Role Of NADPH Oxidase In The Development Of Renal Oxidative Stress Induced By Oxalate And Calcium Oxalate Crystals In Vitro And Vivo

Hyperoxaluria and crystal deposition induce renal oxidative stress (OS) and renal tubular injury which in turn contribute to the development of nephrolithiasis, both mitochondria and NADPH oxidase are considered to be the source of reactive oxygen species (ROS).This study focused on the role of NADPH oxidase in the development of renal oxidative stress induced by oxalate and calcium oxalate crystals in vitro and vivo. Apocynin, losartan and taurine were chosen here to act as the special intervenors (antioxidants). Five related parts of the study were discussed respectively. Results of this study suggest that renal angiotensinⅡand its stimulation of NADPH oxidase may partially account for the development of OS in kidney in a rat model of hyperoxaluria. We also detected evidences that the changes of mitochondria preceded that of the expression of the NADPH oxidase in kidney. In vitro, COM stimulate the expression and activity of NADPH oxidase in macrophage which may also account for the renal OS, since macrophages were seen commonly accumulating in the interstitium of kidney with crystal deposition. Part I: Establishment of a rat model of calcium oxalate nephrolithiasis, and the development of renal oxidative stress in the modelObjective To establish a rat model of calcium oxalate nephrolithiasis, and observe the development of renal oxidative stress (OS) in this model.Methods Animal model of hyperoxaluria and crystal deposition was established in adult male Sprague-Dawley rats by administration of 0.8% ethylene glycol (EG) in drinking water for 4 weeks. At the end of the study, markers for the state of OS, urinary 8-IP and H2O2, and the creatine clearnance and kidney/body weight were examined. Histological examinations of kidneys were performed to check the tubular injury and interstitium injury, as well as the crystal deposition in kidney. Renal tubular ultrastruture changes were analyzed under TEM. The expression of CD68,TGF-βandα-SMA in kidney were evaluated by immunohistochemistry.Results Compared with the control, renal OS developed significantly in rats received EG. Histological examinations of kidneys show renal tubular injury, with increased expression of CD68,TGF-βandα-SMA in kidney.Calcium crystal deposits in kidney were detected evidently.Conclusions Rat model of hyperoxaluria and crystal deposition was established successfully. Renal OS and renal injury, and calcium crystal deposits were detected evidently. Part II: Role of NADPH oxidase in the development of renal oxidative stress in a rat model of calcium oxalate nephrolithiasisObjective To investigate the role of NADPH oxidase in the development of renal oxidative stress (OS) in a rat model of calcium oxalate nephrolithiasis. Methods Rat model of hyperoxaluria and crystal deposition was established by administration of 0.8% ethylene glycol (EG) to the male SD rats in drinking water for 4 weeks. Apocynin, one of the selective NADPH oxidase inhibitor, was also administrated in the experimental group(0.2g·kg-1·d-1). At the end of the study, markers for the state of OS, urinary 8-IP and H2O2, and the kidney/body weight were examined. Immunohistochemistry, RT-PCR and Western blotting analysis were made respectively to evaluate the expression of NADPH oxidase subunits (p47phox, gp91phox, Nox-1, p22phox, Nox-4) in kidneys.Results Compared with the control, OS and renal injury occurred in the rats recived EG, in accordance with the increased expression of NADPH oxidase subunits p47phox, gp91phox, Nox-1 mRNAs and p47phox protein in kidneys. Simutanious treatment with apocynin can partially reduce OS and renal injury, as well as the expression of p47phox and Nox-1 mRNAs and p47phox protein in kidneys.Conclusions The increased expression of NADPH oxidase was suggested to be partially accounted for the development of OS in kidney in this rat model. Part III: AngiotensinⅡstimulate the expression and activity of NADPH oxidase in a rat model of calcium oxalate nephrolithiasisObjective To investigate the roles of angiotensinⅡand NADPH oxidase in the development of renal oxidative stress (OS) in a rat model of calcium oxalate nephrolithiasis.Methods Animal model of hyperoxaluria and crystal deposition was established in adult male Sprague-Dawley rats by administration of 0.8% ethylene glycol (EG) in drinking water for 4 weeks. Simultaneous treatment with apocynin (0.2g·kg-1·d-1) or losartan (30mg·kg-1·d-1) by intragastric administration were performed in rats respectively. At the end of the study, markers for the state of OS, urinary 8-IP and the enzymatic activity of superoxide dismutase (SOD) in kidney homogenates were assessed. The concentration of angiotensinⅡin kidney homogenates was determined using radioimmunoassay method.Expression of NADPH oxidase subunit p47phox protein and mRNA in kidney were localized and evaluated by immunohistochemistry and real time-PCR respectively.Results P47phox express widely in the kidneys of this rat model, including renal cortex, inner medulla and outer medulla.Compared with the control, OS developed significantly in rats received EG, with increased expression of p47phox mRNA in kidneys. Renal angiotensinⅡalso increased significantly. Treatment with apocynin or losartan significantly reduced the excretion of urinary 8-IP, restored the SOD activity, with decrease in the expression of p47phox mRNA in kidney, but the levels of those OS markers in apocynin or losartan treated rats were still higher than that of the normal controls.Conclusions Results suggest that renal AngⅡand its stimulation of NADPH oxidase may partially account for the development of renal OS in this rat model.Part IV: Mitochondria but not NADPH oxidase account for the renal oxidative stress in a rat model of calcium oxalate nephrolithisisObjective Hyperoxaluria and crystal deposition induce oxidative stress (OS) in kidney; both mitochondria and NADPH oxidase are considered to be the source of reactive oxygen species (ROS). Taurine is known as an antioxidant. We investigate the putative source of ROS, as well as the effects of taurine treatment on renal protection in a rat model of calcium oxalate nephrolithisis.Methods Animal model of nephrolithiasis was established in adult male Sprague-Dawley rats by intragastric administration of 2.5% ethylene glycol+2.5% Ammonium Chloride 2ml twice daily, with restriction on intake of drinking water(20ml /per rat daily) for 4 weeks. Simultaneous treatment with taurine (2.0% mixed with the chow) was performed. At the end of the study, indexes of OS and renal injury were assessed. Renal tubular ultrastruture changes were analyzed under TEM. Crystal deposition in kidney was analysed under light microscopy. Expression of NADPH oxidase subunit p47phox protein and mRNA in kidney were localized and evaluated by immunohistochemistry and real time-PCR respectively. AngiotensinⅡin kidney homogenates was determined by radioimmunoassay method. Results Compared with the control, oxidative injury of kidney occurred in rats induced nephrolithiasis. Hyperplasia of mitochondria developed in renal tubular epithelium .The activities of SOD and GSH-Px in mitochondria decreased significantly and the mitochondrial membrane showed oxidative injury. Taurine treatment significantly alleviated oxidative injury of kidney and its mitochondria, restored SOD and GSH-Px activities, with relative slight morphological changes in kidney. We could not detect statistically changes in the renal p47phox mRNA expression, as well as the renal angiotensinⅡin those rats. Conclusions Results suggest that mitochondria but not NADPH oxidase account for the OS in kidney, taurine protected the kidney from oxidative injury by the mitochondrial-linked pathway in this rat model.Part V: Calcium oxalate crystals stimulate the expression and activity of NADPH oxidase in macrophage in vitroObjective The NADPH oxidase was originally discovered in neutrophils, where it is a potent source of millimolar quantities of superoxide during phagocytosis and plays a vital role in nonspecific host defense. Macrophages were seen commonly accumulating around the crystals in the interstitium of kidney, which suggest that macrophage involve in the crystal deposition in kidney.We test our hypothesis that COM crystals stimulate the expression and activity of NADPH oxidase in macrophage which may also account for the renal oxidative stress (OS) and injury.Methods Confluent cultures of macrophage cells (Ana-1) were exposed to COM crystals for 6 h .Markers for the state of oxidative stress, MDA and LDH in the culture medium were measured to investigate the involvement of ROS. Cellular ROS and the activity of NADPH oxidase in macrophage cells were assessed by ?uorescence spectrophotometrical analysis and spectrophotometrical analysis, respectively. We also investigated the effect of the NADPH oxidase inhibitor apocynin on the COM crystal-induced expression of p47phox protein using western blot analysis. Cell-crystal reaction was detected by light microscopy and TEM.Results Exposure of macrophage cells to COM crystals resulted in increased expression of p47phox protein, with increased MDA and LDH in the culture medium. Cellular ROS and the activity of NADPH oxidase in macrophage cells also increased significantly. Treatment with apocynin reduced the crystal-induced expression of p47phox protein, inhibited the activity of NADPH oxidase in macrophage cells, in accordance with decrease of those OS markers. We also detected the phagocytosis of crystal by macrophage under light microscopy and TEM.Conclusions Results indicated that COM crystals stimulate the expression and activity of NADPH oxidase in macrophage .This may be one of the the mechanism of renal interstitium injury induced by crystal deposition.