Nephron Number, Renal Function, and Arterial Pressure in Aged GDNF Heterozygous Mice

Methods

All experiments were approved in advance by Monash University Departments of Physiology and Anatomy and Cell Biology Animal Ethics Committees and were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.

Our GDNF mouse colony was initially established with founders from the laboratory of Dr Heiner Westphal (Laboratory of Mammalian Genes and Development, National Institutes of Health, Bethesda, Md).^10,11 Male GDNF heterozygous mice (129Sv-C57BL/6 hybrid; 6th generation C57BL/6 back-cross) were mated with female C57BL/6 mice. After weaning, tail tissue was obtained from all mice for genotyping by polymerase chain reaction,^10,11,17 and the mice were coded such that the following experiments were carried out in a blinded fashion.

Results

At 14 months of age, there were no significant differences in GFR, renal blood flow, renal vascular resistance, filtration fraction, fractional sodium and potassium excretions, or urinary protein concentration between GDNF heterozygous and wild-type mice (Table 1). In contrast, anesthetized mean arterial pressures were 18 mm Hg higher in GDNF heterozygotes than their wild-type littermates (P<0.001; Table 1). There were no differences in body or kidney weights between the two groups (Table 1).

Renal Histology and Pathology

Kidneys of 14-month-old GDNF heterozygous and wild-type mice both showed evidence of tubulointerstitial pathology. Most wild-type kidneys analyzed showed areas of focal tubular vacuolation (Figure) and mild inflammation around the renal pelvis. GDNF heterozygous kidneys also showed tubular vacuolation; however, the extent of vacuolation was much greater and more widespread than in wild types (Figure). There was no evidence of degeneration within the vacuolated cells as they displayed viable nuclei and no signs of necrosis or apoptosis. Despite significant hypertrophy, the glomeruli of GDNF heterozygous kidneys showed no evidence of sclerosis or hypercellularity (Figure). In addition, of the GDNF heterozygous kidneys analyzed, one showed areas of focal interstitial inflammation and one contained a cortical cyst.

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Light micrographs of 14-month-old wild-type and GDNF heterozygous mice kidneys. There was no evidence of glomerular sclerosis or hypercellularity in either wild-type (A) or GDNF heterozygous kidneys (B). The extent of vacuolation was greater and more widespread in GDNF heterozygous (D) kidneys than in wild types (C). Bar, 50 μm.

Discussion

A low renal nephron number (“reduced nephron endowment”) has been linked with the development of hypertension, glomerulosclerosis, renal failure, and the long-term failure of renal allografts. There are, however, few available animal models with a congenital nephron deficit uncomplicated by changes in birth and adult body weight with which to examine this link. In 1996, three independent research groups generated mice with a homozygous null mutation for GDNF.^10,12,13 The homozygous null mutants died within the first 24 hours of postnatal life as the result of bilateral renal agenesis. The heterozygotes, however, were indistinguishable from wild-type littermates in terms of body weight but had reduced renal mass. Recently, our group published a detailed stereological analysis of glomerular number and volumes in these GDNF heterozygous mice, studying the mice at 30 days of age when kidney development is complete.¹⁷ We found that glomerular number was 30% lower than wild-type littermates, although glomerular volumes were similar at this age.¹⁷

The GDNF heterozygous mouse thus provides a unique animal model with which to examine whether reduced glomerular endowment at birth is a predictor of subsequent development of cardiovascular disease such as essential hypertension later in life. GDNF heterozygous and wild-type mice start to die of natural causes at around 16 months and therefore we chose to study them at 14 months, making the assumption that this was equivalent to late middle age in humans, when the incidence of cardiovascular diseases such as essential hypertension rises markedly in the population.

It has been hypothesized that reductions in glomerular number leads to hypertrophy of the remaining glomeruli with time. This has been well documented in the commonly used 5/6 nephrectomy model. The present study appears to indicate that such hypertrophy also occurs when glomerular numbers are reduced genetically. We previously reported that young GDNF heterozygous mice (30 days old) showed reduced glomerular numbers with no change in mean glomerular volume and thus demonstrated, overall, a reduced total glomerular volume (product of glomerular number and volume). In the current study, we found that by 14 months of age, glomeruli of GDNF heterozygotes were significantly hypertrophied such that the total glomerular volume was no longer different between wild-type and heterozygous littermates. Whole-kidney GFR, renal blood flow, and fractional excretions were also not different between wild-type and GDNF heterozygous littermates. Although micropuncture analysis was not performed in this study, an index of average single nephron function can be calculated by dividing whole-kidney GFR by the number of glomeruli. With 30% fewer nephrons and similar GFR, GDNF heterozygous mice appear to have marked hyperfiltration with 30% greater calculated single nephron GFR values compared with wild-type mice (17.7±1.3 and 13.5±1.2, respectively; P<0.05). Such glomerular hypertrophy and hyperfiltration is often a predictor of glomerular damage and progression to glomerular sclerosis.²³ However, GDNF heterozygous mice showed no evidence of glomerular sclerosis or increased proteinuria at 14 months of age. GDNF heterozygous mice did demonstrate greater proximal tubule vacuolation at 14 months of age compared with wild-type mice. The significance of these changes on tubular reabsorption in the GDNF mice are as yet unclear; however, the fractional excretion of sodium and potassium were not different between wild-type and GDNF heterozygous littermates.

Finally, we found that these old GDNF heterozygous mice had mean arterial pressures that were 18 mm Hg higher than their wild-type littermates. At this time, we are not able to say when arterial pressure became elevated. Gerlai et al,²⁴ however, reported that the mean arterial pressure of 4- to 7-month-old GDNF heterozygous and wild-type mice were not significantly different, suggesting the elevation in pressure observed in the present study occurs subsequent to this. Interestingly, this group also showed plasma creatinine levels of the GDNF heterozygous mice to be 10 times higher than wild-type mice, suggesting that GFR tended to be decreased in younger normotensive GDNF heterozygous mice. These findings are compatible with Brenner’s hypothesis² that a reduced filtration surface area leads to the development of glomerular hypertrophy and hypertension in order to maintain adequate renal function. These findings suggest that the GDNF heterozygous mice may prove to be a useful model of essential hypertension. While anesthesia with Inactin and ketamine has been found to have only mild effects on arterial pressure,²⁵ conscious, sequential blood pressure recordings and renal functions at different ages during the lifespan of the GDNF mice are required to document the time course and accurately determine the degree of the elevation in arterial pressure and changes in glomerular structure and renal function.

A number of experimental models of reduced nephron number have been previously used to study the association between nephron number and blood pressure, each with significant limitations. Rats born to pregnant dams fed a low protein diet do show reduced nephron number; however, they have other major phenotype differences from control mice such as reduced body weight, which confounds simple interpretation of the results.^26–28 With 5/6 nephrectomy, the extent of nephron reduction is much more severe than would be expected in the natural genetic variation in humans. Mice overexpressing human insulin-like growth factor also have reduced nephron endowment with glomerular numbers 20% lower than control mice,²⁹ but at this time there have been no reports on renal function or blood pressure of these mice.

GDNF mice have the advantage that their body weights are similar to their wild-type control mice from birth; indeed the control mice have the added advantage of being littermates, and the gene manipulated has a specific role in nephrogenesis itself. GDNF has been shown to be a potent survival factor for a variety of neuronal populations in vitro and in vivo, and some of these populations are reduced in the GDNF null mutant mice.^12,13 However, apart from a reduction in the number of Aβ-caliber sensory nerve endings in the adult but not neonatal whisker follicle³⁰ and an impairment in learning the position of a hidden platform in a water maze task,²⁴ the GDNF heterozygous mouse appears to have normal-functioning dopaminergic, noradrenergic, and motor systems.^12,13,24 Recently, Shen et al³¹ reported hypoganglionosis of the gastrointestinal tract of GDNF heterozygous mice with up to 1 in 5 GDNF heterozygous mice dying before weaning because of complications resulting from enteric aganglionosis. It is important to note that GDNF heterozygous mice with hypoganglionosis were asymptomatic and thus, given the similar body weights and fecal pellets of GDNF heterozygous and wild-type littermates in the current study, it appears unlikely that enteric hypoganglionosis contributed to the higher blood pressure seen in these 14-month-old GDNF heterozygous mice. Of course, one cannot exclude the possibility that undetected phenotypes might also affect the cardiovascular outcomes in these mice.

Interestingly, an acquired nephron deficit after surgical reduction of renal mass does not always lead to hypertension, even when the reduction is greater than the 30% congenital loss demonstrated in GDNF heterozygous mice. A 50% reduction in glomerular number after unilateral nephrectomy in rats does not lead to the development of hypertension unless the nephrectomy is performed just after birth.^32,33 In humans, adult unilateral nephrectomy does not lead to increased prevalence of hypertension in kidney donors unless these donors have underlying conditions such as obesity or diabetes.^34,35 However, there does appear to be an increased risk of hypertension for patients who had a kidney removed as children because of Wilms tumor.^36,37 Even more dramatic losses in renal mass such as those seen with surgical five-sixths nephrectomy does not always lead to hypertension in rats unless generated by unilateral nephrectomy plus infarction of two thirds of the other kidney.³⁸ Of course, with these surgical models of renal and glomerular deficit, there is an acute and quite dramatic physiological reaction to the loss of renal mass involving the renal sympathetic nervous system and various hormonal systems leading to immediate (minutes) doubling of sodium and potassium excretion and followed by (hours-days) marked elevations in GFR, renal blood flow, and cardiac output, falls in renal vascular resistance, and compensatory growth of the remaining renal tissue in the following days to weeks.^38–43 Such dramatic changes cannot be compared with the situation of inherent nephron deficit in which, one could argue, there is no acute physiological reaction but rather a slow adaptive response to the growing needs of the animal.

In summary, mice heterozygous for the GDNF gene that have 30% fewer nephrons than wild types show elevated arterial pressure, normal GFR, and thus hyperfiltration in old age. Unlike the situation at 1 month, glomeruli of 14-month-old GDNF heterozygous mice are hypertrophied; however, this occurs without evidence of glomerular pathology. Thus, the results found in this low nephron-number mouse, uncomplicated by changes in body weight, are in accord with the hypothesis of Brenner et al² that a reduction in nephron number from birth leads to the development of hypertension and hyperfiltration.