Expression,Regulation And Post-Translational Modification Of IRBIT And L-IRBIT In Rat Kidney

The kidney plays a central role in maintaining the systemic acid-base balance and fluid-electrolyte homeostasis.IRBIT?IP₃R binding protein released with inositol1,4,5-trisphosphate?and L-IRBIT are highly homologous,with a sequence identity up to88%.IRBIT and L-IRBIT have been demonstrated to have a very broad range of biological functions.Particularly,they are closely involved in the regulation of systemic acid-base balance by interacting with a variety of acid-base transporters.In the present study,by using western blotting and immunofluorescence staining,we examined the expression of IRBIT and L-IRBIT in rat kidney.Our immunofluorescence staining showed that IRBIT and L-IRBIT are abundant in the medullary thick ascending limb?mTAL?.We then examined the effect of NH₄Cl and high NaCl intake on the expression of L-IRBIT in rat kidney.NH₄Cl overload induces metabolic acidosis.Compared to the normal control,metabolic acidosis significantly increases the abundance of the phosphorylated?designated as L-IRBIT-P?and the total L-IRBIT in rat kidney.Interestingly,compared to the control,the relative proportion of the phosphorylated L-IRBIT-P in the total L-IRBIT is decreased under metabolic acidosis.Compared to the control,high NaCl intake significantly increases by 1.7 folds the abundance of L-IRBIT in the kidney.However,the level of the phosphorylated L-IRBIT-P is not significantly different from the control.In our study by western blotting,the apparent molecular weights?MW?of IRBIT and L-IRBIT from different sources were consistently higher by about 11 kD than their predicted MW.We isolated IRBIT by immunoprecipitation?IP?followed by SDS-PAGE separation.The band coresponding to IRBIT was cut and subjected to mass spectrometry analysis.Mass-spectra analysis confirmed the presence of IRBIT in the band.Moreover,mass-spectra analysis identified SUMO-like signal from the band.By western blotting with IRBIT or L-IRBIT samples isolated by IP,an antibody against SUMO recognized a band corresponding to the identical position of the band identified by anti-IRBIT or anti-L-IRBIT.Finally,we performed mutagenesis analysis with human IRBIT.However,we identified no SUMOylation sites in human IRBIT.Our study suggests that IRBIT and L-IRBIT play important roles in the kidney.It is likely that IRBIT and L-IRBIT are involved in the adaptive response to metabolic acidosis and high salt insults in the kidney.