Deficiency in Six2 during prenatal development is associated with reduced nephron number, chronic renal failure, and hypertension in Br/+ adult mice

Abstract

The Br/+ mutant mouse displays decreased embryological expression of the homeobox transcription factor Six2, resulting in hertitable renal hypoplasia. The purpose of this study was to characterize the renal physiological consequences of embryonic haploinsuffiency of Six2 by analyzing renal morphology and function in the adult Br heterozygous mutant. Adult Br/+ kidneys weighed 50% less than those from wild-type mice and displayed glomerulopathy. Stereological analysis of renal glomeruli showed that Br/+ kidneys had an average of 88% fewer glomeruli than +/+ kidneys, whereas individual glomeruli in Br/+ mice maintained an average volume increase of 180% compared with normal nephrons. Immunostaining revealed increased levels of endothelin-1 (ET-1), endothelin receptors A (ET(A)) and B (ET(B)), and Na-K-ATPase were present in the dilated renal tubules of mutant mice. Physiological features of chronic renal failure (CRF) including elevated mean arterial pressure, increased plasma creatinine, and dilute urine excretion were measured in Br/+ mutant mice. Electron microscopy of the Br/+ glomeruli revealed pathological alterations such as hypercellularity, extracellular matrix accumulation, and a thick irregular glomerular basement membrane. These results indicate that adult Br/+ mice suffer from CRF associated with reduced nephron number and renal hypoplasia, as well as glomerulopathy. Defects are associated with embryological deficiencies of Six2, suggesting that proper levels of this protein during nephrogenesis are critical for normal glomerular development and adult renal function.

Fig. 1.

Photomicrographs of representative kidneys from sibling littermate +/+ (A, C) and Br/+ (B, D) mice. Note the larger size of the +/+ kidney compared with the smaller, cystic Br/+ kidney. Normal glomeruli (C, arrowheads) display cuboidal epithelia in +/+ animals compared with the 3H1 Br/+ kidney with enlarged glomeruli (D, arrowheads), enlarged tubules (t), and increased cellularity between nephrons (*). Bar = 3.0 mm in A and B; 200 μm in C and D.

Fig. 2.

Real-time quantitative (q)PCR shows Six2 expression in the kidney is highest during embryonic (E) development, which is greatly decreased in Br mutant kidneys. Expression of Six2 in normal newborn and adult kidneys is shown relative to expression of Six2 in E day 13.5 (E13.5) kidneys after being normalized against the measured levels of actin (A). Expression of Six2 in Br/+ and Br/Br E13.5 kidneys is shown relative to expression of Six2 in +/+ E13.5 kidneys after being normalized against the measured levels of actin (B).

Fig. 3.

Stereological studies comparing glomeruli in kidneys from adult Br/+ mice and adult +/+ mice. Data collected included total kidney volume (A), glomeruli density (B), total number of glomeruli per kidney (C), and mean glomeruli volume and mean glomeruli surface area (D). Shown are graphs for the 2 genotypes with the horizontal line representing the mean values.

Fig. 4.

Scattergram showing a correlation between mean arterial pressure (mmHg) and plasma creatinine (mg/dl) as an indicator of reduced glomerular filtration rate (r = 0.59, P < 0.01). Circles represent values recorded for +/+ mice, and squares represent values recorded for Br/+ mice.

Fig. 5.

Immunofluorescent staining of endothelin-1 (ET-1) in kidneys from +/+ (A, C, E) and Br/+ (B, D, F) adult mice. Renal tubules in +/+ mice showed low level background staining for ET-1 (A) compared with intense staining for ET-1 around enlarged mutant tubules (B, arrows). Staining for ET_A (C, arrow) and ET_B (E, arrow) revealed localization in the basolateral epithelia of normal tubules, but in the distended tubules of Br/+ mice, extensive staining was observed throughout the epithelium, particularly along the apical surfaces (D, F, respectively). Bar = 100 μm.

Fig. 6.

Immunofluorescent staining of Na-K-ATPase in kidneys from +/+ (A) and Br/+ (B) adult mice. Na-K-ATPase localizes to the basolateral surface of the normal mouse renal tubule epithelial cells (A, arrows) but becomes highly overexpressed and apically mislocated in the distended renal tubules of Br/+ mice (B, arrows).

Fig. 7.

Light (A, B) and transmission electron micrographs (TEM; C, D, E, F) of renal cortical tissues from age-matched adult +/+ (A, C, E) and Br/+ (B, D, E) mice. Control (+/+) mice (A) exhibit normal glomerular histoarchitectures, whereas massive increases in extracellular matrix (ECM) are seen in the mutants (B). TEM of normal (C) and mutant (D) tissues illustrate the increase in specific cellularity and ECM in the mutants. Relatively high-magnification TEM of normal (E) and mutant (F) mice show a substantial increase in the blood-urine barrier thickness (double-ended arrows) in the mutants. CL, capillary lumen; P, podoctye. A and B, ×600; C, ×3,000; D, ×2,800; E, ×9,200; F, ×9,100.