Detection of Na+ transporter proteins in urine

Abstract

Previous studies have established that the vasopressin-regulated water channel of the collecting duct, aquaporin-2, is excreted in the urine, providing a means for assessment of regulation and dysregulation of aquaporin-2 in humans. This article addresses the hypothesis that membrane transporters from upstream nephron segments are normally detectable in urine. The experiments employed rabbit polyclonal antibodies against the major Na transporters of the proximal tubule (the type 3 Na-H exchanger [NHE3]), the thick ascending limb of Henle's loop (the bumetanide-sensitive Na-K-2Cl cotransporter [NKCC2]), and the distal convoluted tubule (the thiazide-sensitive Na-Cl cotransporter [NCC]) in immunoblotting experiments. All three of these transporters were readily detectable as high molecular weight complexes present in low-density membrane fractions from urine of normal rats. Cross linking studies of NHE3, NKCC2, and NCC revealed that high molecular weight complexes are normally present in renal tissue. The molecular weights of the complexes in urine matched those of the cross-linked complexes in native kidney tissue. The presence in urine of integral membrane proteins representative of each nephron segment raises the possibility that limited or comprehensive proteomic analysis of urine samples may be useful in clinical settings.