Nystatin and valinomycin induce tubuloglomerular feedback

The macula densa expresses a luminal Na+-K+-2Cl(-) cotransporter and a baso lateral Cl- conductance. Although it is known that cotransport of Na+, K+, and Cl- is the first step in tubuloglomerular feedback (TGF), subsequent st eps are unclear. We hypothesized that Na+-K+-2Cl(-) entry via the luminal N a+-K+-2Cl(-) cotransporter elevates intracellular Cl- increases electrogeni c Cl- fflux across the basolateral membrane, and depolarizes the macula den sa, initiating TGF. We perfused afferent arterioles with macula densa attac hed. The macula densa was perfused with solutions containing either 5 mM Na + and 3 mM Cl-(low NaCl) or 80 mM Na+ and 77 mM Cl 2 (high NaCl). When the macula densa perfusate was changed from low to high NaCl, afferent arteriol e diameter decreased from 15.8 +/- 0.8 to 13.1 +/- 0.7 mm (P < 0.05). Addin g 10 <mu>M furosemide to the macula densa lumen blocked TGF. When nystatin, a group I cation ionophore, was added to the macula densa lumen together w ith furosemide in the presence of low NaCl, it induced TGF (from 18.0 +/- 1 .5 to 15.6 +/- 1.6 mm; P = 0.003). When valinomycin, a K+-selective ionopho re, was added to the macula densa lumen together with furosemide in the pre sence of low NaCl containing 5 mM K+, it did not induce TGF. Subsequent add ition of 50 mM KCl to the macula densa perfusate induced TGF (from 21.7 +/- 0.8 to 17.5 +/- 1.3 mm; P = 0.0047; n = 6). Adding 50 mM KCl without valin omycin did not induce TGF. When 5-nitro-2-( 3-phenylpropylamino) benzoic ac id (NPPB; 1 muM), a Cl- channel blocker, was added to the bath, it blocked TGF induced by high NaCl, but did not block TGF induced by valinomycin plus 50 mM KCl. NPPB did not alter afferent arteriole constriction induced by n orepinephrine. We concluded that increased NaCl in the lumen of the macula densa leads to kinflux of Cl- via the Na+-K+-2Cl(-) cotransporter. The acce lerated transport increases intracellular Cl-. The subsequent exit of Cl- a cross the basolateral membrane via Cl- channels in turn leads to depolariza tion of the macula densa and thereby induces TGF.