Autophagic flux defect in diabetic kidney disease results in megamitochondria formation in podocytes

Podocyte injury is an important factor in the pathogenesis of diabetic nephropathy. Podocytes are characterized by large numbers of mitochondria. However, mitochondrial dysfunction as it relates to kidney pathology remains poorly understood. The present study found that podocyte mitochondria in different animal models of diabetes mellitus became elongated with the development of albuminuria, suggesting a change in mitochondrial dynamics. We then treated cells with a combination of glucose, fatty acids, and angiotensin II (GFA) to mimic the diabetic milieu. Cultured podocytes exposed to GFA showed megamitochondria formation and decreased autophagosome degradation. We also found that GFA treatment decreased the binding of the autophagosome to the lysosome. Our results suggest that megamitochondria are common in podocytes during diabetic nephropathy and that insufficient autophagy flux may underlie this effect. These findings have expanded our understanding of the pathogenesis of diabetic nephropathy and identified a potential pharmacological target for treatment.     

Nicorandil as a novel therapy for advanced diabetic nephropathy in the eNOS-deficient mouse

Nicorandil is an orally available drug that can act as a nitric oxide donor, an antioxidant, and an ATP-dependent K channel activator. We hypothesized that it may have a beneficial role in treating diabetic nephropathy. We administered nicorandil to a model of advanced diabetic nephropathy (the streptozotocin-induced diabetes in mice lacking endothelial nitric oxide synthase, eNOSKO); controls included diabetic eNOS KO mice without nicorandil and nondiabetic eNOS KO mice treated with either nicorandil or vehicle. Mice were treated for 8 wk. Histology, blood pressure, and renal function were determined. Additional studies involved examining the effects of nicorandil on cultured human podocytes. Here, we found that nicorandil did not affect blood glucose levels, blood pressure, or systemic endothelial function, but significantly reduced proteinuria and glomerular injury (mesangiolysis and glomerulosclerosis). Nicorandil protected against podocyte loss and podocyte oxidative stress. Studies in cultured podocytes showed that nicorandil likely protects against glucose-mediated oxidant stress via the ATP-dependent K channel as opposed to its NO-stimulating effects. In conclusion, nicorandil may be beneficial in diabetic nephropathy by preserving podocyte function. We recommend clinical trials to determine whether nicorandil may benefit diabetic nephropathy or other conditions associated with podocyte dysfunction.     

Glomerular Endothelial Cell Injury and Damage Precedes That of Podocytes in Adriamycin-Induced Nephropathy

The role of podocytes in the development and progression of glomerular disease has been extensively investigated in the past decade. However, the importance of glomerular endothelial cells in the pathogenesis of proteinuria and glomerulosclerosis has been largely ignored. Recent studies have demonstrated that endothelial nitric oxide synthatase (eNOS) deficiency exacerbates renal injury in anti-GBM and remnant kidney models and accelerates diabetic kidney damage. Increasing evidence also demonstrates the importance of the glomerular endothelium in preventing proteinuria. We hypothesize that endothelial dysfunction can initiate and promote the development and progression of glomerulopathy. Administration of adriamycin (ADR) to C57BL/6 mice, normally an ADR resistant strain, with an eNOS deficiency induced overt proteinuria, severe glomerulosclerosis, interstitial fibrosis and inflammation. We also examined glomerular endothelial cell and podocyte injury in ADR-induced nephropathy in Balb/c mice, an ADR susceptible strain, by immunostaining, TUNEL and Western blotting. Interestingly, down-regulation of eNOS and the appearance of apoptotic glomerular endothelial cells occurred as early as 24 hours after ADR injection, whilst synaptopodin, a functional podocyte marker, was reduced 7 days after ADR injection and coincided with a significant increase in the number of apoptotic podocytes. Furthermore, conditioned media from mouse microvascular endothelial cells over-expressing GFP-eNOS protected podocytes from TNF-α-induced loss of synaptopodin. In conclusion, our study demonstrated that endothelial dysfunction and damage precedes podocyte injury in ADR-induced nephropathy. Glomerular endothelial cells may protect podocytes from inflammatory insult. Understanding the role of glomerular endothelial dysfunction in the development of kidney disease will facilitate in the design of novel strategies to treat kidney disease.     

Autophagy Attenuates Diabetic Glomerular Damage through Protection of Hyperglycemia-Induced Podocyte Injury

Despite the recent attention focused on the important role of autophagy in maintaining podocyte homeostasis, little is known about the changes and mechanisms of autophagy in podocyte dysfunction under diabetic condition. In this study, we investigated the role of autophagy in podocyte biology and its involvement in the pathogenesis of diabetic nephropathy. Podocytes had a high basal level of autophagy. And basal autophagy inhibition either by 3-methyladenenine (3-MA) or by Beclin-1 siRNA was detrimental to its architectural structure. However, under diabetic condition in vivo and under high glucose conditions in vitro, high basal level of autophagy in podocytes became defective and defective autophagy facilitated the podocyte injury. Since the dynamics of endoplasmic reticulum(ER) seemed to play a vital role in regulating the autophagic flux, the results that Salubrinal/Tauroursodeoxycholic acid (TUDCA) could restore defective autophagy further indicated that the evolution of autophagy may be mediated by the changes of cytoprotective output in the ER stress. Finally, we demonstrated in vivo that the autophagy of podocyte was inhibited under diabetic status and TUDCA could improve defective autophagy. Taken together, these data suggested that autophagy might be interrupted due to the failure of ER cytoprotective capacity upon high glucose induced unmitigated stress, and the defective autophagy might accelerate the irreparable progression of diabetic nephropathy.     

Inhibition of mitochondrial fission protects podocytes from albumin-induced cell damage in diabetic kidney disease

AimsIdentifying the mechanisms that underlie progression from endothelial damage to podocyte damage, which leads to massive proteinuria, is an urgent issue that must be clarified to improve renal outcome in diabetic kidney disease (DKD). We aimed to examine the role of dynamin-related protein 1 (Drp1)-mediated regulation of mitochondrial fission in podocytes in the pathogenesis of massive proteinuria in DKD.MethodsDiabetes- or albuminuria-associated changes in mitochondrial morphology in podocytes were examined by electron microscopy. The effects of albumin and other diabetes-related stimuli, including high glucose (HG), on mitochondrial morphology were examined in cultured podocytes. The role of Drp1 in podocyte damage was examined using diabetic podocyte-specific Drp1-deficient mice treated with neuraminidase, which removes endothelial glycocalyx.ResultsNeuraminidase-induced removal of glomerular endothelial glycocalyx in nondiabetic mice led to microalbuminuria without podocyte damage, accompanied by reduced Drp1 expression and mitochondrial elongation in podocytes. In contrast, streptozotocin-induced diabetes significantly exacerbated neuraminidase-induced podocyte damage and albuminuria, and was accompanied by increased Drp1 expression and enhanced mitochondrial fission in podocytes. Cell culture experiments showed that albumin stimulation decreased Drp1 expression and elongated mitochondria, although HG inhibited albumin-associated changes in mitochondrial dynamics, resulting in apoptosis. Podocyte-specific Drp1-deficiency in mice prevented diabetes-related exacerbation of podocyte damage and neuraminidase-induced development of albuminuria. Endothelial dysfunction-induced albumin exposure is cytotoxic to podocytes. Inhibition of mitochondrial fission in podocytes is a cytoprotective mechanism against albumin stimulation, which is impaired under diabetic condition. Inhibition of mitochondrial fission in podocytes may represent a new therapeutic strategy for massive proteinuria in DKD.     

AMPK signalling: Implications for podocyte biology in diabetic nephropathy

Diabetic nephropathy is a major long‐term complication of diabetes mellitus and one of the most common causes of end‐stage renal disease. Thickening of the glomerular basement membrane, glomerular cell hypertrophy and podocyte loss are among the main pathological changes that occur during diabetic nephropathy, resulting in proteinuria. Injury to podocytes, which are a crucial component of the glomerular filtration barrier, seems to play a key role in the development of diabetic nephropathy. Recent studies have suggested that dysregulation of AMP‐activated kinase protein, which is an essential cellular energy sensor, may play a fundamental role in this process. The purpose of this review is to highlight the molecular mechanisms associated with AMP‐activated protein kinase (AMPK) in podocytes that are involved in the pathogenesis of diabetic nephropathy.     

MAD2B promotes podocyte injury through regulating Numb-dependent Notch 1 pathway in diabetic nephropathy

Rationale: Recent studies have demonstrated that the loss of podocyte is a critical event in diabetic nephropathy (DN). Previously, our group have found that the mitotic arrest deficient protein MAD2B was involved in high glucose (HG)-induced podocyte injury by regulating APC/C activity. However, the exact mechanism of MAD2B implicated in podocyte injury is still lacking.Methods: The experiments were conducted by using kidney tissues from streptozotocin (STZ) induced diabetic mice with or without podocyte-specific deletion of MAD2B and the cultured podocytes exposed to different treatments. Glomerular pathological injury was evaluated by periodic acid-Schiff staining and transmission electron microscopy. The endogenous interaction between MAD2B and Numb was discovered by yeast two-hybrid analysis and co-immunoprecipitation assay. The expressions of MAD2B, Numb and related pathway were detected by western blot, immunochemistry and immunofluorescence.Results: The present study revealed that MAD2B was upregulated in diabetic glomeruli and cultured podocytes under hyperglycemic conditions. Podocyte-specific deletion of MAD2B alleviated podocyte injury and renal function deterioration in mice of diabetic nephropathy. Afterwards, MAD2B was found to interact with Numb, which was downregulated in diabetic glomeruli and HG-stimulated cultured podocytes. Interestingly, MAD2B genetic deletion could partly reverse the decline of Numb in podocytes exposed to HG and in diabetic mice, and the expressions of Numb downstream molecules such as NICD and Hes-1 were decreased accordingly. In addition, overexpression of Numb ameliorated HG-induced podocyte injury.Conclusions: The present findings suggest that upregulated MAD2B expression contributes to Numb depletion and activation of Notch 1 signaling pathway, which ultimately leads to podocyte injury during DN progression.     

内皮祖细胞在支架术后再内皮化中应用的研究进展

足细胞是附着在肾小球基底膜外的高度分化的上皮细胞,在维持肾小球滤过屏障完整性及限制血浆蛋白的滤出方面均发挥重要作用。近年来,在糖尿病肾病的研究中发现,足细胞损伤对蛋白尿及肾小球硬化等病理变化均明显相关。本文就糖尿病肾病时足细胞损伤的特点作一简要概述,为以后的相关研究奠定基础。     

NLRP3 inflammasome negatively regulates podocyte autophagy in diabetic nephropathy

Inflammasome mechanisms are recognized as a key pathophysiology of diabetic nephropathy (DN). The nucleotide-oligomerization domain-like receptor 3 (NLRP3) inflammasome has attracted the most attention. Autophagy as a conserved intracellular catabolic pathway plays essential roles in the maintenance of podocytes. Although autophagy was involved in preventing excessive inflammatory responses in kidney diseases, a clear understanding of the regulation of NLRP3 inflammasome on autophagy in glomerular damage in DN is still lacking. In this study, we focused on the effect of the activation of NLRP3 inflammasome on the suppression of podocyte autophagy and aimed to investigate the role of autophagy in podocyte injury in DN. Podocyte autophagy has been confirmed to be inhibited in high-fat diet/streptozotocin (HFD/STZ)-induced DN mice, and NLRP3 has been found to be upregulated in both mice and human DN biopsies and in vitro. Activation of NLRP3 inflammasome exacerbated podocyte autophagy and reduced podocyte nephrin expression, while silencing of NLRP3 efficiently restored podocyte autophagy and ameliorated podocyte injury induced by high glucose. The results showed that NLRP3 was a negative regulator of autophagy and suggested that restoration of podocyte autophagy by inactivation of NLRP3 under high glucose could reduce podocyte injury. Proper modification of autophagy and inflammasome has the potential to benefit the kidney in DN.     

Stabilization of hypoxia-inducible factor 1α by cobalt chloride impairs podocyte morphology and slit-diaphragm function

Glomerular podocytes are the major components of the renal filtration barrier, and altered podocyte permselectivity is a key event in the pathogenesis of proteinuric conditions. Clinical conditions such as ischemia and sleep apnea and extreme physiological conditions such as high-altitude sickness are presented with renal hypoxia and are associated with significant proteinuria. Hypoxia is considered as an etiological factor in the progression of acute renal injury. A sustained increase in hypoxia-inducible factor 1α (HIF1α) is a major adaptive stimulus to the hypoxic conditions. Although the temporal association between hypoxia and proteinuria is known, the mechanism by which hypoxia elicits proteinuria remains to be investigated. Furthermore, stabilization of HIF1α is being considered as a therapeutic option to treat anemia in patients with chronic kidney disease. Therefore, in this study, we induced stabilization of HIF1α in glomerular regions in vivo and in podocytes in vitro upon exposure to cobalt chloride. The elevated HIF1α expression is concurrence with diminished expression of nephrin and podocin, podocyte foot-processes effacement, and significant proteinuria. Podocytes exposed to cobalt chloride lost their arborized morphology and cell-cell connections and also displayed cytoskeletal derangements. Elevation in expression of HIF1α is in concomitance with loss of nephrin and podocin in patients with diabetic nephropathy and chronic kidney disease. In summary, the current study suggests that HIF1α stabilization impairs podocyte function vis-à-vis glomerular permselectivity.     

Liver X receptor activation induces podocyte injury via inhibiting autophagic activity

Podocyte injury plays a key role in the occurrence and development of kidney diseases. Decreased autophagic activity in podocyte is closely related to its injury and the occurrence of proteinuria. Liver X receptors (LXRs), as metabolic nuclear receptors, participate in multiple pathophysiological processes and express in several tissues, including podocytes. Although the functional roles of LXRs in the liver, adipose tissue and intestine are well established; however, the effect of LXRs on podocytes function remains unclear. In this study, we used mouse podocytes cell line to investigate the effects of LXR activation on podocytes autophagy level and related signaling pathway by performing Western blotting, RT-PCR, GFP-mRFP-LC3 transfection, and immunofluorescence staining. Then, we tested this effect in STZ-induced diabetic mice. Transmission electron microscopy and immunohistochemistry were employed to explore the effects of LXR activation on podocytes function and autophagic activity. We found that LXR activation could inhibit autophagic flux through blocking the formation of autophagosome in podocytes in vitro which was possibly achieved by affecting AMPK, mTOR, and SIRT1 signaling pathways. Furthermore, LXR activation in vivo induced autophagy suppression in glomeruli, leading to aggravated podocyte injury. In summary, our findings indicated that activation of LXRs induced autophagy suppression, which in turn contributed to the podocyte injury.

     

足细胞病与相关循环因子的表达

肾小球足细胞的损伤不仅是遗传性肾小球病的发病基础,还在很多后天的肾小球疾病中发挥重要作用。常见的足细胞病以微小病变性肾病(Minimal Change Disease,MCD)和局灶阶段性肾小管硬化(Focal Segmental glomerulosclerosis,FSGS)为主,有实验证明,足细胞损伤同时参与了各种不同类型的肾小球疾病,比如糖尿病肾病,HIV相关肾病,膜性肾病及其他获得性肾小球病等,因此,了解足细胞病损伤的机制尤为重要。临床观察提出局灶节段行肾小球硬化患者体内可能存在一种与大量蛋白尿发生有关的循环因子,相关的动物实验和体外观察同时也证实了循环因子的存在。越来越多的研究支持T细胞功能不全,细胞因子异常释放可能是循环因子的来源之一。如果能对患者循环因子进行检测,借助它来指导治疗方案的选择(免疫抑制剂、血浆置换),可能成为局灶节段行肾小球硬化诊断和治疗中的一个突破。本文以国内外研究文献为基础,对文献资料进行分析、归纳和总结,综述了足细胞病中相关循环因子的表达,探讨足细胞病发生及复发的机理,为足细胞病的定向治疗提供帮助。     

Losartan reverses glomerular podocytes injury induced by AngII via stabilizing the expression of GLUT1

Podocyte impairment is a key pathogenic even in the initiation and development of glomerular diseases associated with proteinuria. The type 2 diabetic patients is characterized by progressive increases in albuminuria which are associated with the development of characteristic histopathological features. Losartan had a benefit in decreasing albuminuria in type 2 diabetic patients,suggesting that losartan may have another effect other than blockade of the traditional renin–angiotensin system (RAS). However, the mechanism has remained undetermined. Glucose transporter 1 (GLUT1) is the predominant basal glucose transporter. In the kidney, GLUT1 was overexpressed predominantly in glomerular mesangial cells and in small vessels, rather than in podocytes. The increased glomerular GLUT1 mimicked diabetes-induced glomerular GLUT1 expression. In this study, we hypothesized that increased GLUT1 expression induced by angiotensinII (AngII) contributes to the progression of podocytes injury, losartan can block the effect of AngII and protect podocytes via stabilizing the expression of GLUT1, our results strongly suggest that losartan has a direct and protective effect on podocytes. This represents a novel mechanism by which losartan may protect podocyte from apoptotic death and improve podocyte function via stabilizing the expression of GLUT1. This finding underlines the crucial role of GLUT1 in the pathogenesis of podocyte injury and proteinuria.     

Nephrotic syndrome and subepithelial deposits in a mouse model of immune-mediated anti-podocyte glomerulonephritis

Subepithelial immune complex deposition in glomerular disease causes local inflammation and proteinuria by podocyte disruption. A rat model of membranous nephropathy, the passive Heymann nephritis, suggests that Abs against specific podocyte Ags cause subepithelial deposit formation and podocyte foot process disruption. In this study, we present a mouse model in which a polyclonal sheep anti-mouse podocyte Ab caused subepithelial immune complex formation. Mice developed a nephrotic syndrome with severe edema, proteinuria, hypoalbuminemia, and elevated cholesterol and triglycerides. Development of proteinuria was biphasic: an initial protein loss was followed by a second massive increase of protein loss beginning at approximately day 10. By histology, podocytes were swollen. Electron microscopy revealed 60-80% podocyte foot process effacement and subepithelial deposits, but no disruption of the glomerular basement membrane. Nephrin and synaptopodin staining was severely disrupted, and podocyte number was reduced in anti-podocyte serum-treated mice, indicating severe podocyte damage. Immunohistochemistry detected the injected anti-podocyte Ab exclusively along the glomerular filtration barrier. Immunoelectron microscopy localized the Ab to podocyte foot processes and the glomerular basement membrane. Similarly, immunohistochemistry localized mouse IgG to the subepithelial space. The third complement component (C3) was detected in a linear staining pattern along the glomerular basement membrane and in the mesangial hinge region. However, C3-deficient mice were not protected from podocyte damage, indicating a complement-independent mechanism. Twenty proteins were identified as possible Ags to the sheep anti-podocyte serum by mass spectrometry. Together, these data establish a reproducible model of immune-mediated podocyte injury in mice with subepithelial immune complex formation.     

Asparaginyl endopeptidase protects against podocyte injury in diabetic nephropathy through cleaving cofilin-1

Podocyte injury and loss are critical events in diabetic nephropathy (DN); however, the underlying molecular mechanisms remain unclear. Here, we demonstrate that asparaginyl endopeptidase (AEP) protects against podocyte injury through modulating the dynamics of the cytoskeleton. AEP was highly upregulated in diabetic glomeruli and hyperglycemic stimuli treated-podocytes; however, AEP gene knockout and its compound inhibitor treatment accelerated DN in streptozotocin-induced diabetic mice, whereas specific induction of AEP in glomerular cells attenuated podocyte injury and renal function deterioration. In vitro, elevated AEP was involved in actin cytoskeleton maintenance and anti-apoptosis effects. Mechanistically, we found that AEP directly cleaved the actin-binding protein cofilin-1 after the asparagine 138 (N138) site. The protein levels of endogenous cofilin-1 1-138 fragments were upregulated in diabetic podocytes, consistent with the changes in AEP levels. Importantly, we found that cofilin-1 1-138 fragments were remarkably unphosphorylated than full-length cofilin-1, indicating the enhanced cytoskeleton maintenance activity of cofilin-1 1-138. Then we validated cofilin-1 1-138 could rescue podocytes from cytoskeleton disarrangement and injury in diabetic conditions. Taken together, our data suggest a protective role of elevated AEP in podocyte injury during DN progression through cleaving cofilin-1 to maintain podocyte cytoskeleton dynamics and defend damage.Subject terms: Cell death, Kidney diseases     

The PPARβ/δ agonist GW0742 modulates signaling pathways associated with cardiac myocyte growth via a non-genomic redox mechanism

The aim was to explore the effects of rapamycin on autophagy and injury of podocytes in streptozocin (STZ)-induced type 1 diabetic mice, and its role in delaying progression of diabetic nephropathy. In this study, male Balb/c mice were divided into three groups: control (n = 12), STZ-induced diabetic (n = 12), and rapamycin-treated diabetic (DM + Rapa) (n = 12), which received intraperitoneal injection of rapamycin (2 mg/kg/48 h) after induction of DM. Levels of urinary albumin (UA), blood urea nitrogen, serum creatinine, and kidney weight/body weight were measured at week 12. Renal pathologic changes, number of podocytes autophagy, and organelles injury were investigated by PAS staining, transmission electron microscopy, and immunofluorescence staining, respectively. Western blot was performed to determine the expression of LC3 (a podocyte autophagy marker), phosphorylated mammalian target of rapamycin, p-p70S6K, bax, and caspase-3 protein. Podocytes count was evaluated by immunofluorescence staining and Wilms tumor 1 immunohistochemistry, and Western blot of nephrin and podocin. The results indicated that rapamycin could reduce the kidney weight/body weight and UA secretion. It could alleviate podocyte foot process fusion, glomerular basement membrane thickening, and matrix accumulation, and increase the number of autophagosomes, and LC3-expressing podocytes. Down-regulation of bax and caspase-3 protein, and up-regulation of nephrin and podocin protein were observed in the glomeruli of diabetic mice after administration of rapamycin. In conclusion, rapamycin can ameliorate renal injury in diabetic mice by increasing the autophagy activity and inhibition of apoptosis of podocytes.