Overexpression of kidney neutral endopeptidase (EC 3.4.24.11) and renal function in experimental cirrhosis

Abstract

Neutral endopeptidase degrades atrial natriuretic peptide (ANP) and bradykinin and may generate endothelin-1 from big-endothelin. In advanced cirrhosis, sodium retention is accompanied by elevated plasma ANP levels, and infusion of ANP causes hypotension, but in normal humans increasing the concentration of ANP through the inhibition of neutral endopeptidase, localized in renal proximal tubule cells, causes natriuresis without any arterial pressure drop. The purpose of this study was the assessment of kidney neutral endopeptidase expression and responses to candoxatrilat (a specific inhibitor of this enzyme) in rats with CCl4-induced cirrhosis. Two groups of control rats (n = 5) were injected with vehicle or 3 mg/kg candoxatrilat. Three groups of cirrhotic rats with ascites (n = 10) received vehicle alone or 3 or 10 mg/kg candoxatrilat. In cirrhotic rats, Western blot analysis revealed a 170% increase in renal neutral endopeptidase protein content (P < 0.03), mainly in the proximal nephron and macula densa, and both candoxatrilat dosages increased plasma ANP levels, urinary volume, and urinary excretion of sodium, ANP, and cGMP compared with vehicle alone (all P < 0.03). Candoxatrilat (10 mg/kg) also reduced tubular solute-free water reabsorption (P < 0.03) in cirrhotic rats, but renal blood flow, arterial pressure, and plasma renin activity were unaffected. Neutral endopeptidase inhibition has natriuretic and aquaretic actions in cirrhosis without any effect on blood pressure and kidney perfusion due to a significant overexpression of this enzyme in renal cortex.

Calcium-dependent diuretic system in preascitic liver cirrhosis

“…saline) has been employed in our laboratory for years. We never experienced any hemorrhagic shock in our rats and uniformly applied in control and cirrhotic animals, this sampling of small amounts of blood never induced significant hypotension or impairment of cardiac function [16,19]. Blood samples withdrawn 60 min after the injection of vehicle or F 0.5 were also used to measure plasma concentrations of vasopressin (AVP), plasma renin activity (PRA), aldosterone (A), norepinephrine (N), and plasma concentration of nitrites and nitrates (NO 2 /NO 3 ).…”

“…Blood was sampled (time 0) by cardiac puncture (0.3 cc) and inulin (IN) 10% (w/v) (Laevosan-Gesellschaft, Linz/Donau, Austria) plus para-aminohippurate (PAH) 20% (w/v) (Nephrotest, BAG Gmbh, Munich, Germany) were administered into the caudal vein as a priming bolus followed by a continuous infusion for 150 min, in order to assess glomerular filtration rate (GFR) and renal plasma flow (RPF) at different times by means of their respective steady-state plasma clearances (CIN and CPAH) [17][18][19]. The steady-state technique has the following advantages over the urine collection clearance method: catheterization of the bladder can be omitted, less analytical work is needed, exact timing of serum and urine samples is unnecessary, and more urine is available for other tests [18].…”

“…For Western blot analysis, membrane fractions were prepared from kidneys removed from five rats in each experimental group (G1-G3) [19]. Blots were incubated with a rabbit polyclonal CaRs antibody (ABR Affinity BioReagents, Golden, CO, USA) and an antibody against b-actin (Sigma, St. Louis, Missouri, USA).…”

“…Blots were incubated with a rabbit polyclonal CaRs antibody (ABR Affinity BioReagents, Golden, CO, USA) and an antibody against b-actin (Sigma, St. Louis, Missouri, USA). Washing and incubation times, along with detection, were performed as standardized [16,19]. Densitometric quantification was performed using b-actin as an internal standard: before any comparison the net intensity of CaR bands in each experiment was normalized to the intensity of the corresponding b-actin band, used as an internal standard in order to evaluate the degree of non-specific protein content in the homogenate.…”

“…Plasma and urinary concentrations of electrolytes and IN, and PAH plasma concentrations were measured as described elsewhere [19,24,25]. Plasma AVP, aldosterone, norepinephirne, and PRA were determined as described elsewhere [13,16,19].…”