Protease stimulation of renal sodium reabsorption in vivo by activation of the collecting duct epithelial sodium channel (ENaC)

Background: Proteases can increase the activity of the epithelial sodium channel (ENaC) by cleaving its α- or γ-subunit. However, evidence so far comes only from studies in vitro in either heterologous expression systems or isolated nephron segments. The present study has tested whether exposure to a luminal protease can alter sodium reabsorption along the rat collecting duct in vivo. Methods: Rats on normal laboratory chow were prepared for renal micropuncture. Late distal tubules of superficial nephrons were microinjected and perfused twice (3 nL min−1 for 3–6 min) with a solution similar to native tubular fluid, but containing 14[C]inulin and 22Na. The first perfusion was either a control solution or solution containing amiloride 1 mM or hydrochlorothiazide (HCTZ ) 1 mM; the second perfusion was either a control solution (time control) or a solution containing chymotrypsin 2 µg mL−1 ± aprotinin 100 µg mL−1 or amiloride 1 mM or HCTZ 1 mM. Urinary recoveries of 14[C]inulin and 22Na were recorded. Results: In time controls, the Na/In ratio did not change significantly (32.2 ± 3.4% versus 34.5 ± 3.1%). In contrast, chymotrypsin reduced the ratio from 33.3 ± 3.8% to 25.5 ± 2.5% (P < 0.05), indicating an increase in sodium reabsorption. When co-injected with chymotrypsin, the protease inhibitor aprotinin abolished the stimulatory effect of chymotrypsin on sodium reabsorption (31.7 ± 3.4% versus 32.1 ± 2.1%), while aprotinin alone had no effect. When chymotrypsin was co-injected with HCTZ, the Na/In ratio decreased from 36.8 ± 2.3% to 28.0 ± 3.4% (P < 0.05), whereas when given with amiloride, there was no change in the ratio (45.8 ± 3.4% versus 45.5 ± 2.3%), indicating that stimulation of sodium reabsorption by chymotrypsin was ENaC-dependent. Conclusions: These findings demonstrate proteolytic activation of ENaC in vivo, and suggest that changes in protease activity of the glomerular filtrate and tubular fluid in health or disease could affect net renal sodium excretion.