DOI: 10.4225/03/5897c7bd62572
title: The development and function of high nephron endowment

description: An increasing amount of evidence suggests that low nephron number is a risk factor for development of chronic cardiovascular disease (particularly hypertension) and chronic kidney disease.  In humans, nephron number varies as much as 13-fold, from approximately 210,000 to 2.7 million (mean approximately 875,000).  While most human and animal studies have focused on the question of low nephron number, few studies have explored whether high nephron number provides protection against the development of hypertension or renal disease.  This has largely been due to the absence of suitable experimental models of augmented nephron number.  However, recent studies have demonstrated that perturbation to transforming growth factor-β2 (TGFβ2) or betaglycan (a TGFβ2 accessory receptor) gene expression is associated with augmented nephron number in heterozygous mice in adulthood.  The experiments described in this thesis were designed to elucidate the developmental mechanisms responsible for augmented nephron endowment in betaglycan heterozygous (betaglycan+/-) mice, to determine the functional consequences of a high nephron endowment, and to attempt to augment nephron number in the normal mouse kidney in vivo through manipulation of TGFβ2 signalling.  The overall hypothesis of this thesis is that mild perturbation of TGFβ2 signalling during metanephric development augments nephron endowment and produces a renal phenotype which is functionally beneficial in the presence of physiological stressors.
To identify the mechanisms responsible for the augmented nephron number in betaglycan+/- mice, metanephroi from embryonic day (e) 11.5-15.5 wildtype, betaglycan+/-, and betaglycan null (betaglycan-/-) embryos were analysed using in vivo and in vitro approaches.  Metanephroi of betaglycan+/- mice exhibited augmented ureteric branching morphogenesis that, in turn, correlated with a 32% increase in nephron endowment at e15.5.  In contrast, betaglycan-/- metanephroi demonstrated impaired ureteric branching morphogenesis and reduced nephron endowment at e15.5 compared to wildtype metanephroi.  Interestingly, the augmented and impaired ureteric branching phenotypes observed in betaglycan+/- and betaglycan-/- metanephroi respectively, appeared similar to that previously reported in Tgfb2 heterozygous (Tgfb2+/-) and null (Tgfb2-/-) embryos.  Finally, differing gene expression profiles (determined using quantitative real-time PCR) in betaglycan+/- and betaglycan-/- metanephroi correlated strongly with their nephron number phenotype.  Transient reduction in the expression of ureteric branching inhibitor Bmp4 at e11.5, coupled with increased branching promoter Gdnf expression at e12.5 may underpin accelerated ureteric branching in betaglycan+/-metanephroi.  Interestingly, Tgfb1 expression was significantly reduced to similar levels in betaglycan+/- and betaglycan-/- metanephroi from e13.5.  However, Tgfb2 expression was reduced in a dose-dependent manner, with betaglycan-/- metanephroi showing a greater reduction in Tgfb2 expression than betaglycan+/- metanephroi.  These findings suggest that manipulation of betaglycan signalling may provide a therapeutic avenue for augmenting nephron endowment.  However, the contrasting mutant phenotypes (betaglycan+/- versus betaglycan-/-) indicate that a greater understanding of the sensitivity of the developing metanephros to TGFβ superfamily signalling is required for these interventions to be successfully and safely developed.
The functional importance of high nephron endowment for the regulation of arterial pressure and renal function was studied using adult Tgfb2+/- mice, which have 60% more nephrons than wildtype mice.  Arterial pressure, heart rate and renal function were determined under baseline, physiologically stimulated and pathological conditions.  Renal function under basal conditions and in response to water deprivation was similar in wildtype and Tgfb2+/- mice.  Acute and chronic high salt diets induced similar renal functional responses in both wildtype and Tgfb2+/- mice.  Marked elevations in arterial pressure were observed in wildtype but not Tgfb2+/- mice when fed a chronic high salt diet.  A 5% NaCl diet induced a mild elevation in mean arterial pressure (MAP; ~5 mmHg) in both genotypes, however when subsequently fed an 8% NaCl diet, wildtype mice displayed a further elevation in MAP (~16 mmHg) which was not observed in Tgfb2+/- mice.  The chronic high salt diet also increased perivascular collagen deposition in wildtype kidneys, but not heterozygous kidneys.  While data from this study have shown Tgfb2+/- mice to be protected from the pathological influences of a chronic high salt diet, it remains unclear whether this is due to augmented nephron endowment or systemic reduction in TGFβ2 expression. 
In the final experiment, an anti-TGFβ2 neutralising antibody was administered to neonatal mouse pups in an attempt to augment nephron endowment in their developing kidneys.  In mice, nephrogenesis continues until 5 days after birth, thus providing a window of postnatal nephrogenesis for intervention.  The anti-TGFβ2 neutralising antibody (at a dose of 5 µg/g body weight or 10 µg/g body weight) was administered to pups via single daily intraperitoneal injections between postnatal (PN) 0 – PN4.  Pups were weighed and monitored until PN 14 at which time tissues were collected.  Total nephron endowment was unchanged in mice that received the antibody.  However, administration of the highest dose of antibody (10 µg/g) correlated with an increase in mean (15%) and total glomerular (12%) volume at PN14.  While nephron endowment remained unchanged following antibody administration, this study provides a framework by which subsequent approaches for augmenting nephron endowment can be developed.
The findings presented in this thesis suggest that perturbations to TGFβ2/betaglycan signalling can result in altered nephron endowment.  Furthermore, the findings demonstrate that the high nephron endowment in Tgfb2+/- mice does not impact upon baseline arterial pressure or renal function.  Of great significance and consistent with the overall hypothesis, the high nephron endowment in Tgfb2+/- mice was associated with a beneficial protective phenotype in the presence of a chronic cardiovascular or renal stressor, namely a chronic high salt diet.  Manipulation of TGFβ2 signalling in the developing metanephros may provide a pathway towards nephron rescue in offspring at risk of developing kidneys with low nephron endowment.