Inhibition of Na+/H+ exchange (NHE) subtypes has been investigated in a study of the mouse fibroblast L cell line (LAP1) transfected with human (h) NHE1, rabbit (rb) NHE2, rat (rt) or human (h) NHE3 as well as an opossum kidney cell line (OK) and porcine renal brush-border membrane vesicles (BBMV). S3226 ¿3-[2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methyl-phenyl]-N-isopro pylidene-2-methyl-acrylamide dihydro-chloride¿ was the most potent and specific NHE3 inhibitor with an IC50 value of 0.02 micromol/l for the human isoform, whereas its IC50 value for hNHE1 and rbNHE2 was 3.6 and approximately = 80 micromol/l, respectively. In contrast, amiloride is a weak NHE3 inhibitor (IC50>100 micromol/l) with a higher affinity to hNHE1 and rbNHE2. Cariporide (4-isopropyl-3-methylsulphonyl-benzoyl-guanidine methane-sulphonate), which has an IC50 for NHE3 of approximately 1 mmol/l, is a highly selective NHE1 inhibitor (0.08 micromol/l). Therefore, S3226 is a novel tool with which to investigate the physiological and pathophysiological roles of NHE3 in animal models.

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We used clearance and free-flow micropuncture techniques to evaluate the influence of several diuretic agents, given both individually and in various combinations, on transport of sodium, potassium, and fluid, and on acidification and ammonium transport, within the distal tubule of the rat kidney. The loop diuretics, furosemide and piretanide, sharply increased fractional delivery of fluid, sodium, and potassium into the distal tubule, and, as a result, sodium reabsorption and potassium secretion were enhanced in this nephron segment. These two drugs also stimulated urinary acidification and increased urinary phosphate, titratable acid, and ammonium excretion. These effects took place both within the loop of Henle and along the distal tubule. Amiloride and triamterene alone inhibited distal tubular sodium reabsorption and potassium secretion, and, when given with one of the loop diuretics, suppressed both the kaliuresis and the increased acid and ammonium excretion induced by the latter agents. Hydrochlorothiazide and tizolemide inhibited sodium reabsorption within the distal tubule, and were associated with a stimulation of potassium secretion within this segment. Addition of one of these two latter distally acting agents to either of the loop diuretics led to a further augmentation of sodium excretion, but to a reduction of potassium excretion, compared to the responses seen after the loop diuretics alone.

Na(+)/H(+) exchanger NHE3 is expressed in the luminal membrane of proximal tubule and thin and thick ascending limb of Henle's loop. To further define its role, the novel NHE3 inhibitor S3226 was employed in micropuncture experiments in nephrons with superficial glomeruli of anesthetized rats. Microperfusion of proximal convoluted tubule with S3226 revealed a dose-dependent inhibition of reabsorption (IC(50) of 4-5 microM) with a maximum inhibition of 30% for fluid and Na(+). During microperfusion of Henle's loop (last superficial proximal to first superficial distal tubular loop), no effect of S3226 (10 or 30 microM) on the reabsorption of fluid or Na(+) was observed. Finally, S3226 (30 microM) left the tubuloglomerular feedback response unaltered as determined by the fall in proximal tubular stop-flow pressure in response to increasing loop of Henle perfusion rate. These studies indicate that NHE3 significantly contributes to fluid and Na(+) reabsorption in proximal convoluted tubule. NHE3 appears not to significantly contribute to fluid or Na(+) reabsorption in the loop of Henle (including the S3 segment of proximal tubule) or macula densa control of nephron filtration.

This study assessed the functional role of Na(+)/H(+) exchanger (NHE) isoforms NHE3 and NHE2 in the proximal tubule, loop of Henle, and distal convoluted tubule of the rat kidney by comparing sensitivity of transport to inhibition by Hoe-694 (an agent known to inhibit NHE2 but not NHE3) and S-3226 (an agent with much higher affinity for NHE3 than NHE2). Rates of transport of fluid (J(v)) and HCO(3)(-) (J(HCO3)) were studied by in situ microperfusion. In the proximal tubule, addition of ethylisopropylamiloride or S-3226 significantly reduced J(v) and J(HCO3), but addition of Hoe-694 caused no significant inhibition. In the loop of Henle, J(HCO3) was also inhibited by S-3226 and not by Hoe-694, although much higher concentrations of S-3226 were required than what was necessary to inhibit transport in the proximal tubule. In contrast, in the distal convoluted tubule, J(HCO3) was inhibited by Hoe-694 but not by S-3226. These results are consistent with the conclusion that NHE2 rather than NHE3 is the predominant isoform responsible for apical membrane Na(+)/H(+) exchange in the distal convoluted tubule, whereas NHE3 is the predominant apical isoform in the proximal tubule and possibly also in the loop of Henle.