Histone Deacetylase 4 Selectively Contribute To Podocyte Injury In Diabetic Nephropathy

Objective:Diabetic nephropathy (DN) is one of the most important microvascular complications, and the leading cause of end stage of renal disease (ESRD). Although the pathogenesis of DN is multifactorial, an increasing number of experimental studies have highlighted the importance of histone deacetylase (HDAC)-mediated epigenetic processes in the development of renal injury. HDACs are a family of enzymes, which compete for the control of acetylation of lysine residues making up the histones with histone acetyltransferase (HAT), determining the post-translational acetylation status of histone and a number of other nonhistone proteins. To date, 18 HDACs identified in mammals are divided into four classes according to their sequence identity and catalytic activity. Class I HDACs (HDAC1,2,3, and 8) share sequence homology with the yeast Rpd3 protein, which contain a nuclear localization signal (NLS) and, with the exception of HDAC3, lack a nuclear export signal (NES). Thus, class I HDACs are mainly located in the nucleus and regulate many gene expressions, such as Thioredoxin binding protein-2 (TBP-2), Myocyte enhancer factor-2 (MEF2), NF-κB, GATA. Class II HDACs (HDAC4,5,6,7,9, and 10) are larger proteins than class I HDACs because they contain additional regulatory domains. Class Ⅱ HDACs contain a NLS and a NES, and are known to’shuttle’ between the cytoplasm and nucleus in a manner regulated by phosphorylation. Class II HDACs are further subdivided into class Ⅱa HDACs (HDAC 4,5,7,9) and class Ⅱb HDACs (HDAC6,10). Function of HDACs different from each other, therefore, it is important to elucidate the functional role of individual HDACs in DN and develop HDAC inhibitors with improved specificity. By targeting only the most relevant HDAC isoform in a particular indication, it may be possible to greatly improve the efficacy by removing certain toxicities that may be associated with the inhibition of multiple isoforms.Autophagy is emerging as an important pathway in many biological processes and diseases. Although studies have shown that autophagy regulates many critical aspects of normal and disease conditions in the kidney, such as tubular injuries, kidney development and aging, genetic diseases associated with the kidney, and DN, the importance of autophagy in the kidney has only just begun to be elucidated. Autophagy has an essential role in stress adaption in renal injuries through removal of protein aggregates and damaged organelles, and promotion of cell survival; autophagy can also contribute to cell death by autophagic cell death. However, how the process of autophagy is altered in the pathogenesis of renal diseases and how this alteration is beneficial or detrimental to renal functions is unclear. It is being increasingly recognized that acetylation can also regulate autophagy. Increased cellular acetylation level by HDAC inhibition in cells promotes autophagy, and knockdown of the histone acetyltransferase KAT2B/p300 induces autophagy in nutrient rich conditions. In this study, we identified for the first time the expression patterns of HDACs in DN and found that HDAC4 selectively contributes to podocyte injury by exacerbating inflammation and suppressing autophagy.Methods:1 Animal studies:1.1 Expression pattern of Zn2+ -dependent HDAC in STZ-induced diabetic nephropathyThirty wild male Sprague-Dawley rats (200g-250 g) were divided into two groups randomly, vehicle group and STZ-induced diabetic rats group. The expression of HDAC were tested by Western blotting (WB), Real-time RT-PCR and immunohistochemistral staining (IHC). Expression of HDAC4 in podocyte were detected by immunofluorescence, synaptopodin antibody was used to mark podocyte and HDAC4 antibody was used to detect the expression of HDAC4 in podocyte.1.2 Expression pattern of HDAC2, HDAC4 and HDAC5 in db/db miceThe expression of HDAC2, HDAC4 and HDAC5 were tested by WB.2 Human renal biopsy samples studiesThe samples of renal biopsies were obtained from Department of Pathology, Shandong University School of Medicine. Control samples were obtained from the healthy kidney poles of individuals who underwent tumor nephrectomies without diabetes or renal disease. Immunohistochemistry technology were used to observe the change of HDAC2, HDAC4 and HDAC5 in the kidney biopsies from normal kidney, DN patients and diabetic patients without nephropathy. Relative mRNA levels of HDAC2, HDAC4 and HDAC5 in the renal biopsies from all the subjects were examed by real time RT-PCR. The correlation between HDAC mRNA levels and estimated glomerular filtration rate (GFR) in all subjects were estimated.3 Studies in vitro:Function of HDAC and mechanism by which HDAC4 contribute to podocyte injury.3.1 Expression level of HDAC2, HDAC4 and HDAC5 in in response to common detrimental factors in DN:Renal cells including human podocyte, human mesengial cell, human glomerular endothelial cell and Human proximal tubule epithelial cells were cultured in vitro. Effects of different stimuli on the expression of HDAC2, HDAC4 and HDAC5 in podocytes, such as high glucose (HG), advanced glycation end-product (AGE), transforming growth factor-β1 (TGF-β1) were carried out by WB.3.2 Effect of HDAC4 silence in podocyte inflammatory response and apoptosis: Podocytes were transfected with shRNA-HDAC4 and pCMV6-HDAC4 plasmids by Lipofectamine 2000; The level of TNF-a, TGF-β1, IL-8 and MCP1 were assessed by ELISA kit; Apoptosis of Podcyte with HDAC4 silence and in the treatment of High glucose were assessed by Flow cytometry.3.3 Role of HDAC4 in regulation of autophagy level in podocyte: The expression level of LC3B and Beclinl were detected by WB; The transmission electron microscopy was used to detect the number of typical autophagosomes; RFP-GFP-LC3 adenovirus construct was used to monitor progression from the autophagosome to autolysosome in this study.3.4 Effect of acitvation of autophagy in podocyte: WB was used to detect the expression level of nephrin and podocin with silence of HDAC4 and in the treatment of Rapamycin; Apoptosis of Podcyte with HDAC4 silence and in the treatment of Rapamycin were assessed by Flow cytometry; Podocyte architectural integrity were assessed by immunofluorescence, F-actin antibody were used to mark the cytoskeleton.3.5 The role of HDAC4 in deacetylation and activation of STAT1:Co-IP was carried out to assess the interaction between HDAC4 and STAT1; Co-IP was carried out to assess the acetylation level of STAT1; Immunofluorescence were used to examined the subcellular localization of STAT1.4. Role of gene silence of HDAC4 in renal injury of diabetic rats:shRNA-HDAC4 or their negative controls were delivered to the rat kidney by means of intraparenchymal injections. A 31G needle was inserted at the lower pole of the kidney parallel to the long axis and was carefully pushed toward the upper pole. As the needle was being slowly removed,100 μ1 of filter-purified lentivirus or phosphate-buffered saline was injected. PAS staining and TEM were used to assess kidney injury. ELISA kit was used to assess the level of proinflammatory mediators. WB was used to detect the expression levl of LC3B, Beclinl and nephrin.Results:1. Animal studies:Expression patterns of HDACs in the kidney from STZ-induced diabetic rats and db/db mice:1.1 Expression patterns of HDACs in streptozotocin-induced diabetic rats:Real time RT-PCR and WB analyses showed that among Zn2+-dependent HDACs, the expressions of HDAC2, HDAC4 and HDAC5 were significantly increased in the kidney from STZ-induced diabetic rats. The levels of HDAC1,3,6,7,8,9,10 and 11 had no significant changes; Our immunohistochemical studies further confirmed the upregulation of HDAC2, HDAC4 and HDAC5 in the kidney from STZ-induced diabetic rats; The immunofluorescence analysis show that the expression level of HDAC4 in podocyte from diabetic rats was significantly increased.1.2 Expression patterns of HDAC2, HDAC4 and HDAC5 in db/db mice: Compared to the sample from db/+ mice, we observed the enhanced renal expressions of HDAC2, HDAC4 and HDAC5 in the db/db mice.2 Human renal biopsy samples studies:Expression patterns of HDAC2,4,5 in human diabetic renal tissues and the correlation between HDAC2,4 and 5 mRNA level and eGFRUpregulation of HDAC2、HDAC4 and HDAC5 was observed in paraffin-embedded sections of human diabetic renal tissues by immunohistochemical staining, but weak staining in the kidney from normal controls or diabetic patients without nephropathy (DM-NN). Real time RT-PCR analysis further confirmed the changes of mRNA levels of HDAC2, HDAC4 and HDAC5 in renal biopsies from DN subjects. HDAC2 expression level was shown an increase tendency, although no statistical difference.3 Studies in vitro: Function of HDAC and mechanism by which HDAC4 contribute to podocyte injury.3.1 Podocyte HDAC4 was upregulated in response to common detrimental factors in DN:Among HDAC2, HDAC4 and HDAC5, we found that HG, AGE, or TGF-β1 increased HDAC4 expression in a concentration dependent manner in podocytes. HG and AGE increased HDAC5 expression in mesangial cells. In addition to glomerular cells, we found that HDAC2 was upregulated in response to HG and AGE in human proximal tubule epithelial cells. Finally, immunofluorescent results further confirmed the increase in podocyte HDAC4 expression in the kidney from STZ-induced diabetic rats in vivo3.2 Gene silencing of HDAC4 reduced inflammatory response, apoptosis in podocytes with HG treatment:To further investigate the role of HDAC4 on podocyte function, gene silencing of HDAC4 by shRNA-HDAC4 or overexpression of HDAC4 by pCMV6-HDAC4 transfection were used in this study. We found that HG-enhanced the production of proinflammatory mediators was attenuated by HDAC4 knockdown, as well as podocyte apoptosis measured by flow cytometry3.3 HDAC4 contributed to HG-inhibited basal autophagy in podocytes:HG reduced the expressions of autophagy related proteins in podocytes, in consistent with the results from overexpression of HDAC4. Gene silencing of HDAC4 restored the levels of autophagy-related proteins by Western blot analysis. The typical autophagosomes with double membranes weresignificantly increased in shRNA-HDAC4 transfected podocytes compared with scramble transfected podocytesunder HG condition by transmission electron microscopy (TEM). We utilizedthe tandem RFP-GFP-LC3 adenovirusconstruct to further confirm autophagy induction byform punctate that represent autophagosome formation. After infection with thetandem construct, we evaluated the successful introduction of this adenovirus showing both fluorescent proteins. In addition to accumulation of LC3, there were morered puncta in shRNA-HDAC4 transfected podocytes than in control cells under HG condition.3.4 Autophagy inhibition by HDAC4 reduced the expressions of podocyte slit diaphragm proteins and disrupted the podocyte architectural integrity:To examine the role of HDAC4-suppressed autophagy, we used the autophagy enhancer rapamycin to restore autophagy as control. Our study showed that HG reduced podocin and nephrin expression, induced apoptosis and thechanges in cytoskeleton distribution, which were alleviated by restoring defective autophagy with low dose of rapamycin as well asby gene silencing of HDAC4.3.5 Activation and translocation of STAT1 was associated with HDAC4-mediated acetylation in response to hyperglycaemia:Co-IP showed an interaction between HDAC4 and STAT1 inpodocytes, We also found that HG-induced STAT1 deacetylation was attenuated by gene silencing of HDAC4. Similarly, overexpression of HDAC4 significantly decreased STAT1 acetylation, indicating that HDAC4 may be required for the deacetylation of STAT1 inpodocytes with HG treatment. Immunofluorescence analysis showed that translocation of STAT1 to nucleus, which is an essential step for STAT1 activation, was associated with HDAC4.4 Gene silencing of HDAC4 ameliorated renal injury in STZ-induced diabetic rats:4.1 In vivo gene silencing of HDAC4 by intrarenallentiviral gene delivery in rats: Confocal immunofluorescent analysis demonstrated GFP was expressed in podocytes after intrarenal delivery of lentivirus. Real-time RT-PCR further confirmed the efficiency of the in vivo gene transfer showing that rats injected with the shRNA-HDAC4 lentivirus markedly reduced HDAC4 mRNA levels 48h after injection and can stably transduce renal cells for at least 3 months.4.2 Gene silencing of HDAC4 ameliorated renal injury in STZ-induced diabetic rats:In vivo gene silencing of HDAC4 ameliorated renal injury as evidenced by reduced albuminuria, mesangial expansion, ameliorated podocyte injury and decreased production of proinflammatory mediators in diabetic rats.4.3 The expressions of autophagy related proteins were recovered by Gene silencing of HDAC4 in the kidney from diabetic rats:Gene silencing of HDAC4 even increased the basal levels of Beclin-1 and LC3Ⅱ/LC3Ⅰ ratio in the kidney.4.4 The expression levels of nephrin were recovered by gene silencing of HDAC4 in the kidney from diabetic rats. Conclusion:1. The present study for the first time investigates the expression patterns of individual HDAC in DN.2. The present study further provides direct evidence that HDAC4 specifically contributes to podocyte injury and demonstrate that HDAC4 is one of critical components of a signal transduction pathway that links renal injury to reduced autophagy in DN, suggesting that it is important to elucidate the role of individual HDACs within diabetic kidney and the development of HDAC inhibitors with improved specificity.