Monoubiquitination Of CD36 By Parkin In Glomerular Podocyte Apoptosis Induced By Hyperlipidemia

Background:With the improvement of people’s living standard, the incidence of diabetes is rising year by year. And Diabetic Nephropathy (DN), as one of the main causes of death of diabetes, has been a serious health problem, respected by people gradually. Diabetic nephropathy is one of the diabetes systemic microvascular lesions, manifesting proteinuria, progressive renal damage, and severe renal failure in end stage. Hyperglycemia as one of the main pathogenesis of DN, could cause the metabolism change (including the increase of the activity of the advanced glycation end products and protein kinase C activity and polyols metabolic abnormalities), hypoxia stress in cells, ROS production and endoplasmic reticulum stress, ultimately leading to the DN. Moreover, DN is always accompanied by hyperlipidemia, which can aggravate kidney damage in patients with kidney disease or lead to health kidney damage.The involving of hyperlipidemia and hyperglycemia contribute to the progression of Chronic kidney disease(CKD) and ultimate renal failure. A large number of studies have found that glomerular podocyte damage and loss is a key link in the process of DN development.Podocyte, as one type of the intrinsic glomerular cells, Constitutes the glomerular filtration barrier with the endothelial cells and basement membrane (GBM) for preventing the loss of plasma protein. A lot of researchs suggest that the glomerular podocyte plays a central and critical role in glomerular injury. Podocyte is vulnerable to many factors, including antigen-antibody reaction for cell membrane antigen, hemodynamic changes, metabolic factors, genetic mutations, poisoning, infection, protein overload, etc. Podocyte damage in early stage manifests foot process fusion, disappear and the adhesion function of podocyte and GBM damage, together with dysfunction of slit diaphragm, leading to glomerular filtration membrane integrity destruction and proteinuria formation. Later, podocytes undergo detachment from the GBM, apoptosis and a significant decrease in the number density, finally leading to glomerular sclerosis. In type 2 diabetic patients, the decrease in the number of podocytes indicates the deterioration of renal function, and podocyte apoptosis is the main cause of decrease in podocyte number.CD36 is a transmembrane protein of the class B scavenger receptor family and is widely expressed in many kinds of cells including macrophages, microvascular endothelial cells, platelets, adipocytes, podocytes, etc. CD36 is involved in multiple biological and pathological processes through interacting with multiple extracellular ligands, including long-chain free fatty acids (FFAs), modified (oxidized) low-density lipoprotein (ox-LDL), advanced glycation end (AGE) products, thrombospondin-1 (TSP-1), and collagens Ⅰ and Ⅳ. Many studies showed that hyperlipidemia-induced apoptosis mediated by CD36 was associated with increased uptake of ox-LDL or fatty acid in macrophages leading to atherosclerotic lesions, in hepatocytes leading to liver damage and fibrosis in obese patients, and in proximal tubular epithelial cells leading to progression of diabetic nephropathy. However, the specific role of CD36 in podocytes apoptosis of DN with hyperlipidemia remains poorly investigated.Parkin protein originally found in Parkinson’s disease (Parkinson diseases, PD), expressed widely in human tissue, including brain tissue and liver tissue. Here, we found Parkin expression in kidney tissue for the first time. Mutations in the Parkin gene on chromosome 6 cause autosomal recessive, juvenile-onset parkinsonism (AR-JP), the most common form of early-onset disease. The Parkin gene encodes a 465 amino acid protein consisting of a ubiquitin-like domain at its N terminus and two RING finger domains separated by an in-between-ring domain at its C terminus. Similar to other RING-box proteins, Parkin can function as an E3 ubiquitin ligase, in cooperation with El-activating and E2-conjugating enzymes, to catalyze the ubiquitination of protein substrates (including polyubiquitination and monoubiquitination). The class B scavenger receptor CD36, as a new substrate of Parkin-mediated ubiquitination, expands a potentially function of Parkin in the regulation of FA metabolism. Parkin increases the amount of CD36 by mediating its mono-ubiquitination to enhance its stability, further leading to increased cellular uptake of fatty acids. We also found high expression of Parkin protein in the kidney tissues of CKD with hyperlipidemia and palmitic acid could induce the upregulation of Parkin in podocytes, parelled with CD36 expression. Moreover, there is a mutual combination between Parkin and CD36, and Parkin could inhibit the degradation of CD36, indicating that Parkin also mediated CD36 monoubiquitination in podocyte, further participating in lipotoxicity of podocyte.In this study, we simulated conditions of high fat in the body by palmitic acid stimulating podocytes. And we observed Parkin and CD36 mRNA and protein expression, the level of lipid deposition in cells, and lipotoxicity, in order to further understand the effects of hyperlipidemia on early kidney damage and provide theoretical basis and intervention.Objective:The aim of the present study was thus to investigate the expression of Parkin and CD36 in kidney tissues in DN patients with hyperlipidemia, and the effect of fatty acid on Parkin and CD36 expression and apoptosis of podocytes using an in vitro model and to elucidate its mechanism.Methods:Paraffin-embedded kidney tissue samples (Ctr=18, DN=20) were collected, and the expression of Parkin and CD36 was measured for immunohistochemistry and immunofluorescence staining. We cultured conditionally immortalized mouse podocytes (MPC5) and treated cells with palmitic acid, a saturated FFA, then measured Parkin and CD36 expression by Real-time PCR, Western blot analysis and immunofluorescence, lipid uptake by Oil red O staining and BODIPY staining, apoptosis by Flow cytometry assay, TUNEL assay and Western blot analysis, and ROS production by DCFH-DA fluorescence staining. All statistical analyses were performed using SPSS 21.0 statistical software. Numeric data were shown as means ± SD. Statistical significance between groups was analyzed by student’s t-test and one-way ANOVA. The difference was statistically significant (p<0.05).Results:(1) The Parkin and CD36 expression in kidney tissue was increased in DN patients with hyperlipidemia, and they were co-localizated both in renal glomerulus and renal tubule. Immunohistochemistry and immunofluorescence showed that the expression of Parkin and CD36 in both renal tubules and glomerulus of DN group increased significantly when compared with the control group, and there was a co-localization of Parkin and CD36. (2) Palmitic acid upregulated Parkin and CD36 expression of podocytes, and they were co-localizated in podocyte. Q-PCR and western blot revealed that Parkin and CD36 were upregulated under palmitic acid reduction. At the same time, double immunofluorescent staining revealed co-localization of Parkin and CD36 in podocyte. Every two groups, the difference was significant (P< 0.05). (3) Palmitic acid promoted lipid uptake of podocytes mediated by CD36. Using Oil red O staining and BODIPY lipid probes, we found the increased lipid accumulation in podocytes incubated with palmitic acid. We pretreated podocytes with SSO followed by treatment with PA, and found that the increased uptake of lipids in podocytes induced by palmitic acid was disrupted, which confirmed high lipid condition promoted lipid uptake into podocytes mediated by CD36. (4) Palmitic acid induced podocyte apoptosis mediated by CD36. To study the effect of palmitate on podocyte apoptosis in vitro, we treated podocytes with palmitic acid. Flow cytometry analysis revealed that apoptosis rates significantly increased when treated with palmitic acid at concentrations of 150 and 300 μM. Then, TUNEL and PI staining was employed to further validate the apoptotic effect of palmitic acid on podocytes. Massively increased apoptotic cells was observed in palmitic acid group in contrast to control group, we investigated the effect of palmitic acid on the activation of caspase-3 by western blotting using specific cleaved-caspase3 antibody, exhibiting that dose-dependently upregulated expression levels of cleaved-caspase-3 in palmitic acid-treated podocytes. To further investigate whether CD36 participated in high lipid inducing podocyte apoptosis, we treated podocytes with palmitic acid after pretreatment of SSO, and found that apoptosis was inhibited, compared with those in the group of palmitic acid treatment. Taken together, these data provided evidence that palmitic acid induced podocyte apoptosis was mediated by CD36. (5) Oxidative stress participated in fatty acid-induced podocyte apoptosis mediated by CD36. We used the ROS-sensitive fluorescent probe 2’,7’-dichlorofluorescein diacetate (DCFH-DA) to monitor cellular oxidative stress and found ROS production was considerably increased in podocytes treated with palmitic acid when compared with control group, and inhibition of CD36 function with SSO decreased the production of ROS induced by pamitate. Furthermore, we found that antioxidant tempol significantly attenuated enhanced ROS production and apoptosis in podocytes treated with palmitic acid. These proved that oxidative stress in podocytes induced by fatty acid was mediated by CD36. (6) Parkin mediated CD36 monoubiquitination and inhibited its degradation. Immunofluorescence and co-immunoprecipitation assay showed that Parkin and CD36 combinated each other, and overexpression of Parkin could decrease CD36 decradation, revealing that Parkin mediated CD36 monoubiquitination in podocyte.Conclusion:Parkin and CD36 mediating fatty acid-induced podocyte apoptosis via oxidative stress might participate in the process of DN.