The Interventive Effects Of Histone Deacetylases Inhibitor VPA On Diabetic Nephropathy In Rat Through Endoplasmic Reticulum Stress Regulation And Its Mechanism

Diabetic nephropathy (DN) is one of the serious complications of diabetesmellitus. It is a major cause of end-stage renal disease (ESRD), which causes the heavysocial and economic burden on families. The pathogenesis of DN is complicated and thereis no effective treatment for the disease.Endoplasmic reticulum (ER) is an important subcellular organelles in eukaryotes,which plays an important role in the folding, transportation and modification of theprotein. Endoplasmic reticulum stress (ERS) has played a key role in maintaining thenormal functions of cells. Recent studies demonstrated that sitimulations such asexcess nutrients, free fatty acid, high glucose and oxidative stress can initiate ERS andthen induced cell apoptosis and tissue damage in the diabetes condition. In order tomaintain the stability of the environment, multiple cellular signaling pathways of ERSare mobilized in order to restore ER homeostasis and to promote cell survival. Thisreaction is called unfolded protein response (UPR). However, should attempts aimedat survival fail, ERS response elicits a final maneuver culminating in apoptosisthrough ER-associated death(ERAD). ERS regulate the crosstalk interaction withother cells damage mechanisms and cause cascading effects.High glucose, redundancy free fatty acid, oxidative stress, excessive nutrition indiabetes mellitus are important factors of ERS. These factors can activate the signaltransduction system of ERS through various pathways. ERS can make unfoldedprotein and misfolded protein accumulated. DN is believed to be one of thecomplications that happened in the late diabetic phase. Excessively strongstimulations will cause ERAD. Thus, interference of ERS will be considered toimprove therapeutic effects of DN.Histone acetylation modification is associated with chromatin level of eukaryoticgene expression regulation. Histone acetylation modification is associated withchromatin remodeling. Histone acetylation modification can regulate the transcriptionof genes. The level of histone acetylation is decided by the dynamic equilibriumbetween histone acetylation enzyme (HDAC) and histone acetyl transferase (HAT).Histone acetylation can regulate the cell proliferation, migration and apoptosis.Therefore, histone acetylation enzyme inhibitors (HDACIs) are widely used in treatment of nerve degeneration, diabetes, cardiovascular disease and autoimmunediseases, etc. Researces have showed that HDACIs have protective effects on kidneytissue and are associated with transcriptions of ERS related genes. Therefore, wehypothesized that HDACIs might relieve diabetic nephropathy cell damage throughregulating ERS. Valproic acid (VPA) is one of the representative drugs ofHDACIs, which is very cheap and has few side effects.The research includes in vitro and in vivo parts. We analyzed the effects of VPAon ERS and apoptosis induced by high glucose in NRK-52E cells. Then, we createddiabetic nephropathy rats models and gave the VPA lavage to detect whether the effectof VPA on diabetic kidney damage is related to ERS and apoptosis. We also explorethat whether the mechanism is associated with regulation of acetylation level of ERSrelated genes.MethodsWe cultured NRK-52E cells and determined VPA concentrations in NRK-52Ecells by MTT assays. The expression of ERS-associated protein GRP78, CHOP and Caspase12were detected by Western blot.The cell apoptosis ratio was determined byAnnexinV-FITC/PI amphophil cell.The expression of P-JNK,ATF4, Bcl-2, Bax,cleaved-Caspase3were detected by Western blot.Wistar rats were injected of STZ to induce diabetic model after two weeks ofadaptive raising, Then the diabetic rats were fed high-fat diets with another22weeks.DN models were determined by the biochemical and pathological examination. Then,the DN rats were gavaged by VPA.8weeks later, all the DN rats were killed and thekidneys were taken. The expression of GRP78, CHOP and Caspase12were detectedby Western blot, immuno-histochemical and immunofluorescence.Cell apoptosis ratiowas determined by Terminal deoxynucleotidyl transferase dUTP nick endlabeling(TUNEL). The expression of P-JNK,ATF4, Bcl-2, Bax, and Caspase3weredetected by Western blot. Serum creatinine, albuminuria and glucose were detected byautomatic biochemistry analyzer. Immunohistochemical staining and TransmissionElectron Microscopy were also carried out to examine the renal tissues. Histone H4total acetylation were detected by Histone H4total acetylation colormetric method kit.The acetylatic levels of histone H4in GRP78and CHOP promoter were detected byChromatin immunoprecipitation assay (CHIP) in vitro and in vivo. ResultsIn vitro experiments showed that VPA enhanced the expressions of GRP78andreduced the expressions of CHOP and Caspase12. These results were indicated thatVPA reduced ERS in NRK-52E cells induced by high glucose. VPA reduced the cellapoptosis ratio in NRK-52E cells induced by high glucose. The expressions of P-JNK,ATF4, Bax, Caspase3were decreased and the expression of Bcl-2was increased. Invivo experiments showed that VPA reduced24hours urinary protein and serumcreatinine level in DN rats, but the blood sugar, weight and the ratio of kidneyweight/body were not significantly effected. The results of histopathologicalexamination showed that the bowman’s capsule cavity gap became narrowed,glomerular basement membrane became thick, matrix broadened, mesangial cellsproliferated in. VPA improved the pathological changes in rats model of diabeticnephropathy. The results of transmission electron microscope technology showed thatglomerular basement membrane became obvious thickened, membrane filtration threelayers structure was unclear and foot process tooth comb was disorder, the gapbecame broadened and mesangial matrix was increased in rats model of diabeticnephropathy. VPA improved the pathological changes in rats model of diabeticnephropathy. All the results showed that VPA relieved diabetic renal injury.Immunohistochemistry and immunofluorescence microscopy results showed that VPAincreased the expressions of GRP78and reduced the expressions of CHOP andCaspase12. Western blot showed that VPA increased the expressions of GRP78andreduce the expressions of CHOP and Caspase12.Conclusions1The24h proteinuria, creatinine, histopathological and ultrastructural changes ofkidneys were examined. And injuries were found in both renal glomerular and tubular.of DN rat models. Apoptosis were found in renal glomerular and tubular. Compared tothe glomerular, apoptosis in tubular was more significant.2VPA can reduce ERS and cell apoptosis in NRK-52E cells induced by high glucose.3VPA can reduce ERS and cell apoptosis and relieve diabetes renal injure in DN rats.4VPA may regulate ERS by regulating the acetylation level of H4in GRP78andCHOP promoters.Inhibitor of histones to acetylation enzyme may be a new choice for treatmentof diabetic nephropathy.