Combining new tools to assess renal function and morphology: a holistic approach to study the effects of aging and a congenital nephron deficit

Recently, new methods for assessing renal function in conscious mice (transcutaneous assessment) and for counting and sizing all glomeruli in whole kidneys (MRI) have been described. In the present study, these methods were used to assess renal structure and function in aging mice, and in mice born with a congenital low-nephron endowment. Age-related nephron loss was analyzed in adult C57BL/6 mice (10–50 wk of age), and congenital nephron deficit was assessed in glial cell line-derived neurotrophic factor heterozygous (GDNF HET)-null mutant mice. Renal function was measured through the transcutaneous quantitation of fluorescein isothiocyanate-sinistrin half-life ( t1/2) in conscious mice. MRI was used to image, count, and size cationic-ferritin labeled glomeruli in whole kidneys ex vivo. Design-based stereology was used to validate the MRI measurements of glomerular number and mean volume. In adult C57BL/6 mice, older age was associated with fewer and larger glomeruli, and a rightward shift in the glomerular size distribution. These changes coincided with a decrease in renal function. GNDF HET mice had a congenital nephron deficit that was associated with glomerular hypertrophy and exacerbated by aging. These findings suggest that glomerular hypertrophy and hyperfiltration are compensatory processes that can occur in conjunction with both age-related nephron loss and congenital nephron deficiency. The combination of measurement of renal function in conscious animals and quantitation of glomerular number, volume, and volume distribution provides a powerful new tool for investigating aspects of renal aging and functional changes.

Umbilical artery histomorphometry: a link between the intrauterine environment and kidney development

Prematurity is a risk factor for hypertension, vascular stiffness, nephron deficit and adult onset cardiorenal disease. The vascular tree and kidneys share morphogenic drivers that promote maturation in utero before 36 weeks of gestation. Vascular elastin accrual terminates after birth leaving collagen to promote vascular stiffness. Our objective was to determine if the histomorphometry of the umbilical artery, an extension of the aorta, parallels nephron mass across gestational age groups. From a cohort of 54 newborns, 32 umbilical cord specimens were adequate for evaluation. The umbilical cord was sectioned, stained with trichrome, and digitalized. Muscular and collagenous areas of the umbilical artery were measured in pixels using the Image J 1.48q software. Total kidney volume was measured by ultrasound and factored by body surface area (TKV/BSA). The umbilical artery total area was significantly greater in term v. preterm infants (9.3±1.3 v. 7.0±2.0 mm2; P<0.05) and increased with gestational age; while the percent muscular and collagen areas were independent of gestational age (R2=0.04; P=ns). Percent muscular area correlated positively with TKV/BSA (r=0.53; P=0.002); while an increase in collagen correlated inversely with kidney mass (r=−0.53; P=0.002). In conclusion, an enhanced % muscular area and presumed vascular elasticity was associated with increased renal mass in all infants. Umbilical artery histomorphometry provides a link between the intrauterine environment, vascular and kidney development.

Development of progressive albuminuria in male Munich Wistar Frömter rats is androgen dependent

Munich Wistar Frömter (MWF) rats develop spontaneous albuminuria that is linked to autosomal genetic loci and inherit a nephron deficit in both female and male animals, respectively. However, albuminuria and kidney damage are clearly more pronounced in males. Here we tested whether androgens and the androgen receptor influence albuminuria in male MWF. We first demonstrated in a pilot study that orchiectomy (Ox) of male MWF led to a significant suppression of urinary albumin excretion (UAE), while continuous testosterone supplementation in MWF Ox led to UAE levels similar to sham-operated (Sham) MWF rats. Subsequently, we performed a comparative main study between male MWF and normal Wistar rats to evaluate the effect of the androgen receptor on UAE development in adult animals up to the age of 18 wk. MWF Sham developed a marked increase in UAE compared with Wistar Sham (48.30 ± 6.16 vs. 0.42 ± 0.08 mg/24 h, P < 0.0001). UAE was significantly lower in MWF Ox compared with MWF Sham (−55%, P < 0.0001). In MWF Ox animals supplemented with testosterone and treated with the androgen receptor antagonist flutamide (OxTF) UAE at 18 wk was even lower compared with MWF Ox (−71%, P < 0.01) and similar to age-matched female MWF. The mRNA expression of renal tubular injury markers Kim1 and NGAL was increased in MWF Sham compared with Wistar Sham ( P < 0.0008, respectively) and expression decreased significantly in MWF OxTF ( P < 0.0004, respectively). Thus, the sexual dimorphism in albuminuria development in MWF can be attributed to testosterone and the androgen receptor in male rats.

Abstract 452: The Degree of Nephron Deficit is a Determinant of the Cardiovascular and Renal Risks Associated With a Reduced Nephron Endowment

A reduced nephron endowment is associated with an increased sensitivity to hypertensive stimuli. We examined whether nephron endowment, and the degree of nephron deficit, impacts on the blood pressure and renal response to chronic angiotensin II (AngII) infusion. To address this aim we used the GDNF heterozygous mouse that, despite the same genotype, produces two distinct levels of nephron deficit due to the role of GDNF in both the initiation of kidney development and induction of nephron formation. GDNF het mice are born with either 2 small kidneys and a 30% nephron deficit (Het2K), or a single small kidney (Het1K) and a 65% nephron deficit compared to wild-type littermates (WT). Sixteen week old male GDNF WT (n=9), Het2K (n=5) and Het1K (n=7) mice were implanted with radiotelemetry probes and baseline pressures recorded. Mice were administered enalapril (10mg/kg/day, drinking water) alone for 3 days, then concomitant with an AngII infusion (600ng/kg/min; s.c. osmotic minipump) for a further 21 days. 24hr albumin and urine excretion were assessed at baseline and at the end of the 21 days of blood pressure recording. Baseline MAP was not different between WT, Het2K and Het1K groups (98.3±1.3, 97.1±1.8, 100.9±1.9mmHg respectively). Enalapril treatment led to a similar fall in MAP of 23mmHg in the 3 groups before the commencement of AngII infusion. AngII led to an immediate rise in MAP in all groups. At the end of the 21day infusion period, MAP of WT and Het2K mice were similar (122±3.3, 120.5±6.1mmHg), but MAP of Het1K mice (139.7±3.6mmHg; P<0.01) was significantly elevated over WT mice. Albumin excretion was not different between the groups at baseline, and AngII infusion did not significantly increase albumin excretion in WT or Het2K mice. However albumin excretion in Het1K mice increased 15 fold (4.86±1.56 to 76.05±30.3ug/24hr; P<0.001). Urine excretion also significantly increased in the Het1K (1.59±1.5 to 3.7±0.59ml/24h; P<0.001) in response to Angiotensin II, though WT and Het2k mice were not affected. AngII infusion in Het1K, but not Het2k mice, led to significant exacerbation of hypertension and albuminuria. Our data suggests that the degree of nephron deficit is an important determinant of the risks associated with a reduced nephron endowment.