Podocyte Endoplasmic Reticulum Stress Research Articles

GDF-15 Suppresses Puromycin Aminonucleoside-Induced Podocyte Injury by Reducing Endoplasmic Reticulum Stress and Glomerular Inflammation.

GDF15, also known as MIC1, is a member of the TGF-beta superfamily. Previous studies reported elevated serum levels of GDF15 in patients with kidney disorder, and its association with kidney disease progression, while other studies identified GDF15 to have protective effects. To investigate the potential protective role of GDF15 on podocytes, we first performed in vitro studies using a Gdf15-deficient podocyte cell line. The lack of GDF15 intensified puromycin aminonucleoside (PAN)-triggered endoplasmic reticulum stress and induced cell death in cultivated podocytes. This was evidenced by elevated expressions of Xbp1 and ER-associated chaperones, alongside AnnexinV/PI staining and LDH release. Additionally, we subjected mice to nephrotoxic PAN treatment. Our observations revealed a noteworthy increase in both GDF15 expression and secretion subsequent to PAN administration. Gdf15 knockout mice displayed a moderate loss of WT1+ cells (podocytes) in the glomeruli compared to wild-type controls. However, this finding could not be substantiated through digital evaluation. The parameters of kidney function, including serum BUN, creatinine, and albumin-creatinine ratio (ACR), were increased in Gdf15 knockout mice as compared to wild-type mice upon PAN treatment. This was associated with an increase in the number of glomerular macrophages, neutrophils, inflammatory cytokines, and chemokines in Gdf15-deficient mice. In summary, our findings unveil a novel renoprotective effect of GDF15 during kidney injury and inflammation by promoting podocyte survival and regulating endoplasmic reticulum stress in podocytes, and, subsequently, the infiltration of inflammatory cells via paracrine effects on surrounding glomerular cells.

CTRP4 attenuates apoptosis and epithelial-mesenchymal transition markers in podocytes through an AMPK/autophagy-dependent pathway

Hyperglycemia, characterized by high blood glucose levels resulting from pancreatic beta cell dysfunction or impaired insulin signaling, is a contributing factor in the development of diabetic nephropathy. This study aimed to investigate the effects of C1q/TNF-related protein 4 (CTRP4), known for its anti-obesity and anti-inflammatory properties in various disease models, on podocyte apoptosis and endoplasmic reticulum (ER) stress in the presence of elevated glucose levels. The expression levels of various proteins in podocytes and adipocytes were evaluated by Western blotting. Autophagosomes in podocytes were stained by MDC. Chromatin condensation in podocytes was examined by Hoechst staining. The research revealed increased expression of CTRP4 in 3T3-L1 adipocytes and CIHP-1 podocytes exposed to high glucose (HG) conditions. Treatment with CTRP4 effectively mitigated HG-induced apoptosis and ER stress and normalized epithelial-to-mesenchymal transition (EMT) markers in CIHP-1 cells. Furthermore, elevated levels of AMPK phosphorylation and autophagy were observed in CIHP-1 cells treated with CTRP4. Silencing of AMPK or the use of 3-methyl adenine (3 MA) reduced the impacts of CTRP4 on apoptosis, EMT markers and ER stress in CIHP-1 cells. In conclusion, these findings suggest that CTRP4 alleviates ER stress in podocytes under hyperglycemic conditions, leading to the suppression of apoptosis and the restoration of EMT through AMPK/autophagy-mediated signaling. These insights provide valuable information for the development of therapeutic strategies for diabetic nephropathy.

Epigenetics and endoplasmic reticulum in podocytopathy during diabetic nephropathy progression.

Proteinuria or nephrotic syndrome are symptoms of podocytopathies, kidney diseases caused by direct or indirect podocyte damage. Human health worldwide is threatened by diabetic nephropathy (DN), the leading cause of end-stage renal disease (ESRD) in the world. DN development and progression are largely dependent on inflammation. The effects of podocyte damage on metabolic disease and inflammatory disorders have been documented. Epigenetic and endoplasmic reticulum (ER) stress are also evident in DN. Targeting inflammation pathway and ER stress in podocytes may be a prospective therapy to prevent the progression of DN. Here, we review the mechanism of epigenetics and ER stress on podocyte inflammation and apoptosis, and discuss the potential amelioration of podocytopathies by regulating epigenetics and ER stress as well as by targeting inflammatory signaling, which provides a theoretical basis for drug development to ameliorate DN.

Berberine alleviates palmitic acid‑induced podocyte apoptosis by reducing reactive oxygen species‑mediated endoplasmic reticulum stress.

Lipid accumulation in podocytes can lead to the destruction of cellular morphology, in addition to cell dysfunction and apoptosis, which is a key factor in the progression of chronic kidney disease (CKD). Berberine (BBR) is an isoquinoline alkaloid extracted from medicinal plants such as Coptis chinensis, which has been reported to have a lipid-lowering effect and prevent CKD progression. Therefore, the present study aimed to investigate the effect of BBR on palmitic acid (PA)-induced podocyte apoptosis and its specific mechanism using an in vitro model. Cell death was measured using the Cell Counting Kit-8 colorimetric assay. Cell apoptotic rate was assessed by flow cytometry. The expression of endoplasmic reticulum (ER) stress- and apoptosis-related proteins was detected by western blotting or immunofluorescence. Reactive oxygen species (ROS) were evaluated by 2′,7′-dichlorofluorescein diacetate fluorescence staining. The results of the present study revealed that BBR treatment decreased PA-induced podocyte apoptosis. In addition, 4-phenylbutyric acid significantly reduced PA-induced cell apoptosis and the expression of ER stress-related proteins, which indicated that ER stress was involved in PA-induced podocyte apoptosis. In addition, N-acetylcysteine inhibited PA-induced excessive ROS production, ER stress and cell apoptosis of podocytes. BBR also significantly reduced PA-induced ROS production and ER stress in podocytes. These results suggested that PA mediated podocyte apoptosis through enhancing ER stress and the production of ROS. In conclusion, BBR may protect against PA-induced podocyte apoptosis, and suppression of ROS-dependent ER stress may be the key mechanism underlying the protective effects of BBR.

Puromycin aminonucleoside triggers apoptosis in podocytes by inducing endoplasmic reticulum stress.

BackgroundPuromycin aminonucleoside (PAN) is a known podocytotoxin. PAN-induced nephrosis is a widely used animal model for studying human idiopathic nephrotic syndrome. Abnormal protein accumulation associated with podocyte-specific endoplasmic reticulum (ER) stress damages cells structurally and functionally, which in turn induces apoptosis and severe proteinuria. In the present study, we investigated the effect of PAN on ER stress and apoptosis in podocytes in vitro.MethodsMouse podocytes were cultured and treated with various concentrations of PAN. ER stress markers were then evaluated by western blotting, and apoptosis was evaluated by fluorescence-activated cell sorting (FACS) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays.ResultsPAN treatment increased ER stress markers such as activating transcription factor (ATF) 6α and caspase-12 in a dose-dependent manner at 12 and 24 hours, respectively. These markers were reduced by chemical chaperones, such as sodium 4-phenylbutyric acid and tauroursodeoxycholic acid. PAN treatment also increased 78 kD glucose-regulated protein (GRP78)/binding immunoglobulin protein (BiP) at the earlier stage of 12 hours. PAN significantly induced podocyte apoptosis in concentration- and time-dependent manners, as seen using FACS and TUNEL assays. This result was improved by Nox4 siRNA, ATF6 siRNA, and chemical chaperones. LY294002, a PI3-kinase inhibitor, significantly boosted ER stress and apoptosis. PAN-induced ER stress increased oxidative stress and subsequently induced apoptosis, and could be mitigated by inhibition of PI3-kinase signaling.ConclusionOur findings suggest that PAN induces ER stress in podocytes mainly through the GRP78/BiP, ATF6α, and caspase-12 pathways, which trigger apoptosis via induction of oxidative stress. This stress is mitigated by inhibiting PI3-kinase signaling.

Endoplasmic reticulum stress and monogenic kidney diseases in precision nephrology.

The advent of next-generation sequencing (NGS) in recent years has led to a rapid discovery of novel or rare genetic variants in human kidney cell genes, which is transforming the risk assessment, diagnosis, and treatment of kidney disease. Mutations may lead to protein misfolding, disruption of protein trafficking, and endoplasmic reticulum (ER) retention. An imbalance between the load of misfolded proteins and the folding capacity of the ER causes ER stress and unfolded protein response. Mutations in nephrin (NPHS1), podocin (NPHS2), laminin β2 (LAMB2), and α-actinin-4 (ACTN4) have been shown to induce ER stress in HEK293 cells and podocytes in hereditary nephrotic syndromes; various founder mutations in collagen IV α chains (COL4A) have been demonstrated to activate podocyte ER stress in collagen IV nephropathies; and mutations in uromodulin (UMOD) have been reported to trigger tubular ER stress in autosomal dominant tubulointerstitial kidney disease. Meanwhile, ER resident protein SEC63 may modify disease severity in autosomal dominant polycystic kidney disease. These findings underscore the importance of ER stress in the pathogenesis of monogenic kidney disease. Recently, we have identified mesencephalic astrocyte-derived neurotrophic factor (MANF) and cysteine-rich with EGF-like domains 2 (CRELD2) as urinary ER stress biomarkers in ER stress-mediated kidney diseases.

Rtn1a-Mediated Endoplasmic Reticulum Stress in Podocyte Injury and Diabetic Nephropathy

We previously reported a critical role of reticulon (RTN) 1A in mediating endoplasmic reticulum (ER) stress in kidney tubular cells and the expression of RTN1A correlates with the renal function and the severity of kidney injury in patients with diabetic nephropathy (DN). Here, we determined the roles of RTN1A and ER stress in podocyte injury and DN. We used db/db mice with early unilateral nephrectomy (Unx) as a murine model of progressive DN and treated mice with tauroursodeoxycholic acid (TUDCA), a specific inhibitor of ER stress. We found increased expression of RTN1A and ER stress markers in the kidney of db/db-Unx mice. Treatment of TUDCA not only attenuated proteinuria and kidney histological changes, but also ameliorated podocyte and glomeruli injury in diabetic mice, which were associated with reduction of RTN1A and ER stress marker expression in the podocytes of TUDCA-treated mice. In vitro, we showed RTN1A mediates albumin-induced ER stress and apoptosis in human podocytes. A positive feedback loop between RTN1A and CHOP was found leading to an enhanced ER stress in podocytes. Our data suggest that ER stress plays a major role in podocyte injury in DN and RTN1A might be a key regulator of ER stress in podocytes.

Mesencephalic Astrocyte-Derived Neurotrophic Factor as a Urine Biomarker for Endoplasmic Reticulum Stress-Related Kidney Diseases.

Endoplasmic reticulum (ER) stress and disrupted proteostasis contribute to the pathogenesis of a variety of glomerular and tubular diseases. Thus, it is imperative to develop noninvasive biomarkers for detecting ER stress in podocytes or tubular cells in the incipient stage of disease, when a kidney biopsy is not yet clinically indicated. Mesencephalic astrocyte-derived neurotrophic factor (MANF) localizes to the ER lumen and is secreted in response to ER stress in several cell types. Here, using mouse models of human nephrotic syndrome caused by mutant laminin β2 protein-induced podocyte ER stress and AKI triggered by tunicamycin- or ischemia-reperfusion-induced tubular ER stress, we examined MANF as a potential urine biomarker for detecting ER stress in podocytes or renal tubular cells. ER stress upregulated MANF expression in podocytes and tubular cells. Notably, urinary MANF excretion concurrent with podocyte or tubular cell ER stress preceded clinical or histologic manifestations of the corresponding disease. Thus, MANF can potentially serve as a urine diagnostic or prognostic biomarker in ER stress-related kidney diseases to help stratify disease risk, predict disease progression, monitor treatment response, and identify subgroups of patients who can be treated with ER stress modulators in a highly targeted manner.

Essential Role of X-Box Binding Protein-1 during Endoplasmic Reticulum Stress in Podocytes.

Podocytes are terminally differentiated epithelial cells that reside along the glomerular filtration barrier. Evidence suggests that after podocyte injury, endoplasmic reticulum stress response is activated, but the molecular mechanisms involved are incompletely defined. In a mouse model, we confirmed that podocyte injury induces endoplasmic reticulum stress response and upregulated unfolded protein response pathways, which have been shown to mitigate damage by preventing the accumulation of misfolded proteins in the endoplasmic reticulum. Furthermore, simultaneous podocyte-specific genetic inactivation of X-box binding protein-1 (Xbp1), a transcription factor activated during endoplasmic reticulum stress and critically involved in the untranslated protein response, and Sec63, a heat shock protein-40 chaperone required for protein folding in the endoplasmic reticulum, resulted in progressive albuminuria, foot process effacement, and histology consistent with ESRD. Finally, loss of both Sec63 and Xbp1 induced apoptosis in podocytes, which associated with activation of the JNK pathway. Collectively, our results indicate that an intact Xbp1 pathway operating to mitigate stress in the endoplasmic reticulum is essential for the maintenance of a normal glomerular filtration barrier.

Angiotensin II induces endoplasmic reticulum stress in podocyte, which would be further augmented by PI3-kinase inhibition.

IntroductionAngiotensin II (Ang II) contributes to the pathological process of vascular structures, including renal glomeruli by hemodynamic and nonhemodynamic direct effects. On renal effects, Ang II plays an important role in the development of proteinuria and glomerulosclerosis by the modification of podocyte molecules and cell survival. In the present study, we investigated the effect of Ang II on endoplasmic reticulum (ER) stress in podocytes.MethodsWe cultured mouse podocytes with increasing doses of Ang II and evaluated ER stress markers by Western blotting.ResultsAng II increased Bip protein, an ER chaperone, in a dose-dependent manner at 24 h, which was ameliorated by losartan, an angiotensin II type 1 receptor antagonist. Ang II also increased ER stress markers, such as phospho-PERK, phospho-eIF2α, and ATF4 proteins of podocyte, significantly in a dose-dependent manner at 24 h. Increased phospho-PERK and ATF4 proteins were further augmented by phosphoinositide 3 (PI3)-kinase inhibitor, LY294002, which suggested that Ang II could induce podocyte ER stress of PERK-eIF2α-ATF4 axis via PI3-kinase pathway.DiscussionThese studies suggest that Ang II could induce podocyte ER stress of PERK-eIF2α-ATF4 axis via PI3-kinase pathway, which would contribute to the development of podocyte injury induced by Ang II, and the augmentation of PI3-kinase would be a therapeutic target.

Expression of calcineurin in podocytes in response to endoplasmic reticulum stress in diabetic nephropathy

To investigate the regulation of calcineurin (CaN) by endoplasmic reticulum stress (ERS) in podocytes in vitro and in vivo at the stage microalbuminuria in diabetic nehropathy (DN). The urinary albumin excretions of C57BLKS/J (Lepr) db/db and db/m mice at the ages of 6, 9, and 12 weeks were measured. The expressions of CaN and synaptopodin of these mice were observed. In immortalized mouse podocytes, the expression of podocyte CaN incubated with different concentrations of paltimate was quantitatively determined by real-time PCR. The changes of CaN incubated with paltimate with or without ursodeoxy-cholic acid (UDCA) were analyzed by confocal microscopy and Western blotting. As urine protein increased, the expression of CaN was enhanced and the expression of synaptopodin was reduced in early stage DN db/db mice potocytes. In immortalized mouse podocytes, as the concentrations of palmitate increased, CaN mRNA increased. By confocal microscopy, the fluorescence intensity of CaN increased in palmitate treatment group. After co-incubation with palmitate and UDCA, the fluorescence intensity decreased. The similar results were shown by Western blotting. At the stage of microalbuminuria in DN, ERS in podocytes up-regulates the expression of CaN.

Na+/H+ exchanger-1 reduces podocyte injury caused by endoplasmic reticulum stress via autophagy activation

Podocyte injury has a critical role in the pathogenesis of proteinuria. Induction of endoplasmic reticulum (ER) stress is thought to lead to podocyte injury; however, no effective strategy for reducing ER stress-induced injury has been identified. We investigated specific mechanisms for reducing podocyte injury caused by ER stress. We found that the induction of ER stress in podocytes was related to cytoskeleton injury and increased proteinuria, which was associated with autophagy activation and downregulation of Na+/H+ exchanger-1 (NHE-1) in the rat model of passive Heymann nephritis. Using mouse podocyte cells (MPCs), we showed that ER stress could lead to podocyte injury accompanied by autophagy activation, and the disturbance of autophagy aggravated cytoskeleton loss under conditions of ER stress. The balance between autophagy activation and ER stress was critical to podocyte survival, in which the efficiency of autophagy could have a pivotal role. Strikingly, the overexpression and small interfering RNA knockdown of NHE-1 results suggested that NHE-1 exerts a protective effect by reducing the loss of synaptopodin in MPCs exposed to ER stress. This protective mechanism involves NHE-1 activation of autophagy via the PI3K/Akt pathway to reduce ER stress injury in podocytes. This mechanism may provide a new pathway to prevent podocyte injury.

Renal protection by low dose irbesartan in diabetic nephropathy is paralleled by a reduction of inflammation, not of endoplasmic reticulum stress

Diabetes can disrupt endoplasmic reticulum (ER) homeostasis which leads to ER stress. ER stress-induced renal apoptosis seems to be involved in the development of diabetic nephropathy. The present study was designed to investigate the contribution of reduced ER stress to the beneficial effects of an angiotensin receptor blocker. Insulin-dependent diabetes mellitus was induced by streptozotocin injections to hypertensive mRen2-transgenic rats. After 2weeks animals were treated with 0.7mg/kg/day irbesartan. Blood glucose, blood pressure and protein excretion were assessed. Expression of ER stress markers was measured by real-time PCR. Immunohistochemistry was performed to detect markers of ER stress, renal damage and infiltrating cells. Glomerulosclerosis and apoptosis were evaluated. Diabetic mRen2-transgenic rats developed renal injury with proteinuria, tubulointerstitial cell proliferation as well as glomerulosclerosis and podocyte injury. Moreover, an increase in inflammation, podocyte ER stress and apoptosis was detected. Irbesartan somewhat lowered blood pressure and reduced proteinuria, tubulointerstitial cell proliferation and glomerulosclerosis. Podocyte damage was ameliorated but markers of ER stress (calnexin, grp78) and apoptosis were not reduced by irbesartan. On the other hand, inflammatory cell infiltration in the tubulointerstitium and the glomerulus was significantly attenuated. We conclude that irbesartan reduced renal damage even in a very low dose. The beneficial effects of low dose irbesartan were paralleled by a reduction of blood pressure and inflammation but not by a reduction of ER stress and apoptosis. Thus, sustained endoplasmic reticulum stress in the kidney does not necessarily lead to increased inflammation and tubulointerstitial or glomerular injury.

A proteome-wide screen identifies valosin-containing protein as an essential regulator of podocyte endoplasmic reticulum stress

To investigate proteins expressed in the renal tissue of the passive Heymann nephritis (pHN) rat model, we prepared pHN rat models with anti-FxA1 serum and analyzed the proteins differentially expressed in the kidney tissue with label-free liquid chromatography- tandem mass spectrometry. We then analyzed in depth the endoplasmic reticulum stress (ERS)-related protein using an online bioinformatics platform. Forty-one differential proteins and their annotations were obtained. Gene Ontology (GO) function analysis showed that 16 proteins were involved in cellular metabolism and 22 were proteins related to catalytic activity, including protein folding or ATPase. Protein-GO networks indicated that VCP could interact with the ERS marker HSPa5, with both involved in a single pathway. On inhibition of podocyte VCP by RNAi under normal conditions, the HSPa5 expression level did not change, but when the cell was subjected to ERS by tunicamycin, HSPa5 expression significantly increased with RNAi of VCP when compared with the tunicamycin-treated group. Our results showed that ERS plays an important role in podocyte injury of membranous nephropathy and is mediated by an HSPa5-VCP signaling pathway, in which the most predominant proteins are those related to cellular metabolism and catalytic activity.

Regulation of podocyte survival and endoplasmic reticulum stress by fatty acids.

Apoptosis of podocytes is considered critical in the pathogenesis of diabetic nephropathy (DN). Free fatty acids (FFAs) are critically involved in the pathogenesis of diabetes mellitus type 2, in particular the regulation of pancreatic β cell survival. The objectives of this study were to elucidate the role of palmitic acid, palmitoleic, and oleic acid in the regulation of podocyte cell death and endoplasmic reticulum (ER) stress. We show that palmitic acid increases podocyte cell death, both apoptosis and necrosis of podocytes, in a dose and time-dependent fashion. Palmitic acid induces podocyte ER stress, leading to an unfolded protein response as reflected by the induction of the ER chaperone immunoglobulin heavy chain binding protein (BiP) and proapoptotic C/EBP homologous protein (CHOP) transcription factor. Of note, the monounsaturated palmitoleic and oleic acid can attenuate the palmitic acid-induced upregulation of CHOP, thereby preventing cell death. Similarly, gene silencing of CHOP protects against palmitic acid-induced podocyte apoptosis. Our results offer a rationale for interventional studies aimed at testing whether dietary shifting of the FFA balance toward unsaturated FFAs can delay the progression of DN.

Involvement of endoplasmic reticulum (ER) stress in podocyte injury induced by excessive protein accumulation

An imbalance between protein load and folding capacity is referred to as endoplasmic reticulum (ER) stress. As a defense mechanism, cells express ER stress inducible chaperons, such as oxygen-regulated proteins 150 (ORP150) and glucose-regulated proteins (GRPs). While ER stress is important in various diseases, a pathophysiologic role for ER stress in kidney disease remains elusive. Here we investigate expression of ER stress proteins in cultured rat podocytes as well as in our recently developed animal model of abnormal protein retention within the ER of podocytes (i.e., megsin transgenic rat). The expression of ER stress inducible proteins (ORP150, GRP78, or GRP94) in cultured podocytes treated with tunicamycin, A23187, SNAP, hypoxia, or hyperglycemia, and the renal tissues or isolated glomeruli from megsin transgenic rats was analyzed by Western blotting analysis, immunohistochemistry, or confocal microscopy. Cultured podocytes demonstrated that treatment with tunicamycin, A23187, and SNAP, but not hypoxia or hyperglycemia, up-regulate expression of ER stress proteins. Extracts of isolated glomeruli from megsin transgenic rats reveal marked up-regulation of ER stress chaperones in podocytes, which was supported by immunohistochemical analysis. Confocal microscopy revealed that ER stress in podocytes was associated with cellular injury. Podocytes of transgenic rats overexpressing a mutant megsin, without the capacity for polymerization within the ER, do not exhibit ER stress or podocyte damage, suggesting a pathogenic role of ER retention of polymerized megsin. This paper implicates a crucial role for the accumulation of excessive proteins in the podocyte ER in the induction of ER stress and associated podocyte injury.