Role of uromodulin and complement activation in the progression of kidney disease.

Uromodulin (UMOD) is a glycoprotein that is selectively expressed on the epithelial cells of the thick ascending limb of Henle's loop and the early distal renal tubule. The present study aimed to investigate whether UMOD was associated with complement activation in patients with renal diseases. In addition, its biological function was examined in vitro. The expression levels of UMOD and complement components, including C1q, C3, C4 and C3a, and membrane attack complex (MAC) in the plasma of patients with IgA nephropathy (IgAN; n=58) and lupus nephritis (LN; n=36) were detected using ELISA, which was used to determine the association between UMOD expression and complement components. In addition, a simulated hypoxia-reoxygenation (H/R) model was used to stimulate UMOD expression in mouse inner medullary collecting duct cells. Additionally, the association between UMOD expression and complement components C1q and C3d at the cellular level was identified using western blotting and immunofluorescence, respectively. It was revealed that the plasma UMOD concentration was significantly decreased in patients with IgAN and LN compared with in healthy controls, and the levels of C3a and MAC were significantly increased in the plasma of patients with IgAN and LN. Furthermore, the plasma levels of C1q, C3 and C4 in patients with LN, but not in patients with IgAN, were significantly decreased compared with in healthy controls. The plasma levels of UMOD were negatively correlated with the plasma C3a and MAC concentrations. However, the plasma levels of UMOD were significantly and positively correlated with the plasma C1q concentration, but not with that of C3 and C4. It was identified that UMOD expression started to increase after 1 h of simulated H/R, and continued to increase at 6 and 12 h. In addition, cells with lower UMOD expression had higher C3d expression in vitro. Collectively, the present results suggested that UMOD was associated with severe complement activation and may be involved in complement-mediated immune protection by inhibiting complement activation in renal disease.

Uromodulin aggravates renal tubulointerstitial injury through activation of the complement pathway in rats.

Uromodulin (Umod) is the most abundant constituent of urine in humans and exclusively found in the kidney tubular epithelium. However, the specific role of Umod in renal tubulointerstitial injury is yet to be understood. The present study was conducted with aim of investigating the potential therapeutic mechanism of Umod in the regulation of renal tubulointerstitial injury. Protein expression of Umod in renal tubular epithelial cells was measured with the conduction of Western blot analysis. Enzyme-linked immunosorbent assay and immunofluorescence assay were performed to detect the complement activation products and the activation products of surface deposition. The expression of C1q, C2, C4, B factor, C3, C5, H factor, CD46, CD55, C3aR, and C5aR were determined with the use of reverse-transcription quantitative polymerase chain reaction and Western blot analyses. Subsequently, the unilateral ureteral obstruction (UUO) rat model was established. Renal tubulointerstitial injury was assessed with the application of hematoxylin-eosin staining and Masson staining in rats. UUO rats and normal rats were injected with si-NC or si-Umod and complement inhibitor. UUO rats were observed to have serious impairment of kidney tubule, renal tubular dilation, and epithelial atrophy, with downregulated Umod and activated complement pathway. Silencing of Umod resulted in the activation of complement system while promoting interstitial fibrosis in renal tubules. Moreover, addition of complement inhibitor significantly alleviated the renal tubule injury and fibrosis. Collectively, our study suggests that silencing of Umod mediates the complement pathway, exacerbating renal tubulointerstitial injury in rats, which provides insight into the development of novel therapeutic agents for renal tubulointerstitial injury.

Interaction of uromodulin and complement factor H enhances C3b inactivation.

Recent studies suggest that uromodulin plays an important role in chronic kidney diseases. It can interact with several complement components, various cytokines and immune system cells. Complement factor H (CFH), as a regulator of the complement alternative pathway, is also associated with various renal diseases. Thus, we have been suggested that uromodulin regulates complement activation by interacting with CFH during tubulointerstitial injury. We detected co-localization of uromodulin and CFH in the renal tubules by using immunofluorescence. Next, we confirmed the binding of uromodulin with CFH in vitro and found that the affinity constant (KD ) of uromodulin binding to CFH was 4.07 × 10(-6) M based on surface plasmon resonance results. The binding sites on CFH were defined as the short consensus repeat (SCR) units SCR1-4, SCR7 and SCR19-20. The uromodulin-CFH interaction enhanced the cofactor activity of CFH for factor I-mediated cleavage of C3b to iC3b. These results indicate that uromodulin plays a role via binding and enhancing the function of CFH.

Implication of urinary complement factor H in the progression of immunoglobulin A nephropathy.

BACKGROUND: After activation, the complement system is involved in the pathogenesis of Immunoglobulin A nephropathy (IgAN). Complement factor H (CFH) is a crucial inhibitory factor of the alternative pathway of the complement system. The study investigated the effects of urinary CFH levels on IgAN progression. METHODS: A total of 351 patients with IgAN participated in this study. They were followed up for an average of 51.8 ± 26.6 months. Renal outcome was defined as a composite endpoint, that included instances of end-stage renal disease (ESRD), ≥ 50% decline in estimated glomerular filtration rate (eGFR) or doubling of plasma creatinine levels. Urinary CFH levels were measured by enzyme-linked immunosorbent assay and calculated as the ratio of urinary CFH over creatinine (uCFH/uCr). RESULTS: In the whole cohort, uCFH/uCr values were associated with disease progression either as continuous [log(uCFH/uCr)] or categorical traits (dichotomous and quartile variables) after adjusting for eGFR, proteinuria, mean arterial blood pressure, histological grading and immunosuppressive therapy in the Cox proportional hazard model. Kaplan-Meier analysis showed that higher uCFH/uCr values at baseline predicted worse renal outcome during follow-up (log-rank, P < 0.001). Receiver operating characteristic curve (ROC) analysis showed that log(uCFH/uCr) had predictive value for renal outcome (area under curve [AUC] = 0.745), and the AUC increased to 0.805 after being incorporated into baseline eGFR and proteinuria. In subgroup analysis with eGFR ≥ 60 mL/min/1.73 m2, log(uCFH/uCr) had better predictive value (AUC = 0.724, P = 0.002) for renal outcome compared to eGFR (AUC = 0.582, P = 0.259) and proteinuria (AUC = 0.615, P = 0.114). CONCLUSIONS: Urinary CFH levels are associated with renal function decline and increased urinary CFH levels are a risk factor for progression of IgA nephropathy.

Novel UMOD mutations in familial juvenile hyperuricemic nephropathy lead to abnormal uromodulin intracellular trafficking.

BACKGROUND: Familial juvenile hyperuricemic nephropathy (FJHN) is an autosomal dominant disorder characterized by hyperuricemia and progressive chronic kidney disease. Uromodulin gene (UMOD) mutations, leading to abnormalities of uromodulin intracellular trafficking contribute to the progress of the disease. METHODS: We did UMOD screening in three Chinese FJHN families. We thus constructed mutant uromodulin express plasmids by site-mutagenesis from wild type uromodulin vector and transfected them into HEK293 (human embryonic kidney) cells. And then we detected uromodulin expression by western blot and observed intracellular distribution by immunofluorescence. RESULTS: We found three heterozygous mutations. Mutation Val109Glu (c.326T/A; p.Val109Glu) and mutation Pro236Gln (c.707C/A; p.Pro236Gln) were newly indentified mutations in two distinct families (family F1 and family F3). Another previously reported UMOD mutation Cys248Trp (c.744C/G; p.Cys248Trp) was detected in family F2. Phenotypes varied both within the same family and between different families. Uromodulin expression is abnormal in the patient biopsy. Functional analysis of mutation showed that mutant types of uromodulin were secreted into the supernatant medium much less when compared with wild type. In mutant type uromodulin transfected cells, intracellular uromodulin localized less in the Golgi apparatus and more in endoplasmic reticulum(ER). CONCLUSIONS: Our results suggested that the novel uromodulin mutations found in the Chinese families lead to misfolded protein, which was retained in the endoplasmic reticulum, finally contributed to the phenotype of FJHN.

[Protective effects and mechanisms of xin'ganbao capsule on STZ induced early kidney injury in diabetic rats].

OBJECTIVE: To discuss the protective effects of Xin'ganbao Capsule (XC) on early kidney injury in streptozocin (STZ)-induced diabetic rats and its mechanisms. METHODS: Twenty-four male Wistar rats were selected to establish STZ induced diabetes mellitus (DM) model. After modeling they were randomly divided into the model group,the XC group (at the daily dose of 0.5 g/kg), and the benazepril group (at the daily dose of 4 mg/kg), 8 in each group. Another 8 rats were chosen as the blank control group. Rats in the model group and the blank control group were administered with equal volume of normal saline by gastrogavage for 8 successive weeks. The blood glucose was monitored by the end of the 4th week and the 8th week. The 24 h urine protein (24 hUP), blood urea nitrogen (BUN), and serum creatinine (SCr) were detected by the end of the 8th week. The transforming growth factor-beta1 (TGF-beta1), laminin (LN), collagen IV (Col-IV) expression were detected using immunohistochemical assay. The mRNA expressions of renal TGF-beta1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), and plasminogen activator inhibitor-1 (PAI-1) were detected using RT-PCR. The pathological changes of the renal tissue were observed by HE, PAS, and Masson stain methods. RESULTS: Compared with the blank control group, hyperglycemia, polydipsia, polyphagia, polyuria, weight loss, emaciation, dry and dim body hair, and irritability appeared in the diabetic rats. After 8 weeks the symptoms of the two medication groups were attenuated. When compared with the blank control group, the 24 hUP, SCr, blood glucose, Col-IV, LN, TGF-beta1 positive expression ratio, the levels of TGF-beta1, TIMP-1, PAI-1 mRNA, the area of glomerular (GA), extracellular matrix (ECM), and ECM/GA all increased in the model group with statistical difference (P<0.01). The pathological changes showed obvious glomerular enlargement, the capillary loop expansion, the proliferation of the mesangial cells, increased mesangial matrix, widen and thicken glomerular basement membrane (GBM), and tubular derangement. The vacuolar degeneration and shedding could be seen in partial epithelial cells. The protein cast could also be seen with infiltration of interstitial inflammatory cells. Compared with the model group, each index of the two medication groups decreased with statistical difference (P<0.01). The pathological changes were less in the two medication groups. The mesangial cells were slightly proliferated and the mesangial matrix slightly increased. The mRNA expressions of SCr and PAI-1 were lower in the XC group than in the benazepril group (P<0.05). There was no statistical difference in the other indices between the two medication groups (P > 0.05). Conclusions XC had some protective effects and anti-glomerulosclerosis effects on early kidney injury in STZ-induced diabetic rats. Its mechanisms might be associated with down-regulating the mRNA expressions of TGF-beta1, TIMP-1, PAI-1, and Col-IV, reducing ECM and urine protein.

[The renal protective effect of xinganbao capsule in adriamycin induced nephropathy rats].

OBJECTIVE: To study the renal protective effect of Xinganbao Capsule on rats with adriamycin induced nephropathy (AIN). METHODS: Forty male SD rats were randomly divided into four groups, i.e., the normal control group (N), the AIN model group (M), the Benazepril group (B),and the Xinganbao Capsule group (X). AIN rat model was established by left unilateral nephrectomy and repeated caudal vein injection of adriamycin. Gastric perfusion of xinganbao Capsule (at the dose of 500 mg/kg per day) and Benazepril (at the dose of 4 mg/kg per day) was given to rats in the X group and the B group respectively one week after nephrectomy. Rats were sacrificed at the 8th week after medication. The 24-h urinary protein excretion (24 h-UP) and blood biochemical indices were determined. Renal tissues were collected for pathological changes under light and electron microscopes. Expressions of fibronection (FN), collagen IV (COL-IV), and osteopontin (OPN) in renal tissues were detected by immunohistochemistry. mRNA levels of transforming growth factor-beta 1 (TGF-beta1), tissue inhibitor of metalloproteinase-1 (TIMP-1), and plasminogen activator inhibitor-1 (PAI-1) were measured by fluorescent Real-time PCR. RESULTS: When compared with the model group, 24 h-UP, blood urea nitrogen (BUN), and serum creatinine (SCr), and blood lipids levels were significantly lowered in the X group. The mesangial matrix percentage was less in the X group than in the M group. Renal FN, COL-IV, and OPN expressions more significantly decreased in the X group than in the M group. Similarly mRNA expressions of TGF-beta1,, TIMP-1, PAl-1 in renal tissues obviously decreased. CONCLUSION: Xinganbao Capsule could exert its renal protective action possibly through reducing the urinary protein excretion, correcting lipid metabolic disturbance, inhibiting excessive accumulation of extracellular matrix, decreasing the expression of fibrosis factors, and improving the pathological damage of kidneys in the AIN rat model.