| Background/Aims:Uromodulin(UMOD),also called Tamm-Horsfall protein,is the most abundant protein in healthy human urine.It is a glycoprotein selectively expressed on the epithelial cells of the thick ascending limb of Henle’s loop and the origin part of distal convoluted tubule.Mutations in the coding region of the UMOD gene can lead to a group of autosomal dominant,progressive kidney diseases collectively called uromodulin-associated kidney disease(UAKD),the main clinical manifestations are familial hyperuricemia and gout,chronic hypertension and eventually progress to end-stage renal disease.Genetic studies have found that single nucleotide polymorphism(SNPs)in the upstream regulatory region of uromodulin are correlated with different risks of hypertension and chronic kidney disease(CKD).Altered uromodulin expression is linked with increased risks of urinary tract infection,kidney stone,hypertension,hyperuricemia and acute and chronic kidney diseases.Our previous research has shown that the level of uromodulin was decreased in urine and associated with low eGFR and interstitial fibrosis/tubular atrophy in patients with IgA nephropathy.Furthermore,the UMOD knockout mice exhibited more serious injury compared with wild-type mice after ischemia-reperfusion injury.These studies have shown that uromodulin plays an important role in the development of kidney diseases.Complement system is composed of more than 30 proteins ubiquitous in circulation,tissue fluid and cell membrane surface,and has a set of fine regulation mechanism.Complement system can be activated by the following three different ways:classical pathway,alternative pathway and mannan-binding lectin(MBL)pathway.The activated complement system can produce a variety of protein fragments with enzyme activity,including complement C3a、complement C5a and membrane attack complex(MAC),etc.,thus mediate a variety of biological effects,such as inflammatory response and immune response.Studies have shown that different types of renal intrinsic cells can produce a variety of complement components and/or complement receptors,such as C2,C3,C4,factor B,factor H,CR1,CR3 and C3aR.The kidney is one of the most sensitive organs for abnormal complement activation.Many studies have confirmed that abnormal complement activation exists in a variety of renal diseases,including lupus nephritis,IgA nephropathy,diabetic nephropathy,membranous proliferative nephropathy and so on.Our previous study found that complement H factor level in urine is related to renal tubulointerstitial injury.Braun et al found that complement H factor knockout mice caused renal tubulointerstitial damage due to over-activation of complement.These studies show that complement activation is involved in the pathophysiological process of a variety of renal diseases.Uromodulin is a protein that specifically expressed in renal tubular epithelial cells.the changes of its expression and secretion are closely related to the development of many kinds of renal diseases.the specific physiological function and mechanism have been concerned by researchers.Previous studies have shown that uromodulin plays an important role in regulating water and salt transport,participating concentration in urine,protecting the formation of kidney stones and preventing urinary tract infection.In recent years,with the deepening of research,the role of uromodulin in immune regulation has been gradually recognized.It has been found that uromodulin can bind to inflammatory factors and immune molecules such as IgG,IL-1,IL2,IL-8 and TNF-a to play an immunomodulatory role.In addition,uromodulin can also bind to complement Clq and inhibit the activation of complement classical pathway.Our previous study has found that in complement H factor knockout mice,the secretion of uromodulin decreased,and the expression level of mRNA in kidney tissue decreased.Our previous studies have shown that uromodulin can significantly enhance the function of complement H factor to cleave C3b and inhibit complement over activation.Previous studies and our result suggest that uromodulin may be related to complement activation-mediated renal disease.however,there are few studies on the relationship between uromodulin and complement activity,especially,the effect of plasma uromodulin on complement activation in patients with renal disease has not been reported.Therefore,In this study,we aimed to investigate whether plasma uromodulin is associated with complement activation in patients with renal diseases.In addition,we used the renal tubular epithelial cells to explore its biological function in vitro.Methods:We examined the expression of uromodulin and complement components,Clq,C3,C4,C3a and mac in the plasma of IgA nephropathy(IgAN,n=58)and lupus nephritis(LN,n=36)patients using Elisa to determine the association between uromodulin level and complement components in patients.In addition,we used a simulated hypoxia-reoxygenation(H/R)model by hypoxia for 1 h,6 h and 12 h respectively and then reoxygenation for 24 h to stimulate the expression of uromodulin in mouse inner medullary collecting duct cells(mIMCD3),and detected the relationship between uromodulin expression and complements C1q and C3d at the cellular level using western blotting and immunofluorescence,respectively.Results:We found that the plasma uromodulin concentration was markedly decreased in patients with IgAN and LN compared with the healthy controls(all P<0.0001),c3a and mac were shown to be increased in plasma of patients with IgAN and LN(all P<0.05).The plasma levels of C1q,C3 and C4 in patients with LN were Significantly decreased compared with healthy controls,Statistical significance was observed between LN and healthy controls(P=P<0.05,P<0.0001,P<0.0001 respectively),whereas,no significant difference was observed in the plasma levels of C1q,C3 and C4 between the IgAN and healthy controls(P=0.443,P=0.055,respectively).The plasma level of uromodulin was negative correlated with the concentration of the plasma C3a and mac(r=-0.410;p<0.001;r=-0.301;p=0.003,respectively).However,the plasma level of uromodulin was positive correlated with the concentration of the plasma C1q(r=0.258,p=0.016),a positive correlation between uromodulin expression and C3 and C4 was also observed,although not statistically significant(all P>0.05).Uromodulin expression started to increase after 1 h of simulated H/R,and continued to increase at 6h and 12 h.The results showed that the expression of complement C1q was decreased at 1h.reached the peak at 6h,and began to decrease at 12h.Immunofluorescence results showed that the expression of activation product C3d was increased after 1h and decreased after 6h.Finally,the spearman correlation analysis between plasma uromodulin concentration and renal function biomarkers found that plasma uromodulin concentration was positively correlated with eGFR and negatively correlated with serum creatinine,blood urea nitrogen and cystatin C.We also analyzed the correlation between the concentration of plasma uromodulin and renal pathological features in the disease group,and found that the low level of plasma uromodulin concentration in patients with IgA nephropathy was related to renal tubulointerstitial fibrosis,and the low level of plasma uromodulin concentration in LN patients was related to the activity of the disease.Conclusions:1.The concentration of plasma uromodulin was decreased in patients with IgAN and LN,and it was correlated with complement activation,suggesting that uromodulin may have a certain effect on complement activation in renal disease.2.The expression of uromodulin was continuously increased from 1h to 12h under the stimulation of hypoxia and reoxygenation in vitro,and the increased uromodulin may have a certain effect on the ability of renal tubular epithelial cells to synthesize local complements.3.Low levels of plasma uromodulin in patients with IgAN and LN are associated with decreased renal function. |
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