Disruption of the endothelin A receptor in the nephron causes mild fluid volume expansion

Abstract

Background: Endothelin, via endothelin A receptors (ETA), exerts multiple pathologic effects that contribute to disease pathogenesis throughout the body. ETA antagonists ameliorate many experimental diseases and have been extensively utilized in clinical trials. The utility of ETA blockers has been greatly limited, however, by fluid retention, sometimes leading to heart failure or death. To begin to examine this issue, the effect of genetic disruption of ETA in the nephron on blood pressure and salt handling was determined.

Methods: Mice were generated with doxycycline-inducible nephron-specific ETA deletion using Pax8-rtTA and LC-1 transgenes on the background of homozygous loxP-flanked ETA alleles. Arterial pressure, Na metabolism and measures of body fluid volume status (hematocrit and impedance plethysmography) were assessed.

Results: Absence of nephron ETA did not alter arterial pressure whether mice were ingesting a normal or high Na diet. Nephron ETA disruption did not detectably affect 24 hr Na excretion or urine volume regardless of Na intake. However, mice with nephron ETA knockout that were fed a high Na diet had mild fluid retention as evidenced by an increase in body weight and a fall in hematocrit.

Conclusions: Genetic deletion of nephron ETA causes very modest fluid retention that does not alter arterial pressure. Nephron ETA, under normal conditions, likely do not play a major role in regulation of Na excretion or systemic hemodynamics.

Figure 1

Schema of conditional KO of the ENDRA gene. The Pax8 promoter drives expression of rtTA which requires doxycycline to activate the bicistronic Cre recombinase and luciferase-expressing transgene. Cre is expressed specifically in the nephron but not in glomeruli. ETA receptor KO mice are homozygous for the floxed ENDRA gene and heterozygous for the two transgenes.

Figure 2

Specificity and sites of ETA receptor KO. Panel A shows a representative blot (N = 6 each gender) of PCR for ENDRA gene recombination, demonstrating Cre activity in liver and kidney. Panel B shows a representative blot (N = 8) of PCR for ENDRA gene recombination in microdissected thick ascending limb (TAL), proximal tubule (PT), cortical collecting duct (CCD) and glomeruli from Pax8-rtTA/LC1/floxed ETA (iETA) mice treated with doxycycline (DOX) (separate mice shown in each lane). Negative (homozygous floxed ETA (control)) and positive (iETA + DOX) PCR of whole kidney DNA are shown. Panel C shows real-time PCR of ETA mRNA (normalized to GAPDH mRNA) in cortex and inner medulla from control + DOX and iETA + DOX mice (N = 8 each group). *p < 0.05 vs. control + DOX.

Figure 3

Representative photomicrographs of kidneys from Pax8-rtTA/LC1/floxed ETA (iETA) mice before and after doxycycline (DOX) treatment. Panel A shows cortex and Panel B shows medulla prior to DOX treatment; Panel C shows cortex after DOX treatment. DOX-treated iETA medullary staining is not shown since ETA immunostaining was not apparent in untreated iETA mice. Arrows indicate blood vessels. Staining was performed on 6 different mice of each genotype. Images are 200X.

Figure 4

Systolic (Panel A) and diastolic (Panel B) BP in control and Pax8-rtTA/LC1/floxed ETA (iETA) mice before and after doxycycline (DOX) treatment. Animals were fed a normal (0.3%) Na diet for 3 days followed by a high (3.2%) Na diet for 5–7 days. DOX was given for 11 days, then BP recordings continued 5 days after discontinuation of DOX. BP was obtained by continuous radiotelemetry. Daily BP values are shown as the average of the every 10-minute recordings throughout that 24 hr period. N = 12 each data point.

Figure 5

Urine volume (Panel A), urinary Na excretion (Panel B) and creatinine clearance (Panel C) in control and Pax8-rtTA/LC1/floxed ETA (iETA) mice before and after doxycycline (DOX) treatment. Animals were fed a normal (0.3%) Na diet for 3 days followed by a high (3.2%) Na diet for 7 days. Urine (24 hr) was obtained on the third day of starting normal or high Na diets. Note that within each panel, open or closed bars represent the same mice, i.e., mice were sequentially given a normal and high Na diet, treated with DOX, and the diets repeated. N = 8 each data point.

Figure 6

Change in body volume status in Pax8-rtTA/LC1/floxed ETA (iETA) mice comparing before and after vehicle or doxycycline (DOX) treatment. Animals were fed a normal (0.3%) Na diet for 7 days followed by a high (3.2%) Na diet for 7 days, then treated with DOX, and the diets repeated; measurements were obtained on the 7th day of each diet. Panel A shows change in body weight, Panels B and C show changes in total body water and extracellular fluid volume, respectively, as assessed by impedance plethysmography, and Panel D shows the change in hematocrit. N = 8 each data point. *p < 0.05 vs. –DOX, same diet.