Hyposmolality stimulates apical membrane Na(+)/H(+) exchange and HCO(3)(-) absorption in renal thick ascending limb

Abstract

The regulation of epithelial Na(+)/H(+) exchangers (NHEs) by hyposmolality is poorly understood. In the renal medullary thick ascending limb (MTAL), transepithelial bicarbonate (HCO(3)(-)) absorption is mediated by apical membrane Na(+)/H(+) exchange, attributable to NHE3. In the present study we examined the effects of hyposmolality on apical Na(+)/H(+) exchange activity and HCO(3)(-) absorption in the MTAL of the rat. In MTAL perfused in vitro with 25 mM HCO(3)(-) solutions, decreasing osmolality in the lumen and bath by removal of either mannitol or sodium chloride significantly increased HCO(3)(-) absorption. The responses to lumen addition of the inhibitors ethylisopropyl amiloride, amiloride, or HOE 694 are consistent with hyposmotic stimulation of apical NHE3 activity and provide no evidence for a role for apical NHE2 in HCO(3)(-) absorption. Hyposmolality increased apical Na(+)/H(+) exchange activity over the pH(i) range 6.5-7.5 due to an increase in V(max). Pretreatment with either tyrosine kinase inhibitors or with the tyrosine phosphatase inhibitor molybdate completely blocked stimulation of HCO(3)(-) absorption by hyposmolality. These results demonstrate that hyposmolality increases HCO(3)(-) absorption in the MTAL through a novel stimulation of apical membrane Na(+)/H(+) exchange and provide the first evidence that NHE3 is regulated by hyposmotic stress. Stimulation of apical Na(+)/H(+) exchange activity in renal cells by a decrease in osmolality may contribute to such pathophysiological processes as urine acidification by diuretics, diuretic resistance, and renal sodium retention in edematous states.

Figure 1

Hyposmolality stimulates HCO₃^– absorption in the MTAL. Osmolality in lumen and bath solutions was decreased from 295 mosmol/kg H₂O (Control) to 245 mosmol/kg H₂O (Hypo) by removal of either 50 mM mannitol (a) or 25 mM NaCl (b) from the control solution. JHCO₃^–, absolute rate of HCO₃^– absorption. Data points are average values for single tubules. Lines connect paired measurements made in the same tubule. P values are for experimental period (Hypo) versus either initial or recovery period (Control) by ANOVA. Mean values are presented in Results.

Figure 2

Bath hyposmolality is necessary for stimulation of HCO₃^– absorption. After an initial control period, hyposmolality was produced in the lumen alone (50 mM mannitol removed). The bath was then made hyposmotic in the continued presence of a hyposmotic lumen. JHCO₃^–, data points, and lines as in Figure 1. P value is for lumen plus bath versus either period of lumen alone (ANOVA). P = NS, control versus lumen. Mean values are presented in Results.

Figure 3

Furosemide has no effect on stimulation of HCO₃^– absorption by hyposmolality. Hyposmolality was produced by removal of 50 mM mannitol from lumen and bath solutions. Furosemide (Furos; 10^–4 M) was present in the lumen throughout the experiments. JHCO₃^–, data points, lines, and P value are as in Figure 1. Mean values are presented in Results.

Figure 4

Effects of lumen EIPA, amiloride, and HOE 694 on HCO₃^– absorption in hyposmotic solutions. MTAL were perfused and bathed in hyposmotic solution (50 mM mannitol removed) and then 50 μM EIPA (a) and 50 μM amiloride or 100 μM HOE 694 (b) was added to the tubule lumen. JHCO₃^–, data points, lines, and P values are as in Figure 1. Mean values are presented in Results.

Figure 5

Hyposmolality stimulates apical membrane Na⁺/H⁺ exchange activity by increasing V_max. MTAL were studied in isosmotic (filled circles) or hyposmotic (50 mM mannitol removed; open circles) solutions. Apical Na⁺/H⁺exchange rates (JNa/H) were determined at various pH_i values from rates of pH_i recovery measured after addition of Na⁺ to the tubule lumen (see Methods). Data points are from 10 tubules in isosmotic solution and 12 tubules in hyposmotic solution. Lines and kinetic parameters are from least-squares fits to the Hill equation.

Figure 6

EIPA inhibits apical membrane Na⁺/H⁺ exchange activity. MTAL were studied in isosmotic (a) or hyposmotic (50 mM mannitol removed) (b) solutions. Apical Na⁺/H⁺ exchange rates (JNa/H) were determined from rates of pH_i recovery after lumen Na⁺ addition in the absence and presence of 50 μM lumen EIPA. Data points are values for single tubules. Data without EIPA are from Figure 5; n = 4 for each condition. Mean values are presented in Results. Values below figure panels are pH_i ± SE at which JNa/H was determined.

Figure 7

Tyrosine kinase inhibitors block stimulation of HCO₃^– absorption by hyposmolality. Hyposmolality was produced by removal of 50 mM mannitol from lumen and bath solutions. Genistein (7 μM, filled circles) or herbimycin A (1 μM, open circles) was present in the bath throughout the experiments. JHCO₃^–, data points, lines, and P value are as in Figure 1. Mean values are presented in Results.