The Roles Of [Ca～(2+)]i, Oxidative Stress, And Osteopontin In Fluoride-induced Renal Damages And Proteomic Analysis Of Fluorotic Rat Kidney

Fluoride is a widely distributed nephrotoxin with exposure potentially resulting from environmental fluorine excess. Kidney is the main excretory organ of fluorine. The remarkable renal pathological changes were observed in rats treated by sodium fluoride (NaF), and the major damage induced by fluoride was renal tubules. Significant increase of fluoride caused renal fibrosis and renal tubules dilatation. The proximal renal tubular cells degeneration and necrosis associated with proliferation were observed when lower dose of fluoride was administered. A series of experiments was conducted to study the mechanism of NaF on renal tubules lesion. Different oral dose of NaF in aqueous solution was given to Wistar rats and cultured renal tubular cells in vitro to observe morphological and functional changes of renal cells. This study sought to observed the following problems kidney injury caused by fluoride toxicity by means of pathological, biochemical, molecular biological and proteomical techniques: (1) To study the effects of NaF renal apoptosis and proliferation, and evaluate oncogene bax and bcl-2 change in the renal cell; (2) To measure the change of renal intracellular calcium in rat renal epithelial cells and Ca2+-ATPase. (3) The role of oxidative stress in renal injury mechanism induced by over-dose fluoride. (4) The expression and roles of osteopontin(OPN) and matrixmetalloproteinase-2 (MMP-2) in kidney lesion of rats treated with NaF; (5) To find out the renal proteomic differential expression in fluorosis rats.The pathological examination showed that renal epithelial cells degenerated, and appeared intensely eosinophilic cytoplasm with dense nuclear chromatin fragments. Notable renal apoptosis ws observed in fluoride-treated group compared to the control through Flow Cytometry and TUNEL techaniques. The results of MTT method In vitro culture renal tubular cells showed that the proliferous activity changed from sightly to dramatically intensed in response tofluoride concentration from 0.1mg/L to 15mg/L, but renal tubular cells proliferation obviously decreased in the 25mg F7L group. The mRNA level of bax showed a significant increase in renal tubular cells exposed to NaF and the strong relationship to fluoride dose. The mRNA level of bcl-2 gradually descended in renal tubular cells along with increase of fluoride concentration and showed a low correlation coefficient to administered fluoride. These results revealed that renal tubules apoptosis was the fundamental mode in fluorosis renal injury. The effect of fluoride on the kidney presented a trend of "complete dose-response" relationship.It was reported that fluoride caused renal calcification, which was deduced by an increase in parathyroid hormone (PTH) secretion and a subsequent large increase in renal calcium. Herein, we examined the effect of NaF on serum total calcium (t[Ca]) .ionized calcium(l[Ca]) and renal [Ca2+]i and Ca2+-ATPase. It showed that t[Ca] and i[Ca] in serum decreased in low calcium+ fluoride group, and the i[Ca] decreased to statistical significance. All these results confirmed that renal injury were more exacerbated in low calcium+ fluoride environment than that of single fluoride group. We examined the renal [Ca2+]i using a Ca2+ probe, fura-2. Fluoride effectively increased the concentration of [Ca2+]i in renal epithelial cells. In order to confirm the mechanism of calcium accumulation in renal epithelial cells, we examined the activity of Ca2+-ATPase in renal tissue. NaF stimulated Ca2+-ATPase in the 100mg F7L, but the activity of Ca2+-ATPase obviously decreased in low calcium + fluoride group. The Ca2+-ATPase was detected in renal tubular cells. The results showed minimal dose (1-5 mg F/L) stimulated activity of Ca2+-ATPase, however, high- dose fluoride inhibited activity of this enzyme. Our results partially explained calcium accumulation in renal epithelial cells induced by fluoride through inhibiting the activity of Ca2+-ATPase.The study was to explored the effect of NaF on oxidative stress of kidney. The activity of r...