| A disintegrin-like and metalloprotease with thrombospondin type1repeats13(ADAMTS13) is a specific VWF-cleaving protease. Severe deficiency of this proteaseleads to the excessive accumulation of ultralarge VWF multimers and platelet aggregationwith microthrombus formation. This is the main cause of thrombotic thrombocytopenicpurpura (TTP).Previous studies had revealed that ADAMTS13activity was often reduced in otherdiseases, such as cardiovascular disease, chronic liver disease, disseminated intravascularcoagulation and chronic kidney disease (CKD). ADAMTS13is synthesized and secretedby hepatic stellate and endothelial cells. In the human kidney, ADAMTS13is found inpodocytes and kidney tubular epithelial cells. It is unclear about the function ofnephrogenic ADAMTS13.Recent studies had suggested that ADAMTS13activity was decreased in sepsis andassociated with renal function. In cultured hepatic stellate cells and endothelial cells,inflammatory cytokines were involved in the synthesis, release of ADAMTS13. Theseresults could explain the reason of ADAMTS13decreased in sepsis, but it is hard toexplain the relation between ADAMTS13and renal function. We hypothesized thatADAMTS13not only changed in plasma but also in local kidney. Whether theinflammatory cytokines directly inhibit on renal local ADAMTS13?Previous studies indicated that following the induction of ischemic or toxic renalinjury, dramatic increases in renal cortical cholesterol content result. HMG-CoA(3-hydroxy-3-methylglutaryl coenzyme A) reductase inhibitors or statins, which lowerblood cholesterol levels and reduce inflammatory plaque formation in atherosclerosis,exert anti-inflammatory and antiproliferative actions. Statins seem to exert benefits even insettings of infection.Our study discusses the relationships among VWF:Ag, ADAMTS13activity,inflammatory factors and renal function from clinical type, pathological type and cell levels. We hope to supply new method for clinical treatment and theory evidence throughinvestigating the effects of inflammatory factors and simvastatin on expression onpodocytes and renal tubular epithelial cells.PART1Study of ADAMTS13activity and inflammatory factors inchronic kidney diseaseMethods: Plasma levels of VWF:Ag, TNF-α, IL-4and IL-6were measured by usingELISA kits. The VWF-cleaving activity of ADAMTS13in plasma was assayed by usingthe FRETS–VWF73ELISA kits.Results:1. Compared to the healthy volunteers (controls), CKD (include chronicglomerular nephritis (CGN), nephrotic syndrome (NS), lupus nephritis (LN)) patientsexhibited higher levels of VWF:Ag, significantly higher VWF/ADAMTS13ratio andmarkedly lower levels of ADAMTS13activity. Furthermore, the VWF:Ag levels andADAMTS13activity in the NS group were lower than those in the CGN and LNgroups (p<0.05). However, the VWF/ADAMTS13ratios in the three CKD groupsexhibited no clear difference.2. TNF-α levels were positively correlated with the VWF:Ag levels (r=0.242,p=0.013) and negatively correlated with the glomerular filtration rate (GFR, r=-0.193,p=0.049). IL-6levels were positively correlated with the VWF:Ag levels (r=0.196,p=0.046) but not correlated with GFR. A significant negative correlation was observedbetween the VWF:Ag levels and GFR (r=-0.194, p=0.048). The VWF:Ag levels werenot correlated with the ADAMTS13activity in CKD patients. ADAMTS13activity inCKD patients was negatively correlated with the CHOL levels (r=-0.2, p=0.042).SLEDAI in LN patients was positively correlated with the levels of TNF-α (r=0.354,p=0.023) and VWF (r=0.472, p=0.002), but did not correlate with ADAMTS13activity and the VWF/ADAMTS13ratio.3. The plasma VWF:Ag levels, ADAMTS13activity and their ratio did not showany clear difference between the different pathological types of CKD.PART2VWF and ADAMTS13expression in renal tissueMethods: Expression of VWF and ADAMTS13in renal tissue was analyzed byimmunohistochemistry.Results: ADAMTS13and VWF expressed in renal tissue. There were no significant differentbetween CKD group and normal group. According to pathological types, ADAMTS13expression in membranous nephropathy (MN) group is decreased than in non-MN group(p<0.05). VWF expression appears no difference in the different group.PART3The study of ADAMTS13expression in podocytesMethods: ADAMTS13mRNA levels in podocytes are measured by PCR and proteinlevels are detected by western blot. The VWF-cleaving activity of ADAMTS13in cellsupernatant is assayed using the FRETS–VWF73ELISA kits. To determine the presence ofHMG-CoA reductase on podocytes, flow cytometry is used.Results:1. Podocytes were treated with increasing concentrations of TNF-α, IL-4, IL-6and simvastatin for24h respectively. ADAMTS13mRNA levels in IL-4–treated groupwere significantly lower than in control group. ADAMTS13mRNA levels inIL-6–treated group were also significantly decreased. The levels of ADAMTS13mRNA were not different in the podocytes treated with either0.1μmol/L or1μmol/Lsimvastatin but were significantly higher in the podocytes treated with10μmol/L thanin control group. TNF-α had no effect on ADAMTS13mRNA levels.2. When podocytes were treated with10ng/mL IL-4and increasingconcentrations of simvastatin (0,0.1,1,10μmol/L) for24h, ADAMTS13mRNAlevels significantly increased in a dose-dependent manner. The combined treatment of100ng/mL IL-6and increasing simvastatin concentrations (0,0.1,1,10μmol/L) for24h also demonstrated the similar results.3. We measured ADAMTS13protein levels in podocytes supernatants andlysates by western blot. ADAMTS13protein expressed in cell supernants and lysates(190kDa). ADAMTS13protein expressions were decreased in a dose-dependentmanner in the IL-6treated podocytes. In comparison with untreated group, only in IL-6100ng/Ml group ADAMTS13protein levels in cell supernatant were significantlydecreased, and its expression in lysates were significantly decreased in both IL-610ng/mL and100ng/mL groups. TNF-α or IL-4group didn’t show significant effect onADAMTS13protein expression. When podocytes were treated with increasingconcentrations of simvastatin, ADAMTS13protein levels significantly increased in a dose-dependent manner in both cell supernatants and lysates. The results were similarin podocytes cell supernatant and lysates when podocytes were co-cultured with IL-6(100ng/mL) and simvastatin (0,0.1,1,10μmol/L).4. We measured the HMC-CoA reductase (HMGCR) expression in podocytes byflow cytometry and get the negative results.5. We detected the ADAMTS13activity in podocytes cell supernants. Theresults in control group was9.86±0.67(%). There were no differents among variousgroups.The study of ADAMTS13expression in human renal tubular epithelialMethods: ADAMTS13mRNA levels in human renal tubular epithelial (HK-2) aremeasured by PCR and protein levels are detected by western blot.Results:In HK-2cell line, ADAMTS13mRNA in IL-6group (1,10,100ng/mL) decreased(0.78-fold;0.54-fold, P<0.01;0.34-fold, P<0.01) in a dose-dependent manner. InTNF-α(0.1,1,10ng/mL) group, ADAMTS13mRNA level also decreased (1.08-folds;0.62-fold, p<0.05;0.33-fold, p<0.01) than in control group. When HK-2were treated withsimvastatin and the results increased in a dose-dependent manner (10μmol/L2.10-folds,p<0.01). ADAMTS13mRNA levels significantly increased in a dose-dependent manner.The combined treatment of100ng/mL IL-6or10ng/mL TNF-α co-cultured withincreasing simvastatin concentrations for24h also resulted in a dose-dependent increase inthe ADAMTS13mRNA levels. We collected the cell supernatants in all groups. Wedetected the ADAMTS13protein by western blot and got negative results. The positivecontrol and β-actin were observed.Conclusions: In brief, plasma ADAMTS13activity decreased and VWF:Ag increasedin chronic kidney disease patients. Different inflammatory factors appeared differenteffects on expression and secretion of ADAMTS13on podocytes and HK-2. Simvastatinincreases the expression on podocytes and HK-2. Our study supply some evidences oninflammatory disease and chronic kidney disease... |
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