In vivo studies were conducted in Na-replete anesthetized male Wistar rats with denervated kidneys. Intrarenal injections of angiotensin-(1-7) [ANG-(1-7) at > 1 nmol/kg produced a shallow dose-dependent decrease in renal blood flow that was mediated by the AT1-type ANG II receptor. A constant intrarenal infusion of ANG-(1-7) at 0.1 and 1 nmol.min-1.kg-1 had minimal effects on renal blood flow and blood pressure and resulted in an elevated urinary excretion of Na and water compared with the time-control saline-infused group. To determine whether ANG-(1-7) may have a direct action on tubular epithelium to inhibit Na reabsorption, we examined the effect of ANG-(1-7) on transport-dependent O2 consumption (Qo2) in fresh suspensions of rat proximal tubules in vitro. ANG-(1-7) inhibited Qo2 in a concentration-dependent fashion with a threshold concentration of approximately 100 pM. Stimulating Na-K-adenosinetriphosphatase (Na-K-ATPase) activity with nystatin caused a leftward shift of the inhibitory concentration-response curve to ANG-(1-7). The 22% inhibition of Qo2 by 1 pM ANG-(1-7) was abolished by pretreatment with 5 mM ouabain (Na-K-ATPase inhibitor), unaltered by pretreatment with 1 microM PD-123319 (AT2 receptor antagonist), partially attenuated by 1 microM losartan (AT1 receptor antagonist), and abolished by 1 microM [Sar1, Thr8]ANG II (nonselective ANG receptor antagonist). Together these findings indicate that ANG-(1-7) has biological activity in the kidney and, at nonvasoconstrictor doses, results in increased Na and water excretion in vivo. One site of action is the proximal tubule, where ANG-(1-7) can inhibit an ouabain-sensitive Na-K-ATPase exit step in cellular Na transport. This novel inhibitory action of ANG-(1-7) appears to be mediated by an AT1 receptor (minor component) and a non-AT1, non-AT2 ANG receptor (major component).

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We modified and improved enzyme digestion and density gradient separation procedures to obtain fractions of proximal and distal renal tubules with high yield and viability. Kidneys from two anesthetized adult Wistar rats were flushed with Krebs-Henseleit buffer (KHB) and then perfused in situ with recirculated KHB containing collagenase and hyaluronidase at 125 mmHg. Cortices were excised, minced, and incubated in KHB containing enzymes for 35 min at 37 degrees C. Dissociated tubules were removed at 10-min intervals, rinsed, and placed in KHB containing 10% calf serum, vitamins, and amino acids at 4 degrees C. Separation was achieved by suspending the tissue in 45% isosmotic Percoll layered over an undiluted Percoll cushion and centrifuging. Proximal tubules sedimented near the cushion. Distal segments were isolated in the uppermost bands of a second 35% Percoll separation. Viability was greater than 95% as measured by lactate dehydrogenase leakage and quantitated by oxygen consumption and ATP content. Basal oxygen consumption was greater than 33 nmol O2 X min-1 X mg protein-1 in all fractions and was stimulated by succinate and inhibited by amiloride and ouabain. Basal ATP content averaged 9.7 nmol/mg ATP. An average 3.3-fold separation for the proximal fraction and 24.5-fold separation for the distal fraction was assessed by the enrichment of six specific enzyme markers, with several of the markers indicating separations up to 32-fold. Isolated tubules also displayed functional responses to parathyroid hormone and vasopressin. Distal, but not proximal, segments demonstrated significantly increased adenosine 3',5'-cyclic monophosphate formation with vasopressin.

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