Increased cGMP phosphodiesterase activity mediates renal resistance to ANP in rats with bile duct ligation

Abstract

Background. Liver disease resulting from common bile duct ligation (CBDL) causes abnormal sodium metabolism that is manifested by resistance to the natriuretic action of atrial natriuretic peptide (ANP). This resistance is corrected both in vitro and in vivo by zaprinast, a selective inhibitor of a guanosine cyclic-3′-5′-monophosphate (cGMP)-specific phosphodiesterase (PDES). Several other PDEs with affinity for cGMP are expressed in kidney and could also be involved in this response. Methods. We measured cGMP hydrolysis in inner medullary collecting duct (IMCD) cell homogenates from kidneys of sham-operated and CBDL rats and quantitated the amount of PDE5 protein by Western blotting and immunoprecipitation studies. We also characterized ANP responsiveness in vivo of kidneys of anesthetized sham and CBDL rats by measuring sodium excretion before and after volume expansion (VE). Results. Kinetic analysis of PDE5 activity in homogenates of IMCD cells isolated from kidneys of sham-operated rats indicated a Vmax of 85.3 ± 1.7 versus 157 ± 2.9 pmol/mg/min from CBDL rats (P < 0.01), without a difference in Km. Enzyme activity was inhibited competitively by 1,3-dimethyl-6-(2-propoxy-5-methanesulfonylamidophenyl)pyrazol[3,4d] -pyrimidin-4-(5H)-one (DMPPO), a potent and specific inhibitor of PDE5, with an apparent K, of 4.5 ± 0.7 and 4.9 ± 0.7 nmol/L and an IC50 of 6.1 ± 0.8 and 8.7 ± 0.7 nmol/L in sham and CBDL rats, respectively (P = NS). DMPPO exhibited very poor inhibitory activity against the calcium-calmodulin-dependent PDE1 in IMCD homogenates from sham rats (Ki 1.3 ± 0.1 μmol/L and IC50 1.9 ± 0.2 μmol/L). Western analysis using an antiserum made against bovine lung PDE5 revealed a twofold increase in PDE5 protein in cytosolic extracts from IMCD of CBDL rat kidneys compared with sham-operated controls, and immunoprecipitation studies indicated that the increase in PDE5 protein accounted for the observed increase in cGMP hydrolysis. DMPPO (10 nmol/L) normalized the blunted ANP-dependent cGMP accumulation by IMCD cells from CBDL rats in vitro. Intrarenal infusion of DMPPO (0.5 nmol/min) in CBDL rats corrected both the impaired natriuretic response to VE and the blunted VE-related increase in urinary cGMP excretion from the infused, but not the contralateral kidney. Conclusion. These results demonstrate that renal resistance to ANP in CBDL rats is accompanied by heightened activity of PDE5, which is due largely to an increase in PDE5 protein. Other PDEs could contribute only a minor part to the enhanced cGMP hydrolysis observed in kidneys of CBDL rats. This PDE5-dependent ANP resistance may represent an important contributor to the sodium retention of liver disease.