Detection Of Autoantibodies Against B1/B2 Subunits Of V-H~+-ATPase In Sera Of Patients With Renal Tubular Acidosis

ObjectiveAutoimmunity plays an important role in renal tubular acidosis(RTA).The presence of autoantibodies against renal tubular epithelial cells has been found in patients with RTA,but the detailed description of these autoantibodies is current lacking.Our aims of this study was to detect a presence or absence of autoantibodies against B1 and/or B2 subunits of vacuolar H~+-adenosine triphosphatease(v-H~+-ATPase) in sera of patients diagnosed with RTA.MethodsEleven untreated patients diagnosed with RTA(10 of these cases were associated with Sjogren syndrome),eight untreated patients with simple Sjogren syndrome(sSS)and eight healthy controls(HC)were enrolled in the study.Part of the fasting whole blood was taken out for measuring immuno-function by flow cytometry, and other serum was for detecting serum biochemistry,and autoantibodies presence or absence by indirect immunofluorescence assay.Firstly,to detect a presence or absence of serum autoantibodies against renal tubular epithelial cells and observe the expression of B 1 subunit and B2 subunits of v-H~+-ATPase in normal renal tissue of human,paraffin sections of normal human renal tissue were incubated with sera of RTA,sSS,HC or anti- B1 or B2 subunits of v-H~+-ATPase antibodies overnight at 4℃,then autoantibodies against renal tubular epithelial cells in sera were conjugated with fluorescein isothiocyanate(FITC,green color)-labeled goat-anti-human immunoglobulin G(IgG),while B1 or B2 subunits of V-H~+-ATPase were conjugated with tetramethylrhodamine isothiocyanate(TRITC, red color)-labeled rabbit-anti-goat IgG.Nuclei were stained with 4,6-diamino-2-phenyl indole(DAPI,blue color).For each positive sample,the sera or anti-B1 or B2 subunits of V-H~+-ATPase antibodies were diluted to repeat the trial until negative results were obtained.To confirm a presence or absence of autoantibodies against B1 and B2 subunits of v-H~+-ATPase in sera of RTA,paraffin sections of normal human renal tissue were firstly incubated with sera of RTA,sSS or HC overnight at 4℃,and with goat-anti-v-H~+-ATPase B1 subunit antibodies followed later overnight at 4℃.Then,the sections were incubated with TRITC-labeled rabbit-anti-goat secondary antibodies for B subunit of v-H~+- ATPase assay,and with FITC-labeled goat-anti-human secondary antibodies for autoantibodies against renal tubular epithelial cells.Finally,nuclei were stained with DAPI.For each positive sample,we diluted the sera and repeated the trial until negative.Similarly,detection of autoantibodies against B2 subunit of v-H~+-ATPase was performed by the same protocol as that against B1 subunit of v-H~+-ATPase assay described previously.ResultsResults of general indicators:Compared with patients with sSS and HC,results in sera of patients with RTA showed that serum chloride increased,CO2 decreased and urine pH increased.And results of flow cytometry,IG and complements implied immune disorders.Results of autoantibodies against renal tubular epithelial cells:The sera of 6 cases of all RTA patients showed green fluorescence staining in renal tubular epithelial cells,additional experiments showed that the staining intensity was also reduced following the sera of patients with RTA were diluted.Moreover,none of those sera from patients with either sSS or HC showed green fluorescence in renal tubular epithelial cells,which indicated no autoantibodies against renal tubular epithelial cells in sera of patients with sSS and HC.The results indicated the presence of autoantibodies against renal tubular epithelial cells in sera of patients with RTA.Results of distribution characteristics of B1 and B2 subunits of v-H~+-ATPase in normal human renal tissue:Red fluorescence representing B1 subunit of v-H~+-ATPase distributed specifically in intercalated cells,while B2 subunit distributed continuously in the cavosurface of renal tubular epithelial cells.Assay results of autoantibodies against B1 and B2 subunits of v-H~+-ATPase: when sera were incubated prior to anti-B1 subunit antibodies,the red fluorescence intensity showed a decrease in 6 cases of the 11 patients with RTA,which implied that blocking effect of the sera of patients with RTA to anti-B1 subunit of v-H~+-ATPase antibodies was occurred.The sera-induced blocking effects were reduced following the sera of patients with RTA diluted.While none of sera from these patients with either sSS or HC showed the blocking effect like that.The assay results of autoantibodies against B2 subunit of v-H~+-ATPase were as the same as that of B1 subunit.ConclusionThere are autoantibodies against renal tubular epithelial cells in sera of patients with RTA,and the positive ratio is 6/11.There are autoantibodies against B1 and B2 subunits of v-H~+-ATPase in sera of patients with RTA,and the positive ratio are both 6/11.Autoantibodies against B1 and B2 subunits of v-H~+-ATPase are two kinds of autoantibodies against renal tubular epithelial cells in sera of patients with RTA.