The Study Of Histone Deacetylase Inhibitor SAHA Against Renal Calcium Oxalate Crystal And Related Mechanism

Background and ObjectiveWith the improvement of modern living standard and the change of life style, diseases related to environment or metabolism are increasing prominent. As one of the urological common diseases, kidney stones, bring patients pain, operative suffering [1] and heavy economic burden, also bring long-term deterioration effect of kidney function [2]. So it is crucial to prevent and treat renal calculi at the early stage. The most common type of renal calculi is calcium-containing stone, and calcium oxalate stones is approximately eighty percent [3]. The early state of kidney stone presents renal crystallization, which is the focus of research currently. The pathophysiological process of calcium oxalate crystal formation [4] are as follows: crystallization activates nicotinamide adenine dinucleotide phosphate oxidase(NADPH), induce the activation of cyclophilin D, open mPTP, collapse mitochondrion, the production of ROS increase, oxidative stress standard advance, cytochrome C is released by mitochondrion, activate Caspase3, those result in renal epithelial cell injury and apoptosis, change the distribution of the cell membrane phosphatidyl serine, promote crystal-cell adhesion, renal crystallization are formed largely. Therefore, it is valid to reduce renal crystal and alleviate kidney injury-induced by crystal through antioxidant and anti-apoptosis.Corresponding with genetic diseases, epigenetic diseases whose definition refer to the sequence of DNA doesn’t change but protein express disorderly [5] play an important role in environmental-related diseases. Histone modification is one of epigenetics, including histone phosphorylation, ubiquitination, methylation and acetylation [6], histone acetylation/deacetylation play a large part in regulation of gene transcription and activation. Together, histone acetyltransferase and deacetylase regulate the dynamic abundance of histone acetylation/deacetylation, amount of recent studies also find that inhibition of histone deacetylase can protect renal function, including improve renal fibrosis, suppress inflammation and reduce apoptosis [7], what’s more, there is a better research progress in acute kidney injury [8] of which advance the recovery of renal function.The development of most diseases and oxidative stress is inextricably linked, inhibition of oxidative stress level can retard the progression and reverse geterioration of certain diseases. Nuclear transcription factor(red cell source 2) associated factor 2(Nrf2) is a key protective transcription factors who mediated by the antioxidant response, and it plays a role mainly in transcriptional activation of antioxidant proteins. In physiological conditions, Nrf2 is coupled with Keap1 cytoplasm protein, when the body is subjected to oxidative stress injury, reactive oxygen species(Reactive Oxygen Species, ROS) products increased, prompting Keap1-Nrf2 uncoupling, Nrf2 rapidly run into the nucleus, then start the antioxidant protein expression. And during the process of crystal formation, with the stone factors gradually stronger than inhibition of stone, antioxidant capacity of body itself gradually is weakened, at the time,we can block the further growth of crystal if the body against oxidative stress support from outside. Research shows that, histone deacetylase inhibitors(HDACI) intervention could activate expression and up-regulation of Nrf2 and its downstream protein which play a neuroprotective effect in cerebral ischemia reperfusion animal model [9].Base on that histone deacetylase inhibitiors can play the anti-fibrosis, anti-inflammatory and antioxidant functions, then combined with the pathogenesis of renal crystallization,our team make the calcium oxalate crystal model of male mice induced by glyoxylate by intraperitoneal injection of suberoylanilide hydroxamic acid( SAHA), so that to verify protection effect of SAHA on renal injury caused by calcium oxalate crystal, further to investigate the molecular mechanism related, and provide a theoretical basis for clinical prevention and treatment of kidney injury induced by crystal. Research contents and Methods 一、Observe the protection effect of SAHA on the mice who got kidney injury induced by calcium oxalate crystalTwenty-four 6-week-old male ICR mice, weight 20-25 grams, and fed in the animal room of grade SPF. A week after adaptive feeding, they were divided into 4 groups: normal control group(group Control, A.) B. glyoxylate + saline group(group Saline) C. glyoxylate +DMSO group(group DMSO) D. glyoxylate +SAHA group(SAHA group), 6 mice each group, A group, no treatment, B, C,D group, the mice of three groups were treated with respectively DMSO, SAHA, Saline by intraperitoneal injection of 50mg/kg/d, 6 hours later, the three groups were treated with glyoxylate intraperitoneal injection of 100mg/kg/d, the above processing continuous 7 days,then all mice were put into metabolic cages, keep 24-hour urine, venous blood by inner canthus, then use saline to ventricular perfusion, one side of kidney tissue is put in EP tube and stored at-80 degrees C refrigerator, preparing for the detection of calcium content, MDA, SOD, GSH content or activity, the other side of kidney tissue soaked in formalin in order to make paraffin slice, for Von Kossa staining and immunohistochemistry staining.1 Detect the calcium content of kidney tissue, urinary calcium / creatinine ratio, observe Von Kossa staining and analysis of the semi quantitative, evaluate the effect of SAHA on calcium oxalate crystal formation.2 Detect serum creatinine, blood urea nitrogen(automatic biochemical analyzer), analysis of blood KIM1 and urinary KIM1 expression by ELISA method(kidney injury molecular 1), verify the effect of SAHA on renal injury induced by crystallization. 二、The possible mechanism of SAHA in reducing renal injury1. Detect MDA, SOD, GSH content or activity(colorimetric method) of four groups to evaluate the SAHA effect on oxidase stress level in model mice.2. Observe and compare OPN(osteopontin) and the expression of CD44 of kidneys in.all groups.3. Detect the effect of SAHA on the apoptosis of tubular epithelial cells, such as the observation of renal tissue TUNEL staining, the parallel semi quantitative analysis, and Western blot to detect Caspase-3 protein expression. Results一、SAHA reduces kidney injury induced by calcium oxalate crystallization1. Compared with the control group, SAHA decreased the content of calcium in mouse kidney tissue, reduced urinary calcium / creatinine ratio, reduced deposition of calcium oxalate crystal(Von Kossa results), so significantly reduced the formation of calcium oxalate crystal.2. The serum creatinine, blood urea nitrogen decreased significantly in the SAHA group, urinary Kim1 excretion was significantly reduced, and suggested SAHA has obvious protective effects on renal function.二、SAHA effectively inhibits the levels of oxidative stress by renal calcium oxalate crystallization, reduces apoptosis of tubular epithelial cells1.MDA expression of SAHA group in mouse kidney tissue significantly decreased compared with the saline control group and DMSO control group, while the expression of SOD, GSH had no difference obviously.2.Tubular epithelial cell apoptosis in SAHA group were significantly decreased, and Caspase-3 expression decreased obviously.3. Expression level of OPN and CD44 down-regulation in SAHA group significantly.Conclusion1. SAHA significantly reduced the calcium oxalate crystal formation of kidney tissue model mice.2. SAHA can significantly improve the renal function of calcium oxalate crystallization model mice3. SAHA can obviously inhibit the oxidative stress level4. SAHA can obviously reduce the apoptosis of renal tubular epithelial cells5. SAHA down-regulation the expression of OPN, CD44 in renal tissue of model miceFor the first time, our team applied histone deacetylase inhibitor SAHA to renal calcium oxalate crystals of mice model, found that SAHA can reduce the formation of crystal of kidney, reduce renal injury, and the protective mechanism are involved in anti-fibrosis, inhibit oxidative stress level, improve the tubular epithelial cell apoptosis, down-regulation expression of OPN and CD44, this may help for the prevention and treatment of renal calculi especially refractory, recurrent renal calculi,and provides treatment strategies and new targets.