Study On The Role Of Urotensin â…¡ In Aristolochia Manshuriensis-induced Acute Renal Damage In Rats And Its Mechanism

UrotensinⅡ(UⅡ) is the most potent vasoconstrictor peptide identified to date. UⅡwidely exists in the cardiovascular system, kidney, liver, brain and so on from mollusks tomammals. Accumulating evidences indicate that UⅡcould up-regulate the expression ofcollagenⅠ,Ⅲ, FN and PAI-1, decrease the expression and activity of MMP-1, promete thesynthesis and release of ET, and increase the expression of TGF-β1 in both gene and proteinlevels. All of the above show its effects on regulation of active substances and extracellularmatrix (ECM) synthesis and metabolism beyond its effects on vascular tension. In addition,UⅡacts as a mitogen and promotes the proliferation and DNA synthesis of vascular smoothmuscle cells, cardiac fibroblasts, mesangial cells and other cells. Most of above-mentioned roles of UⅡare mediated by its specific receptor GPR14. UⅡcombined with its receptorcan trigger various biological e ffects that play an important pathophysiological role in thedevelopment and progression of primary hypertension, congestive heart failure, renaldiseases, cancer and other dise ases. Although the mechanism of UⅡin these diseases hasnot been clarif ied, it is likely that UⅡma y become a new target for clinical treatment ofcertain diseases after the specific UⅡreceptor antagonist is discovered.Aristolochia ma nshuriensis (AM) is the wood st em of Northeast China Aristolochicwithout rough skin. It is commonly used one of the Chinese herbs and its main componentis aristolochic acid (AA). In the recent years, the cases of renal injury induced by Chineseherbs containing AA were reported. This kind of renal injury was named as aristolochic acidnephropathy (AAN). Extensive researches showed that AAN is one of the common causesof acute and chronic renal failure with tubulointerstitial damage as the main characteristicsof AAN. High dosage of AM leads to acute renal toxicity with the pathologicalmanifestations of epithelial cell degeneration and necrosis, collapsed, and the nude tubularbasement membrane with a few inflammatory cell infiltration and without the tubular cellregeneration. It should be noted that after withdrawal of the medicine containing AA for along term even though, the irreversible renal damage could still advance. There is noeffective therapy for AAN till now. Both UⅡand its receptor are abundantly expressed in kidney. In healthy kidney,UⅡand its recepto r primarily exist in the epithelial cells of the renal tubules and collectingtubules, especially in the distal convoluted tubules. UⅡis secreted and excreted by kidneyand it acts as a growth factor for the renal epithelial cells by autocrine and (or) paracrineways. UⅡparticipates in the renal vasodilation and for mation of urinary sodium.UⅡindirectly affects renal function via the pituitary-adrenal axis, thus infuences thewater-sa lt glucose and fat metabolism. The recent studies showed that expression of UⅡandGPR14 mRNA was significantly increased in patients with diabetic nephropathy; UⅡmRNA was 45 times of that of the control group, while GPR14 mRNA was nearly 2000times of that of the control group; both UⅡand GPR14 were observed mainly in thetubular epithelial cells. All of the above results suggest that UⅡis associated withphysiopathology and pathogenesis of the kidney diseases. Till now, the role of UⅡinAM-induced acute renal injury has not been reported.In the present study, the acute renal damage model were prepared in rats using AMwater decoction by gavage, and cultured rat renal tubular epithelial cells were exposed tothe medium containing AA. In the present experiments, the biochemical, morphological,immunohistochemical staining, ELISA, RT-PCR and Western blot techniques wereemployed to invesigetet the following issues:①the contents of both UⅡand its receptor inAM-induced acute renal damage in rats;②the relationship of UⅡwith EGF, IGF, TGF-β1,ET, and AngⅡin this acute renal ;③the effect of UⅡon ECM, such as the synthesis ofColⅠ, ColⅢand fibronectin, and the synthesis and release of PAI, TIMP-1 and MMP-2in the acute renal damage induced by AM. The main findings of this study are as follows:1. The urinary NAG content was significantly increased after the administration ofAM (20 g/kg·d) for 15 days, and the urinary RBP content was obviously elevated after AMadministration for 25 days.2. The UⅡcontent in serum was significantly increased in rats with acute renaldama ge after the administrati on of AM for 15 days and UⅡcontent in urine was markedlyincreased af ter AM admi nistration for 25 days,. UⅡcontents in serum and urine weregradually increased with the prolongated administration of AM, and kept parallel to thedegree of renal tubule damage.3. The expression of mRNA and protein levels of UⅡin the renal tissue with acuterenal damage was obviously elevated after AM administration for 15 days. The expressionchanges of mRNA and protein of GPR14 were found in the renal tissues after AMadministration for 7 days, which was earlier than those of U II. Both changes were parallelwith the degree of the tubular cell damage.4. Immunohistochemical detection showed that the area and intensity of immunologicstaining for EGF, IGF, TGF-β, ET and AngⅡwere increased in the renal tissue after AMadministration. Also, the area and intensity of FN, PAI-1 and TIMP-1 were significantlyelevated.5. Zymography analysis revealed that MMP-2 activity was markedly decreased in therat renal tissue after AM-treated for 7 days, then reduced gradually with the extension ofexperiment. In vitro study showed MMP-2 activity was decreased in cultured rat renaltubular epithelial cells exposed to AA. 6. The 10-10 ~10 -8 mol/L of UⅡcould stimulate cell proliferation andbromine-deoxy-uridine incorporation in a conc entration-dependent manner in cultured ratrenal tubular cells. The secretion of ColⅠ, FN and TIMP-1 was promot ed by 10 -8 mol/L ofUⅡin cultured tubular epithelial cells.7. mRNA and protei n expression of UⅡand GPR14 was stimulated by UⅡincultured rat renal tubular cells, and mRNA and protein expression of GPR14 wasup-regulated in tubular cells exposed to AA.8. The protein contents of EGF, TGFβ, ET-1, AngⅡ, UⅡ, ColⅠ, ColⅢ, PAI-1 andTIMP-1 were elevated while IGF decreased in supernatant of cultured tubular epithelialcells treated with AA. 10-8 mol/L of UⅡpromoted AA-induced the secretion of IGF andColⅢin the supernatant of the cultured cells.The above results may provide new experimental evidences for treatment of AAN,blockage of the roles of UⅡmay ameliorate renal damage, decelerate the process of AAN,and thus UⅡmay become a new target for therapy of AAN.The main conclusions are as followed:1. The contents of UⅡin serum and urine are significantly increased in the rats withacute renal damage induced by AM, the expression of gene and protein level of UⅡandGPR14 are markedly elevated in the renal tissues of rats with acute renal damage inducedby AM, and keep parallel with the degree of the tubular damage. 2. IGF release from the renal tissues is promoted by UⅡin rats with acute renaldamage induced by AM, thus affects the regeneration and reparation of necrotic tubules.3. UⅡelevates the content of ColⅢin the renal tissues of rats with acute renaldamage induced by AM, thereby promotes the renal fibrosis.4. UⅡin certain concentration ranges stimulates the proliferation and DNA synthesisin the renal tubular cells, also the secretion of ColⅠ, FN and TIMP-1 is promoted.The main innovations of this study are: it is proved that the contents of UⅡsignificantly are increased in serum and urine of the rat with acute renal damage induced byAM; and that both mRNA and protein of U II and GPR14 increase obviously in these renaltissues; ; UⅡstimulates release of IGF from the renal tissues in rats with acute renal damageinduced by AM; UⅡpromote IGF protein expression in the renal tissues in rats with acuterenal damage induced by AM; UⅡincreases the content of ColⅢin the renal tissues inrats with acute renal damage induced by AM; UⅡpromotes the proliferation and DNAsynthesis in the rat renal tubular cells in vitro, and promotes the secretion of ColⅠ, FN andTIMP-1; UⅡup-regulates mRNA and protein expression of GPR14 in tubular epithelialcells exposed to AA.