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Intracellular albumin overload elicits endoplasmic reticulum stress and PKC-delta/p38 MAPK pathway activation to induce podocyte apoptosis.

ABSTRACT: Podocyte injury is closely related to proteinuria and the progression of chronic kidney disease (CKD). Currently, there is no conclusive understanding about the mechanisms involved in albumin overload and podocyte apoptosis response. In this study, we sought to explore the ways by which intracellular albumin can mediate podocyte apoptosis. Here, immortalized mouse podocytes were treated with bovine serum albumin (BSA) at different times and concentrations, in the presence or absence of SB203580 (0.1 µM, inhibitor of mitogen-activated-protein kinase - p38MAPK). Using immunofluorescence images, flow cytometry and immunoblotting, we observed a time-dependent intracellular accumulation of fluorescent albumin-FITC-BSA, followed by concentration-and time-dependent effect of intracellular albumin overload on podocyte apoptosis, which was mediated by increased expression of the chaperone glucose-regulated-protein 78 (GRP 78) and phosphorylated inositol-requiring enzyme 1 alpha (pIRE1-α), as well as protein kinase C delta (PKC-δ), p38MAPK and cleaved caspase 12 expression. SB203580 prevented the cleavage of caspase 12 and the albumin-mediated podocyte apoptosis. These results suggest that intracellular albumin overload is associated with endoplasmic reticulum (ER) stress and upregulation of PKC-δ/p38MAPK/caspase 12 pathway, which may be a target for future therapeutic of albumin-induced podocyte apoptosis.

SUBMITTER: Goncalves GL

PROVIDER: S-EPMC6301950 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Intracellular albumin overload elicits endoplasmic reticulum stress and PKC-delta/p38 MAPK pathway activation to induce podocyte apoptosis.

Gonçalves Guilherme Lopes GL Costa-Pessoa Juliana Martins JM Thieme Karina K Lins Bruna Bezerra BB Oliveira-Souza Maria M

Scientific reports 20181220 1

Podocyte injury is closely related to proteinuria and the progression of chronic kidney disease (CKD). Currently, there is no conclusive understanding about the mechanisms involved in albumin overload and podocyte apoptosis response. In this study, we sought to explore the ways by which intracellular albumin can mediate podocyte apoptosis. Here, immortalized mouse podocytes were treated with bovine serum albumin (BSA) at different times and concentrations, in the presence or absence of SB203580 ...[more]

PMID: 30573754

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Project description:Degenerative disc disease is associated with increased expression of pro-inflammatory cytokines in the intervertebral disc (IVD). However, it is not completely clear how inflammation arises in the IVD and which cellular compartments are involved in this process. Recently, the endoplasmic reticulum (ER) has emerged as a possible modulator of inflammation in age-related disorders. In addition, ER stress has been associated with the microenvironment of degenerated IVDs. Therefore, the aim of this study was to analyze the effects of ER stress on inflammatory responses in degenerated human IVDs and associated molecular mechanisms. Gene expression of ER stress marker GRP78 and pro-inflammatory cytokines IL-6, IL-8, IL-1β, and TNF-α was analyzed in human surgical IVD samples (n = 51, Pfirrmann grade 2-5). The expression of GRP78 positively correlated with the degeneration grade in lumbar IVDs and IL-6, but not with IL-1β and TNF-α. Another set of human surgical IVD samples (n = 25) was used to prepare primary cell cultures. ER stress inducer thapsigargin (Tg, 100 and 500 nM) activated gene and protein expression of IL-6 and induced phosphorylation of p38 MAPK. Both inhibition of p38 MAPK by SB203580 (10 µM) and knockdown of ER stress effector CCAAT-enhancer-binding protein homologous protein (CHOP) reduced gene and protein expression of IL-6 in Tg-treated cells. Furthermore, the effects of an inflammatory microenvironment on ER stress were tested. TNF-α (5 and 10 ng/mL) did not activate ER stress, while IL-1β (5 and 10 ng/mL) activated gene and protein expression of GRP78, but did not influence [Ca2+]i flux and expression of CHOP, indicating that pro-inflammatory cytokines alone may not induce ER stress in vivo. This study showed that IL-6 release in the IVD can be initiated following ER stress and that ER stress mediates IL-6 release through p38 MAPK and CHOP. Therapeutic targeting of ER stress response may reduce the consequences of the harsh microenvironment in degenerated IVD.

Project description:Study questionDoes metformin inhibit excessive androgen-induced endoplasmic reticulum (ER) stress in mouse granulosa cells (GCs) in vivo and in vitro?Summary answerMetformin inhibits testosterone-induced ER stress and unfolded protein response (UPR) activation by suppressing p38 MAPK phosphorylation in ovarian GCs.What is known alreadyPolycystic ovary syndrome (PCOS) is associated with hyperandrogenism. Excessive testosterone induces ER stress and UPR activation in human cumulus cells, leading to cell apoptosis. Metformin has potential inhibitory effects on ER stress and UPR activation, as demonstrated in human pancreatic beta cells and obese mice.Study design, size, durationCumulus cells and follicular fluid were collected from 25 women with PCOS and 25 controls at our IVF centre. A dihydrotestosterone (DHT)-induced PCOS mouse model was constructed and treated with or without metformin. Primary mouse GCs and cumulus-oocyte complexes (COCs) were cultured with testosterone, metformin, a p38 MAPK inhibitor, or p38 MAPK small interfering RNA.Participants/materials, setting, methodsThe levels of UPR sensor proteins and UPR-related genes were measured in cumulus cells from PCOS and control patients by real-time quantitative PCR (qPCR) and western blot. The ovaries, oocytes, GCs and COCs were collected from PCOS mice treated with metformin and controls. The expressions of ER stress markers and p38 MAPK phosphorylation were assessed by qPCR, western blot and immunofluorescence. A subsequent in vitro analysis with primary cultured GCs and COCs was used to confirm the influence of metformin on ER stress activation by qPCR and western blot. Finally, the effects of ER stress activation on GCs and COCs in relation to LH responsiveness were examined by qPCR and COC expansion.Main results and the role of chanceThe expression of the ER stress markers GRP78, CHOP and XBP1s in the cumulus cells was higher in PCOS patients than in control patients, as were the levels of the UPR sensor proteins p-IRE1α, p-EIF2α and GRP78. Compared to those of control mice, the ovaries, GCs and COCs of DHT-treated PCOS mice showed increased levels of ER stress marker genes and proteins. Hyperandrogenism in PCOS mouse ovaries also induced p38 MAPK phosphorylation in COCs and GCs. Metformin inhibited ER stress activation was associated with decreased p-p38 MAPK levels. In vitro experiments, testosterone-induced ER stress was mitigated by metformin or p38 MAPK inhibition in primary cultured GCs and COCs. COCs expanded rapidly in the presence of testosterone during LH administration, and ovulation-related genes, namely, Areg, Ereg, Ptgs2, Sult1e1, Ptx3 and Tnfaip6, were strongly expressed in the COCs and GCs. These effects were reversed by treatment with metformin, an ER stress inhibitor or by knockdown of p38 MAPK.Limitations, reasons for cautionThe number of PCOS patients in this study was small.Wider implications of the findingsThis study provides further evidence for metformin as a PCOS treatment.Study funding/competing interest(s)This study was funded by the National Key Research and Developmental Program of China (2018YFC1004800), the Key Research and Development Program of Zhejiang Province (2017C03022), the Zhejiang Province Medical Science and Technology Plan Project (2017KY085, 2018KY457), the National Natural Science Foundation of China (31701260, 81401264, 81701514), and the Special Funds for Clinical Medical Research of the Chinese Medical Association (16020320648). The authors report no conflict of interest in this work and have nothing to disclose.Trial registration numberN/A.

Project description:BackgroundPrevious studies have found that Hugan Qingzhi tablet (HQT) has significant lipid-lowering and antioxidant effects on non-alcoholic fatty liver disease (NAFLD). Moreover, the results of proteomic analysis confirmed that various proteins in endoplasmic reticulum stress (ERS) pathway were activated and recovered by HQT. However, its mechanism remains confused. The purpose of this study was to explore the effects of HQT-medicated serum on hepatic ERS and its relevant mechanisms.MethodsL02 cells were induced by Free Fatty Acid (FFA) for 24 h to establish a model of hepatic ERS and pretreated with the drug-medicated rat serum for 24 h. Accumulation of intracellular lipid was evaluated using Oil Red O staining and Triglyceride detection kit. The morphological changes of ER were observed by TEM. PKC-δ was silenced by specific siRNA. Western blot and RT-qPCR were applied to detect the expression of markers related to ERS, calcium disorder, steatosis and insulin resistance. The fluorescence of Ca2+ influx was recorded using fluorescence spectrophotometer.ResultsHQT-medicated serum significantly decreased the intracellular TG content. Furthermore, it caused significant reduction in the expression of ERS markers and an improvement in ER structure of L02 cells. PKC-δ was activated into phosphorylated PKC-δ in FFA-induced L02 hepatocytes while these changes can be reversed by HQT-medicated serum. Silencing PKC-δ in L02 cells can restore the expression and activity of SERCA2 in ER and down-regulate the expression of IP3R protein to maintain intracellular calcium homeostasis, so as to relieve FFA-induced ERS and its lipid accumulation and insulin resistance.ConclusionsThe results concluded that HQT-medicated serum exerts protective effects against hepatic ERS, steatosis and insulin resistance in FFA-induced L02 hepatocyte. And its potential mechanism might be down-regulating the activation of PKC-δ and stabilization of intracellular calcium.

Dataset Information

Intracellular albumin overload elicits endoplasmic reticulum stress and PKC-delta/p38 MAPK pathway activation to induce podocyte apoptosis.

Publications

Intracellular albumin overload elicits endoplasmic reticulum stress and PKC-delta/p38 MAPK pathway activation to induce podocyte apoptosis.

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