, could attenuate insulin resistance induced podocytes injury via autophagy activation. The present study deepens our understanding of the role of autophagy in the pathogenesis of DN.

Prediction of the molecular mechanisms and potential therapeutic targets for diabetic nephropathy by bioinformatics methods.

In this study, we aimed to explore the molecular mechanisms of and genetic factors influencing diabetic nephropathy (DN). Gene expression profiles associated with DN were obtained from the GEO database (Accession no. GSE20844). The differentially expressed genes (DEGs) between diabetic mice and non-diabetic mice were screened. Subsequently, the DEGs were subjected to functional and pathway analysis. The protein-protein interaction (PPI) network was constructed and the transcription factors (TFs) were screened among the DEGs. A total of 92 upregulated and 118 downregulated genes were screened. Pathway analysis revealed that the p53 signaling pathway, the transforming growth factor (TGF)-β signaling pathway and the mitogen-activated protein kinase (MAPK) signaling pathway were significantly enriched by upregulated genes. Serpine1 (also known as plasminogen activator inhibitor-1), early growth response 1 (Egr1) and Mdk were found to be significant nodes in the PPI network by three methods. A total of 12 TFs were found to be differentially expressed, of which nuclear receptor subfamily 4, group A, member 1 (Nr4a1) and peroxisome proliferator-activated receptor gamma (Pparg) were found to have multiple interactions with other DEGs. We demonstrated that the p53 signaling pathway, the TGF-β signaling pathway and the MAPK signaling pathway were dysregulated in the diabetic mice. The significant nodes (Serpine1, Egr1 and Mdk) and differentially expressed TFs (Nr4a1 and Pparg) may provide a novel avenue for the targeted therapy of DN.

CD36 is involved in high glucose-induced epithelial to mesenchymal transition in renal tubular epithelial cells

The epithelial-to-mesenchymal transition (EMT) plays an important role in the progression of diabetic nephropathy. Our recent study showed that ROS mediated high glucose (HG)-induced EMT in renal tubular epithelial cells. CD36, a class-B scavenger receptor, has been reported to mediate the production of ROS in chronic kidney disease. In the present study, we examined the effect of inhibition of CD36 with CD36 siRNA or sulfosuccinimidyl-oleate (SSO), a CD36 antagonist, on HG-induced EMT in HK-2 cells. HG induced CD36 expression in a time-dependent manner in HK-2 cells. HG was shown to induce EMT at 72 h. This was blocked by knockdown of CD36 or treatment with SSO. Meanwhile, we also found that knockdown of CD36 or treatment with SSO inhibited HG-induced ROS generation, activation of ERK1/2 and Smad2, expression of TGF-β1 and synthesis of fibronectin. These data suggest that inhibition of CD36 prevented HG-induced EMT in HK-2 cells, highlighting CD36 as a potential therapeutic target for diabetic nephropathy.

Effects of ROS-relative NF-κB signaling on high glucose-induced TLR4 and MCP-1 expression in podocyte injury.

High glucose (HG) induced inflammation is central to progression in diabetic nephropathy (DN). Recent studies have suggested that nuclear factor-kappa B (NF-κB) signaling activation is associated with DN, and podocyte damage may be involved in orchestrating these effects. Therefore, the aim of this study was to investigate the effects of NF-κB signaling on podocytes under HG conditions. The effects of HG and NF-κB signaling on podocytes were assessed by CCK-8 assay, cellular NF-κB translocation assay, measurement of reactive oxygen species (ROS) and Western blot analysis. We found that HG reduced cell viability, activated NF-κB signaling and up-regulated toll-like receptor 4 (TLR4) and monocyte chemoattractant protein-1 (MCP-1). In these cells, NF-κB inhibition with ammonium pyrrolidinethiocarbamate (PDTC) resulted in effectively constraining TLR4 and MCP-1 up-regulation, indicating that protective effects associated with the inhibition of NF-κB were linked to TLR4 and MCP-1 down-regulation in podocytes. Furthermore, HG significantly increased the production of intracellular ROS. Pretreatment with N-acetyl-l-cysteine (NAC) significantly inhibited intracellular ROS generation and increased cell viability, accompanied by a significant NF-κB inhibition and suppression of TLR4 and inflammatory cytokine MCP-1 expression. Collectively, our novel data suggest that HG induces the over-experssion of TLR-4 and MCP-1 through a NF-κB-dependent signaling. NF-κB-mediated increased inflammation is possibly via ROS and contributes to the cell injury. These results may provide potential therapeutic target for diabetic nephropathy in the future.

ASK1 Inhibitor Halts Progression of Diabetic Nephropathy in Nos3-Deficient Mice.

p38 mitogen-activated protein kinase (MAPK) signaling promotes diabetic kidney injury. Apoptosis signal-regulating kinase (ASK)1 is one of the upstream kinases in the p38 MAPK-signaling pathway, which is activated by inflammation and oxidative stress, suggesting a possible role for ASK1 in diabetic nephropathy. In this study, we examined whether a selective ASK1 inhibitor can prevent the induction and progression of diabetic nephropathy in mice. Diabetes was induced in hypertensive endothelial nitric oxide synthase (Nos3)-deficient mice by five low-dose streptozotocin (STZ) injections. Groups of diabetic Nos3(-/-) mice received ASK1 inhibitor (GS-444217 delivered in chow) as an early intervention (2-8 weeks after STZ) or late intervention (weeks 8-15 after STZ). Control diabetic and nondiabetic Nos3(-/-) mice received normal chow. Treatment with GS-444217 abrogated p38 MAPK activation in diabetic kidneys but had no effect upon hypertension in Nos3(-/-) mice. Early intervention with GS-444217 significantly inhibited diabetic glomerulosclerosis and reduced renal dysfunction but had no effect on the development of albuminuria. Late intervention with GS-444217 improved renal function and halted the progression of glomerulosclerosis, renal inflammation, and tubular injury despite having no effect on established albuminuria. In conclusion, this study identifies ASK1 as a new therapeutic target in diabetic nephropathy to reduce renal inflammation and fibrosis independent of blood pressure control.

Heme oxygenase-1 enhances autophagy in podocytes as a protective mechanism against high glucose-induced apoptosis

Injury and loss of podocytes play vital roles in diabetic nephropathy progression. Emerging evidence suggests autophagy, which is induced by multiple stressors including hyperglycemia, plays a protective role. Meanwhile, heme oxygenase-1 (HO-1) possesses powerful anti-apoptotic properties. Therefore, we investigated the impact of autophagy on podocyte apoptosis under diabetic conditions and its association with HO-1. Mouse podocytes were cultured in vitro; apoptosis was detected by flow cytometry. Transmission electron microscopy and biochemical autophagic flux assays were used to measure the autophagy markers microtubule-associated protein 1 light chain 3-II (LC3-II) and beclin-1. LC3-II and beclin-1 expression peaked 12–24h after exposing podocytes to high glucose. Inhibition of autophagy with 3-methyladenine or Beclin-1 siRNAs or Atg 5 siRNAs sensitized cells to apoptosis, suggesting autophagy is a survival mechanism. HO-1 inactivation inhibited autophagy, which aggravated podocyte injury in vitro. Hemin-induced autophagy also protected podocytes from hyperglycemia in vitro and was abrogated by HO-1 siRNA. Adenosine monophosphate-activated protein kinase phosphorylation was higher in hemin-treated and lower in HO-1 siRNA-treated podocytes. Suppression of AMPK activity reversed HO-1-mediated Beclin-1 upregulation and autophagy, indicating HO-1-mediated autophagy is AMPK dependent. These findings suggest HO-1 induction and regulation of autophagy are potential therapeutic targets for diabetic nephropathy.

Human antigen R: A novel therapeutic target for diabetic nephropathy?

Human antigen R: A novel therapeutic target for diabetic nephropathy?