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Fibrosis and loss of functional capillary surface area may contribute to renal tissue hypoxia in a range of kidney diseases. However, there is limited quantitative information on the impact of kidney disease on the barriers to oxygen diffusion from cortical peritubular capillaries (PTCs) to kidney epithelial tubules. Here, we used stereological methods to quantify changes in total cortical PTC length and surface area, PTC length and surface densities, and diffusion distances between PTCs and kidney tubules in adenine-induced kidney injury. After 7 days of oral gavage of adenine (100 mg), plasma creatinine was 3.5-fold greater than in vehicle-treated rats, while total kidney weight was 83% greater. The total length of PTCs was similar in adenine-treated (1.47 ± 0.23 km (mean ± standard deviation)) to vehicle-treated (1.24 ± 0.24 km) rats, as was the surface density of PTCs (0.025 ± 0.002 vs. 0.024 ± 0.004 μm 2 /μm 3 ). The total surface area of PTCs was 69% greater in adenine-treated than vehicle-treated rats. However, the length density of PTCs was 28% less in adenine-treated than vehicle-treated rats. Diffusion distances, from PTCs to the basal membrane of the nearest renal tubule (108%), and to the mid-point of the cytoplasmic height of the nearest tubular epithelial cell (57%), were markedly increased. These findings indicate that, in adenine-induced kidney injury, expansion of the renal cortical interstitium increases the distance required for diffusion of oxygen from PTCs to tubules, rendering the kidney cortex susceptible to hypoxia., (© 2024 The Author(s). The Anatomical Record published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Background: Low nephron number has a direct impact on the development of hypertension and chronic kidney disease later in life. While intrauterine growth restriction caused by maternal low protein diet (LPD) is thought to be a significant cause of reduced nephron endowment in impoverished communities, its influence on the cellular and molecular processes which drive nephron formation are poorly understood., Methods: We conducted a comprehensive characterization of the impact of LPD on kidney development using tomographic and confocal imaging to quantify changes in branching morphogenesis and the cellular and morphological features of nephrogenic niches across development. These analyses were paired with single-cell RNA sequencing to dissect the transcriptional changes that LPD imposes during renal development to affect nephron number., Results: Single cell analysis at E14.5 and P0 revealed differences in the expression of genes and pathways involved in metabolism, cell cycle, epigenetic regulators and reciprocal inductive signals in most cell types analyzed, yielding imbalances and shifts in cellular energy production and cellular trajectories. In the nephron progenitor cells, LPD impeded cellular commitment and differentiation towards pre-tubular and renal vesicle structures. Confocal microscopy revealed a reduction in the number of pre-tubular aggregates and proliferation in nephron progenitor cells. We also found changes in branching morphogenesis, with a reduction in cell proliferation in the ureteric tips as well as reduced tip and tip parent lengths by optical projection tomography which causes patterning defects., Conclusions: This unique profiling demonstrates how a fetal programming defect leads to low nephron endowment which is intricately linked to changes in both branching morphogenesis and the commitment of nephron progenitor cells. The commitment of progenitor cells is pivotal for nephron formation and is significantly influenced by nutritional factors, with a low protein diet driving alterations in this program which directly results in a reduced nephron endowment., Significance Statement: While a mother's diet can negatively impact the number of nephrons in the kidneys of her offspring, the root cellular and molecular drivers of these deficits have not been rigorously explored. In this study we use advanced imaging and gene expression analysis in mouse models to define how a maternal low protein diet, analogous to that of impoverished communities, results in reduced nephron endowment. We find that low protein diet has pleiotropic effects on metabolism and the normal developmental programs of gene expression. These profoundly impact the process of branching morphogenesis necessary to establish niches for nephron generation and change cell behaviors which regulate how and when nephron progenitor cells commit to differentiation.

Corticosteroid therapy, often in combination with inhibition of the renin-angiotensin system, is first-line therapy for primary focal and segmental glomerulosclerosis (FSGS) with nephrotic-range proteinuria. However, the response to treatment is variable, and therefore new approaches to indicate the response to therapy are required. Podocyte depletion is a hallmark of early FSGS, and here we investigated whether podocyte number, density and/or size in diagnostic biopsies and/or the degree of glomerulosclerosis could indicate the clinical response to first-line therapy. In this retrospective single center cohort study, 19 participants (13 responders, 6 non-responders) were included. Biopsies obtained at diagnosis were prepared for analysis of podocyte number, density and size using design-based stereology. Renal function and proteinuria were assessed 6 months after therapy commenced. Responders and non-responders had similar levels of proteinuria at the time of biopsy and similar kidney function. Patients who did not respond to treatment at 6 months had a significantly higher percentage of glomeruli with global sclerosis than responders ( p  < 0.05) and glomerulosclerotic index ( p  < 0.05). Podocyte number per glomerulus in responders was 279 (203-507; median, IQR), 50% greater than that of non-responders (186, 118-310; p < 0.05). These findings suggest that primary FSGS patients with higher podocyte number per glomerulus and less advanced glomerulosclerosis are more likely to respond to first-line therapy at 6 months. A podocyte number less than approximately 216 per glomerulus, a GSI greater than 1 and percentage global sclerosis greater than approximately 20% are associated with a lack of response to therapy. Larger, prospective studies are warranted to confirm whether these parameters may help inform therapeutic decision making at the time of diagnosis of primary FSGS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 de Zoysa, Haruhara, Nikolic-Paterson, Kerr, Ling, Gazzard, Puelles, Bertram and Cullen-McEwen.)

Maternal protein restriction is often associated with structural and functional sequelae in offspring, particularly affecting growth and renal-cardiovascular function. However, there is little understanding as to whether hypertension and kidney disease occur because of a primary nephron deficit or whether controlling postnatal growth can result in normal renal-cardiovascular phenotypes. To investigate this, female Sprague-Dawley rats were fed either a low-protein (LP, 8.4% protein) or normal-protein (NP, 19.4% protein) diet prior to mating and until offspring were weaned at postnatal day (PN) 21. Offspring were then fed a non 'growth' (4.6% fat) which ensured that catch-up growth did not occur. Offspring growth was determined by weight and dual energy X-ray absorptiometry. Nephron number was determined at PN21 using the disector-fractionator method. Kidney function was measured at PN180 and PN360 using clearance methods. Blood pressure was measured at PN360 using radio-telemetry. Body weight was similar at PN1, but by PN21 LP offspring were 39% smaller than controls (P diet < 0.001). This difference was due to proportional changes in lean muscle, fat, and bone content. LP offspring remained smaller than NP offspring until PN360. In LP offspring, nephron number was 26% less in males and 17% less in females, than NP controls (P diet < 0.0004). Kidney function was similar across dietary groups and sexes at PN180 and PN360. Blood pressure was similar in LP and NP offspring at PN360. These findings suggest that remaining on a slow growth trajectory after exposure to a suboptimal intrauterine environment does not lead to the development of kidney dysfunction and hypertension.

Podocytes are terminally differentiated epithelial cells in glomeruli. Podocyte injury and loss are features of many diseases leading to chronic kidney disease (CKD). The developmental origins of health and disease hypothesis propose an adverse intrauterine environment can lead to CKD later in life, especially when a second postnatal challenge is experienced. The aim of this study was to examine whether a suboptimal maternal environment would result in reduced podocyte endowment, increasing susceptibility to diabetes-induced renal injury. Female C57BL/6 mice were fed a low protein diet (LPD) to induce growth restriction or a normal protein diet (NPD) from 3 weeks before mating until weaning (postnatal Day 21, P21) when nephron and podocyte endowment were assessed in one male and one female offspring per litter. Littermates were administered streptozotocin or vehicle at 6 weeks of age. Urinary albumin excretion, glomerular size, and podometrics were assessed following 18 weeks of hyperglycemia. LPD offspring were growth restricted and had lower nephron and podocyte number at P21. However, by 24 weeks the podocyte deficit was no longer evident and despite low nephron endowment neither albuminuria nor glomerulosclerosis were observed. Podocyte number was unaffected by 18 weeks of hyperglycemia in NPD and LPD offspring. Diabetes increased glomerular volume reducing podocyte density, with more pronounced effects in LPD offspring. LPD and NPD diabetic offspring developed mild albuminuria with LPD demonstrating an earlier onset. LPD offspring also developed glomerular pathology. These findings indicate that growth-restricted LPD offspring with low nephron number and normalized podocyte endowment were more susceptible to alterations in glomerular volume and podocyte density leading to more rapid onset of albuminuria and renal injury than NPD offspring., (© 2023 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Podocyte loss and resultant nephron loss are common processes in the development of glomerulosclerosis and chronic kidney disease. While the cortical distribution of glomerulosclerosis is known to be non-uniform, the relationship between the numbers of non-sclerotic glomeruli (NSG), podometrics and zonal differences in podometrics remain incompletely understood. To help define this, we studied autopsy kidneys from 50 adults with median age 68 years and median eGFR 73.5 mL/min/1.73m 2 without apparent glomerular disease in a cross-sectional analysis. The number of NSG per kidney was estimated using the physical dissector/fractionator combination, while podometrics were estimated using model-based stereology. The number of NSG per kidney was directly correlated with podocyte number per tuft and podocyte density. Each additional 100,000 NSG per kidney was associated with 26 more podocytes per glomerulus and 16 podocytes per 10 6 μm 3 increase in podocyte density. These associations were independent of clinical factors and cortical zone. While podocyte number per glomerulus was similar in the three zones, superficial glomeruli were the smallest and had the highest podocyte density but smallest podocytes. Increasing age and hypertension were associated with lower podocyte number, with age mostly affecting superficial glomeruli, and hypertension mostly affecting juxtamedullary glomeruli. Thus, in this first study to report a direct correlation between the number of NSG and podometrics, we suggest that podocyte number is decreasing in NSG of individuals losing nephrons. However, another possible interpretation may be that more nephrons might protect against further podocyte loss., (Copyright © 2022 International Society of Nephrology. All rights reserved.)

Progressive podocyte loss is a feature of healthy ageing. While previous studies have reported age-related changes in podocyte number, density and size and associations with proteinuria and glomerulosclerosis, few studies have examined how the response of remaining podocytes to podocyte depletion changes with age. Mild podocyte depletion was induced in Pod Cre iDTR mice aged 1, 6, 12 and 18 months via intraperitoneal administration of diphtheria toxin. Control mice received intraperitoneal vehicle. Podometrics, proteinuria and glomerular pathology were assessed, together with podocyte expression of p-rp-S6, a phosphorylation target that represents activity of the mammalian target of rapamycin (mTOR). Podocyte number per glomerulus did not change in control mice in the 18-month time period examined. However, control mice at 18 months had the largest podocytes and the lowest podocyte density. Podocyte depletion at 1, 6 and 12 months resulted in mild albuminuria but no glomerulosclerosis, whereas similar levels of podocyte depletion at 18 months resulted in both albuminuria and glomerulosclerosis. Following podocyte depletion at 6 and 12 months, the number of p-rp-S6 positive podocytes increased significantly, and this was associated with an adaptive increase in podocyte volume. However, at 18 months of age, remaining podocytes were unable to further elevate mTOR expression or undergo hypertrophic adaptation in response to mild podocyte depletion, resulting in marked glomerular pathology. These findings demonstrate the importance of mTORC1-mediated podocyte hypertrophy in both physiological (ageing) and adaptive settings, highlighting a functional limit to podocyte hypertrophy reached under physiological conditions., (© 2022. The Author(s).)

Low birth weight is a risk factor for chronic kidney disease, whereas adult podocyte depletion is a key event in the pathogenesis of glomerulosclerosis. However, whether low birth weight due to poor maternal nutrition is associated with low podocyte endowment and glomerulosclerosis in later life is not known. Female Sprague-Dawley rats were fed a normal-protein diet (NPD; 20%) or low-protein diet (LPD; 8%), to induce low birth weight, from 3 wk before mating until postnatal day 21 (PN21), when kidneys from some male offspring were taken for quantitation of podocyte number and density in whole glomeruli using immunolabeling, tissue clearing, and confocal microscopy. The remaining offspring were fed a normal- or high-fat diet until 6 mo to induce catch-up growth and excessive weight gain, respectively. At PN21, podocyte number per glomerulus was 15% lower in low birth weight (LPD) than normal birth weight (NPD) offspring, with this deficit greater in outer glomeruli. Surprisingly, podocyte number in LPD offspring increased in outer glomeruli between PN21 and 6 mo, although an overall 9% podocyte deficit persisted. Postnatal fat feeding to LPD offspring did not alter podometric indexes or result in glomerular pathology at 6 mo, whereas fat feeding in NPD offspring was associated with far greater body and fat mass as well as podocyte loss, reduced podocyte density, albuminuria, and glomerulosclerosis. This is the first report that maternal diet can influence podocyte endowment. Our findings provide new insights into the impact of low birth weight, podocyte endowment, and postnatal weight on podometrics and kidney health in adulthood. NEW & NOTEWORTHY The present study shows, for the first time, that low birth weight as a result of maternal nutrition is associated with low podocyte endowment. However, a mild podocyte deficit at birth did not result in glomerular pathology in adulthood. In contrast, postnatal podocyte loss in combination with excessive body weight led to albuminuria and glomerulosclerosis. Taken together, these findings provide new insights into the associations between birth weight, podocyte indexes, postnatal weight, and glomerular pathology.

Background: Podocyte depletion, low nephron number, aging, and hypertension are associated with glomerulosclerosis and CKD. However, the relationship between podometrics and nephron number has not previously been examined., Methods: To investigate podometrics and nephron number in healthy Japanese individuals, a population characterized by a relatively low nephron number, we immunostained single paraffin sections from 30 Japanese living-kidney donors (median age, 57 years) with podocyte-specific markers and analyzed images obtained with confocal microscopy. We used model-based stereology to estimate podometrics, and a combined enhanced-computed tomography/biopsy-specimen stereology method to estimate nephron number., Results: The median number of nonsclerotic nephrons per kidney was 659,000 (interquartile range [IQR], 564,000-825,000). The median podocyte number and podocyte density were 518 (IQR, 428-601) per tuft and 219 (IQR, 180-253) per 10 6 μ m 3 , respectively; these values are similar to those previously reported for other races. Total podocyte number per kidney (obtained by multiplying the individual number of nonsclerotic glomeruli by podocyte number per glomerulus) was 376 million (IQR, 259-449 million) and ranged 7.4-fold between donors. On average, these healthy kidneys lost 5.63 million podocytes per kidney per year, with most of this loss associated with glomerular loss resulting from global glomerulosclerosis, rather than podocyte loss from healthy glomeruli. Hypertension was associated with lower podocyte density and larger podocyte volume, independent of age., Conclusions: Estimation of the number of nephrons, podocytes, and other podometric parameters in individual kidneys provides new insights into the relationships between these parameters, age, and hypertension in the kidney. This approach might be of considerable value in evaluating the kidney in health and disease., (Copyright © 2021 by the American Society of Nephrology.)

Alcohol during pregnancy can impair fetal development and result in offspring with neurodevelopmental deficits. Less is known about how low to moderate alcohol exposure can affect other organs, such as the kidney. Here, the effects of moderate ethanol exposure throughout pregnancy on kidney development were examined using a rat model. Rats were fed a liquid diet containing 6% ethanol (vol/vol) or control (0% ethanol) throughout pregnancy. Kidneys were collected at embryonic day (E) 20 or postnatal day (PN) 30 and total glomerular (nephron) number determined using unbiased stereology. Kidney function was examined in offspring at 8 and 19 months. At E20, fetuses exposed to ethanol had fewer nephrons with increased apoptosis. Alcohol exposure caused kidney dysregulation of pro- (Bax) and anti- (Bcl-2) apoptotic factors, and reduced expression of the cell proliferation marker, Ki67. Prenatal alcohol decreased expression of Gdnf and Tgfb1, important regulators of branching morphogenesis, in male fetuses. At PN30, kidney volume and nephron number were lower in offspring exposed to prenatal alcohol. Urine flow and osmolality were normal in offspring exposed to alcohol however sodium excretion tended to be lower in females prenatally exposed to alcohol. Findings suggest exposure to moderate levels of alcohol during pregnancy results in impaired kidney development and leads to a permanent nephron deficit. Although the impact on adult kidney function was relatively minor, these data highlight that even at moderate levels, alcohol consumption during pregnancy can have deleterious long-term outcomes and should be avoided., (© 2020 American Association for Anatomy.)

Fetal hypoxia is a common complication of pregnancy. We have previously reported that maternal hypoxia in late gestation in mice gives rise to male offspring with reduced nephron number, while females have normal nephron number. Male offspring later develop proteinuria and renal pathology, including glomerular pathology, whereas female offspring are unaffected. Given the central role of podocyte depletion in glomerular and renal pathology, we examined whether maternal hypoxia resulted in low podocyte endowment in offspring. Pregnant CD1 mice were allocated at embryonic day 14.5 to normoxic (21% oxygen) or hypoxic (12% oxygen) conditions. At postnatal day 21, kidneys from mice were immersion fixed, and one mid-hilar slice per kidney was immunostained with antibodies directed against p57 and synaptopodin for podocyte identification. Slices were cleared and imaged with a multiphoton microscope for podometric analysis. Male hypoxic offspring had significantly lower birth weight, nephron number, and podocyte endowment than normoxic male offspring (podocyte number; normoxic 62.86 ± 2.26 podocytes per glomerulus, hypoxic 53.38 ± 2.25; p < .01, mean ± SEM). In contrast, hypoxic female offspring had low birth weight but their nephron and podocyte endowment was the same as normoxic female offspring (podocyte number; normoxic 62.38 ± 1.86 podocytes per glomerulus, hypoxic 61.81 ± 1.80; p = .88). To the best of our knowledge, this is the first report of developmentally programmed low podocyte endowment. Given the well-known association between podocyte depletion in adulthood and glomerular pathology, we postulate that podocyte endowment may place offspring at risk of renal disease in adulthood, and explain the greater vulnerability of male offspring., (© 2020 American Association for Anatomy.)

A maternal low protein (LP) diet in rodents often results in low nephron endowment and renal pathophysiology in adult life, with outcomes often differing between male and female offspring. Precisely how a maternal LP diet results in low nephron endowment is unknown. We conducted morphological and molecular studies of branching morphogenesis and nephrogenesis to identify mechanisms and timepoints that might give rise to low nephron endowment. Sprague-Dawley rats were fed a normal protein (19.4% protein, NP) or LP (9% protein) diet for 3 weeks prior to mating and throughout gestation. Embryonic day 14.25 (E14.25) kidneys from males and females were either cultured for 2 days after which branching morphogenesis was quantified, or frozen for gene expression analysis. Real-time PCR was used to quantify expression of key nephrogenesis and branching morphogenesis genes at E14.25 and 17.25. At E17.25, nephron number was determined in fixed tissue. There was no effect of either maternal diet or sex on branching morphogenesis. Nephron number at E17.25 was 14% lower in male and female LP offspring than in NP controls. At E14.25 expression levels of genes involved in branching morphogenesis (Gfrα1, Bmp4, Gdnf) and nephrogenesis (Hnf4a, Pax2, Wnt4) were similar in the dietary groups, but significant differences between sexes were identified. At E17.25, expression of Gfrα1, Gdnf, Bmp4, Pax2 and Six2 was lower in LP offspring than NP offspring, in both male and female offspring. These findings provide new insights into how a LP diet leads to low nephron endowment and renal sexual dimorphism., (© 2020 American Association for Anatomy.)

The cellular origins of glomerulosclerosis involve activation of parietal epithelial cells (PECs) and progressive podocyte depletion. While mammalian target of rapamycin-mediated (mTOR-mediated) podocyte hypertrophy is recognized as an important signaling pathway in the context of glomerular disease, the role of podocyte hypertrophy as a compensatory mechanism preventing PEC activation and glomerulosclerosis remains poorly understood. In this study, we show that glomerular mTOR and PEC activation-related genes were both upregulated and intercorrelated in biopsies from patients with focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy, suggesting both compensatory and pathological roles. Advanced morphometric analyses in murine and human tissues identified podocyte hypertrophy as a compensatory mechanism aiming to regulate glomerular functional integrity in response to somatic growth, podocyte depletion, and even glomerulosclerosis - all of this in the absence of detectable podocyte regeneration. In mice, pharmacological inhibition of mTOR signaling during acute podocyte loss impaired hypertrophy of remaining podocytes, resulting in unexpected albuminuria, PEC activation, and glomerulosclerosis. Exacerbated and persistent podocyte hypertrophy enabled a vicious cycle of podocyte loss and PEC activation, suggesting a limit to its beneficial effects. In summary, our data highlight a critical protective role of mTOR-mediated podocyte hypertrophy following podocyte loss in order to preserve glomerular integrity, preventing PEC activation and glomerulosclerosis.

Aims: The worldwide prevalence of gestational diabetes mellitus (GDM) is increasing. Studies in rodent models indicate that hyperglycaemia during pregnancy alters kidney development, yet few studies have examined if this is so in humans. The objective of this study was to evaluate the association of treated GDM with foetal kidney size., Materials and Methods: Participants were recruited from an Australian tertiary hospital, and clinical data were collected from women without GDM and women diagnosed and treated for GDM and their offspring. Participants underwent an obstetric ultrasound at 32-34 weeks gestation for foetal biometry and foetal kidney volume measurement., Results: Sixty-four non-GDM and 64 GDM women participated in the study. Thirty percent of GDM women were diagnosed with fasting hyperglycaemia, while 89% had an elevated 2-hour glucose level. Maternal age, weight and body mass index were similar in women with and without GDM. Estimated foetal weight, foetal kidney dimensions, total foetal kidney volume and birth weight were similar in offspring of women with and without GDM., Conclusions: We conclude that a period of mild hyperglycaemia prior to diagnosis of GDM and treatment initiation, which coincides with a period of rapid nephron formation and kidney growth, does not alter kidney size at 32-34 weeks gestation., Competing Interests: The authors declare they have no conflict of interest., (© 2019 The Authors. Endocrinology, Diabetes & Metabolism published by John Wiley & Sons Ltd.)

Glomerular filtration rate (GFR) declines with age such that the prevalence of chronic kidney disease is much higher in the elderly. SIRT1 is the leading member of the sirtuin family of NAD + -dependent lysine deacetylases that mediate the health span extending properties of caloric restriction. Since reduction in energy intake has also been shown to decrease age-related kidney disease in rodents, we hypothesized that a diminution in SIRT1 activity would accelerate the GFR decline and structural injury with age. To test this hypothesis, we compared changes in the kidney structure and function in control mice and mice that carry a point mutation at a conserved histidine (H355Y) of SIRT1 that renders the enzyme catalytically inactive. Taking advantage of this mouse model along with the disector/fractionator technique for glomerular counting and direct measurements of GFR by inulin clearance, we assessed the impact of SIRT1 inactivity on kidney aging. At 14 months of age, SIRT1 catalytically inactive (Sirt1 Y/Y ) mice had lower GFRs and fewer glomeruli than their wild-type (Sirt1 +/+ ) counterparts. This was not, however, due to either accelerated GFR decline or increased glomerulosclerosis and loss, but rather to reduced glomerular endowment in Sirt1 Y/Y mice. Moreover, the compensatory glomerular hypertrophy and elevated single nephron GFR that customarily accompany reduction in nephron number were absent in Sirt1 Y/Y mice. These findings suggest a role for SIRT1 not only in determining nephron endowment but also in orchestrating the response to it., (© 2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

A normal endowment of nephrons in the mammalian kidney requires a balance of nephron progenitor self-renewal and differentiation throughout development. Here, we provide evidence for a novel action of ureteric branch tip-derived Wnt11 in progenitor cell organization and interactions within the nephrogenic niche, ultimately determining nephron endowment. In Wnt11 mutants, nephron progenitors dispersed from their restricted niche, intermixing with interstitial progenitors. Nephron progenitor differentiation was accelerated, kidneys were significantly smaller, and the nephron progenitor pool was prematurely exhausted, halving the final nephron count. Interestingly, RNA-seq revealed no significant differences in gene expression. Live imaging of nephron progenitors showed that in the absence of Wnt11 they lose stable attachments to the ureteric branch tips, continuously detaching and reattaching. Further, the polarized distribution of several markers within nephron progenitors is disrupted. Together these data highlight the importance of Wnt11 signaling in directing nephron progenitor behavior which determines a normal nephrogenic program., Competing Interests: LO, AC, KS, NL, PW, LC, AJ, AA, OM, JB, IS, ML, AM No competing interests declared, (© 2018, O'Brien et al.)

The ovarian reserve of primordial follicle oocytes is formed during in utero development and represents the entire supply of oocytes available to sustain female fertility. Maternal undernutrition during pregnancy and lactation diminishes offspring ovarian reserve in rats. In mice, maternal oocyte maturation is also susceptible to undernutrition, causing impaired offspring cardiovascular function. We aimed to determine whether programming of the ovarian reserve is impacted in offspring when maternal undernutrition extends from preconception oocyte development through to weaning. C57BL6/J female mice were fed normal protein (20%) or low-protein (8%) diet during preconception, pregnancy and lactation periods. Maternal ovaries were harvested at weaning and offspring ovaries were collected at postnatal day (PN)21 and 24 weeks of age. Total follicle estimates were obtained by histologically sampling one ovary per animal (n = 5/group). There was no impact of diet on maternal follicle numbers. However, in offspring, maternal protein restriction significantly depleted primordial follicles by 37% at PN21 and 51% at 24 weeks (P < 0.05). There were no effects of diet on other follicle classes. Histological analysis showed no differences in the proportion of proliferative follicles (pH3 positive), but increased atresia (cleaved caspase-3-positive, or TUNEL-positive) was detected in ovaries of protein-restricted offspring at both ages (P < 0.05). Our data show that maternal diet during the preconception period, in utero development and early life has significant impacts on follicle endowment and markers of follicle health later in life. This highlights the need for further investigation into the importance of maternal preconception diet for offspring reproductive development and health., (2018 Society for Reproduction and Fertility)

It has been suggested that low nephron number contributes to glomerular hypertension and hyperperfusion injury in progressive chronic kidney disease (CKD). The incidence of CKD in Japan is among the highest in the world, but the reasons remain unclear. We estimated total nephron (glomerular) number (NglomTOTAL) as well as numbers of nonsclerosed (NglomNSG) and globally sclerosed glomeruli (NglomGSG), and the mean volume of nonsclerosed glomeruli (VglomNSG) in Japanese normotensive, hypertensive, and CKD subjects and investigated associations between these parameters and estimated glomerular filtration rate (eGFR). Autopsy kidneys from age-matched Japanese men (9 normotensives, 9 hypertensives, 9 CKD) had nephron number and VglomNSG estimated using disector/fractionator stereology. Subject eGFR, single-nephron eGFR (SNeGFR), and the ratio SNeGFR/VglomNSG were calculated. NglomNSG in Japanese with hypertension (392,108 ± 87,605; P < 0.001) and CKD (268,043 ± 106,968; P < 0.001) was less than in normotensives (640,399 ± 160,016). eGFR was directly correlated with NglomNSG (r = 0.70, P < 0.001) and inversely correlated with VglomNSG (r = -0.53, P < 0.01). SNeGFR was higher in hypertensives than normotensives (P = 0.03), but was similar in normotensives and CKD, while the ratio SNeGFR/VglomNSG was similar in normotensives and hypertensives but markedly reduced in CKD. Nephron number in Japanese with hypertension or CKD was low. This results in a higher SNeGFR in hypertensives compared with normotensive and CKD subjects, but lowered SNeGFR/VglomNSG in CKD subjects, suggesting that changes in GFR are accommodated by glomerular hypertrophy rather than glomerular hypertension. These findings suggest glomerular hypertrophy is a dominant factor in maintenance of GFR under conditions of low nephron number.

Background: Acute kidney injury affects ~70% of asphyxiated newborns, and increases their risk of developing chronic kidney disease later in life. Acute kidney injury is driven by renal oxygen deprivation during asphyxia, thus we hypothesized that creatine administered antenatally would protect the kidney from the long-term effects of birth asphyxia., Methods: Pregnant spiny mice were fed standard chow or chow supplemented with 5% creatine from 20-d gestation (midgestation). One day prior to term (37-d gestation), pups were delivered by caesarean or subjected to intrauterine asphyxia. Litters were allocated to one of two time-points. Kidneys were collected at 1 mo of age to estimate nephron number (stereology). Renal function (excretory profile and glomerular filtration rate) was measured at 3 mo of age, and kidneys then collected for assessment of glomerulosclerosis., Results: Compared with controls, at 1 mo of age male (but not female) birth-asphyxia offspring had 20% fewer nephrons (P < 0.05). At 3 mo of age male birth-asphyxia offspring had 31% lower glomerular filtration rate (P < 0.05) and greater glomerular collagen IV content (P < 0.01). Antenatal creatine prevented these renal injuries arising from birth asphyxia., Conclusion: Maternal creatine supplementation during pregnancy may be an effective prophylactic to prevent birth asphyxia induced acute kidney injury and the emergence of chronic kidney disease.

Background: Animal studies report a nephron deficit in offspring exposed to maternal diabetes, yet are limited to models of severe hyperglycaemia which do not reflect the typical clinical condition and which are associated with foetal growth restriction that may confound nephron endowment. We aimed to assess renal morphology and function in offspring of leptin receptor deficient mice (Lepr db /+) and hypothesized that exposure to impaired maternal glucose tolerance (IGT) would be detrimental to the developing kidney., Methods: Nephron endowment was assessed in offspring of C57BKS/J Lepr db /+ and +/+ mice at embryonic day (E)18 and postnatal day (PN)21 using design-based stereology. Transcutaneous measurement of renal function and total glomerular volume were assessed in 6-month-old offspring. Only +/+ offspring of Lepr db /+ dams were analysed., Results: Compared with +/+ dams, Lepr db /+ dams had a 20% and 35% decrease in glucose tolerance prior to pregnancy and at E17.5 respectively. Offspring of IGT Lepr db /+ dams had approximately 15% fewer nephrons at E18.5 and PN21 than offspring of +/+ dams. There was no difference in offspring bodyweight. Despite normal renal function, total glomerular volume was 13% greater in 6-month-old offspring of IGT Lepr db /+ dams than in +/+ offspring., Conclusions: IGT throughout gestation resulted in a nephron deficit that was established early in renal development. Maternal IGT was associated with glomerular hypertrophy in adult offspring, likely a compensatory response to maintain normal renal function. Given the increasing prevalence of IGT, monitoring glucose from early in gestation may be important to prevent altered kidney morphology. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Podocyte depletion is sufficient for the development of numerous glomerular diseases and can be absolute (loss of podocytes) or relative (reduced number of podocytes per volume of glomerulus). Commonly used methods to quantify podocyte depletion introduce bias, whereas gold standard stereologic methodologies are time consuming and impractical. We developed a novel approach for assessing podocyte depletion in whole glomeruli that combines immunofluorescence, optical clearing, confocal microscopy, and three-dimensional analysis. We validated this method in a transgenic mouse model of selective podocyte depletion, in which we determined dose-dependent alterations in several quantitative indices of podocyte depletion. This new approach provides a quantitative tool for the comprehensive and time-efficient analysis of podocyte depletion in whole glomeruli., (Copyright © 2016 by the American Society of Nephrology.)

Increasing consumption of a high fat 'Western' diet has led to a growing number of pregnancies complicated by maternal obesity. Maternal overnutrition and obesity have health implications for offspring, yet little is known about their effects on offspring kidney development and renal function. Female C57Bl6 mice were fed a high fat diet (HFD, 21% fat) or matched normal fat diet (NFD, 6% fat) for 6 weeks prior to pregnancy and throughout gestation and lactation. HFD dams were overweight and glucose intolerant prior to mating but not in late gestation. Offspring of NFD and HFD dams had similar body weights at embryonic day (E)15.5, E18.5 and at postnatal day (PN)21. HFD offspring had normal ureteric tree development and nephron number at E15.5. However, using unbiased stereology, kidneys of HFD offspring were found to have 20-25% more nephrons than offspring of NFD dams at E18.5 and PN21. Offspring of HFD dams with body weight and glucose profiles similar to NFD dams prior to pregnancy also had an elevated nephron endowment. At 9 months of age, adult offspring of HFD dams displayed mild fasting hyperglycaemia but similar body weights to NFD offspring. Renal function and morphology, measured by transcutaneous clearance of FITC-sinistrin and stereology respectively, were normal. This study demonstrates that maternal fat feeding augments offspring nephron endowment with no long-term consequences for offspring renal health. Future studies assessing the effects of a chronic stressor on adult mice with augmented nephron number are warranted, as are studies investigating the molecular mechanisms that result in high nephron endowment., Competing Interests: The authors have declared that no competing interests exist.

Podocyte depletion plays a major role in the development and progression of glomerulosclerosis. Many kidney diseases are more common in older age and often coexist with hypertension. We hypothesized that podocyte depletion develops in association with older age and is exacerbated by hypertension. Kidneys from 19 adult Caucasian American males without overt renal disease were collected at autopsy in Mississippi. Demographic data were obtained from medical and autopsy records. Subjects were categorized by age and hypertension as potential independent and additive contributors to podocyte depletion. Design-based stereology was used to estimate individual glomerular volume and total podocyte number per glomerulus, which allowed the calculation of podocyte density (number per volume). Podocyte depletion was defined as a reduction in podocyte number (absolute depletion) or podocyte density (relative depletion). The cortical location of glomeruli (outer or inner cortex) and presence of parietal podocytes were also recorded. Older age was an independent contributor to both absolute and relative podocyte depletion, featuring glomerular hypertrophy, podocyte loss, and thus reduced podocyte density. Hypertension was an independent contributor to relative podocyte depletion by exacerbating glomerular hypertrophy, mostly in glomeruli from the inner cortex. However, hypertension was not associated with podocyte loss. Absolute and relative podocyte depletion were exacerbated by the combination of older age and hypertension. The proportion of glomeruli with parietal podocytes increased with age but not with hypertension alone. These findings demonstrate that older age and hypertension are independent and additive contributors to podocyte depletion in white American men without kidney disease., (Copyright © 2016 the American Physiological Society.)

Increases in glomerular size occur with normal body growth and in many pathologic conditions. In this study, we determined associations between glomerular size and numbers of glomerular resident cells, with a particular focus on podocytes. Kidneys from 16 male Caucasian-Americans without overt renal disease, including 4 children (≤3 years old) to define baseline values of early life and 12 adults (≥18 years old), were collected at autopsy in Jackson, Mississippi. We used a combination of immunohistochemistry, confocal microscopy, and design-based stereology to estimate individual glomerular volume (IGV) and numbers of podocytes, nonepithelial cells (NECs; tuft cells other than podocytes), and parietal epithelial cells (PECs). Podocyte density was calculated. Data are reported as medians and interquartile ranges (IQRs). Glomeruli from children were small and contained 452 podocytes (IQR=335-502), 389 NECs (IQR=265-498), and 146 PECs (IQR=111-206). Adult glomeruli contained significantly more cells than glomeruli from children, including 558 podocytes (IQR=431-746; P<0.01), 1383 NECs (IQR=998-2042; P<0.001), and 367 PECs (IQR=309-673; P<0.001). However, large adult glomeruli showed markedly lower podocyte density (183 podocytes per 10(6) µm(3)) than small glomeruli from adults and children (932 podocytes per 10(6) µm(3); P<0.001). In conclusion, large adult glomeruli contained more podocytes than small glomeruli from children and adults, raising questions about the origin of these podocytes. The increased number of podocytes in large glomeruli does not match the increase in glomerular size observed in adults, resulting in relative podocyte depletion. This may render hypertrophic glomeruli susceptible to pathology., (Copyright © 2015 by the American Society of Nephrology.)

Indomethacin and ibuprofen are administered to close a patent ductus arteriosus (PDA) during active glomerulogenesis. Light and electron microscopic glomerular changes with no change in glomerular number were seen following indomethacin and ibuprofen treatment during glomerulogenesis at 14 days after birth in a neonatal rat model. This present study aimed to determine whether longstanding renal structural changes are present at 30 days and 6 mo (equivalent to human adulthood). Rat pups were administered indomethacin or ibuprofen antenatally on days 18-20 (0.5 mg·kg(-1)·dose(-1) indomethacin; 10 mg·kg(-1)·dose(-1) ibuprofen) or postnatally intraperitoneally from day 1 to 3 or day 1 to 5 (0.2 mg·kg(-1)·dose(-1) indomethacin; 10 mg·kg(-1)·dose(-1) ibuprofen). Control groups received no treatment or normal saline intraperitoneally. Pups were killed at 30 days of age and 6 mo of age. Tissue blocks from right kidneys were prepared for light and electron microscopic examination, while total glomerular number was determined in left kidneys using unbiased stereology. Eight pups were included in each group from 14 maternal rats. At 30 days and 6 mo, there were persistent electron microscopy abnormalities of the glomerular basement membrane in those receiving postnatal indomethacin and ibuprofen. There were no significant light microscopy findings at 30 days or 6 mo. At 6 mo, there were significantly fewer glomeruli in those receiving postnatal indomethacin but not ibuprofen (P = 0.003). In conclusion, indomethacin administered during glomerulogenesis appears to reduce the number of glomeruli in adulthood. Alternative options for closing a PDA should be considered including ibuprofen as well as emerging therapies such as paracetamol., (Copyright © 2014 the American Physiological Society.)

A developmental insult that restricts growth in the first generation has the potential to program disease in subsequent generations. The aim of this study was to ascertain transgenerational growth and cardio-renal effects, via the maternal line, in a rat model of utero-placental insufficiency. Bilateral uterine vessel ligation or sham surgery (offspring termed first generation; F1 Restricted and Control, respectively) was performed in WKY rats. F1 Restricted and Control females were mated with normal males to produce second generation (F2) offspring (Restricted and Control) studied from fetal (embryonic Day 20) to adult (12 months) life. F2 Restricted male and female fetuses had reduced (P<0.05) nephron number (down 15-22%) but this deficit was not sustained postnatally and levels were similar to Controls at Day 35. F2 Restricted males, but not females, developed elevated (+16mmHg, P<0.05) systolic blood pressure at 6 months of age, which was sustained to 9 months. This was not explained by alterations to intra-renal or plasma components of the renin-angiotensin system. In a rat model of utero-placental insufficiency, we report alterations to F2 kidney development and sex-specific adult hypertension. This study demonstrates that low birthweight can have far-reaching effects that extend into the next generation.

Nephron number (N(glom)) and size (V(glom)) are correlated with risk for chronic cardiovascular and kidney disease and may be predictive of renal allograft viability. Unfortunately, there are no techniques to assess N(glom) and V(glom) in intact kidneys. This work demonstrates the use of cationized ferritin (CF) as a magnetic resonance imaging (MRI) contrast agent to measure N(glom) and V(glom) in viable human kidneys donated to science. The kidneys were obtained from patients with varying levels of cardiovascular and renal disease. CF was intravenously injected into three viable human kidneys. A fourth control kidney was perfused with saline. After fixation, immunofluorescence and electron microscopy confirmed binding of CF to the glomerulus. The intact kidneys were imaged with three-dimensional MRI and CF-labeled glomeruli appeared as punctate spots. Custom software identified, counted, and measured the apparent volumes of CF-labeled glomeruli, with an ~6% false positive rate. These measurements were comparable to stereological estimates. The MRI-based technique yielded a novel whole kidney distribution of glomerular volumes. Histopathology demonstrated that the distribution of CF-labeled glomeruli may be predictive of glomerular and vascular disease. Variations in CF distribution were quantified using image texture analyses, which be a useful marker of glomerular sclerosis. This is the first report of direct measurement of glomerular number and volume in intact human kidneys., (Copyright © 2014 the American Physiological Society.)

Adverse conditions in utero can have transgenerational effects, in the absence of a subsequent insult. We aimed to investigate the contribution of the maternal pregnancy environment vs. germ line effects in mediating alterations to cardiorenal and metabolic physiology in offspring from mothers born small. Uteroplacental insufficiency was induced by bilateral uterine artery and vein ligation (Restricted group) or sham surgery (Control group) in Wistar-Kyoto rats. Restricted and control female offspring (F1) were mated with either breeder males (embryo donor) or vasectomized males (embryo recipient). Embryo transfer was performed at embryonic day (E) 1, whereby second-generation (F2) embryos gestated (donor-in-recipient) in either a control (Cont-in-Cont, Rest-in-Cont) or restricted (Cont-in-Rest, Rest-in-Rest) mother. In male and female offspring, glomerular number and size were measured at postnatal day (PN) 35, and systolic blood pressure, glucose control, insulin sensitivity, and pancreatic β-cell mass were measured in separate sibling cohorts at 6 mo. Rest-in-Rest offspring were hypothesized to have similar characteristics (reduced growth, altered metabolic control, and hypertension) to non-embryo-transferred Rest, such that embryo transfer would not be a confounding experimental influence. However, embryo-transferred Rest-in-Rest offspring underwent accelerated growth during the peripubertal phase, followed by slowed growth between 2 and 3 mo of age compared with non-embryo-transferred Rest groups. Furthermore, renal function and insulin response to a glucose load were different to respective non-embryo-transferred groups. Our data demonstrate the long-term effects of in vitro embryo manipulation, which confounded the utility of this approach in delineating between the maternal pregnancy environment and germ line effects that drive transgenerational outcomes.

The total number of glomeruli (nephrons) in a kidney is an important microanatomical parameter for at least three reasons: it provides an index of the success/extent of nephrogenesis and can thereby provide insights into the roles of specific genes and feto-maternal environmental factors in nephrogenesis; low nephron number has been linked to an increased risk of cardiovascular and renal disease in adulthood; and knowledge of quantitative kidney microanatomy can illuminate our understanding of physiological mechanisms in health and disease. A range of methods has been used to count glomeruli in kidneys over the past 100 years, with design-based stereology (the physical disector/fractionator combination) considered the gold standard. However, this approach is labor-intensive and expensive, and therefore is not utilized by most laboratories. A new method for counting and sizing every glomerulus in the kidney has recently been described. This method involves in vivo labeling of glomeruli with cationic ferritin, and then magnetic resonance imaging (MRI) of the ex vivo kidney. Values are obtained in one sixth of the time of disector-based approaches. This new MRI method holds great promise for studies of glomerular number and size ex vivo and in vivo.

The structure of the ureteric tree in developing mouse and rat kidneys has previously been quantified in two dimensions. While this type of analysis may provide evidence of changes in ureteric growth, these measurements are effectively inaccurate, as the ureteric tree is a three-dimensional (3D) object. Here we describe a method for measuring the ureteric tree in three dimensions. This technique involves (1) culture of the metanephric kidney at embryonic day 12 (mouse) or 14 (rat), (2) whole-mount immunofluorescence to selectively stain ureteric tree epithelium, (3) confocal microscopy to obtain a complete Z series through the ureteric tree, and (4) image analysis algorithms to binarize, skeletonize, and measure individual branch lengths in 3D. This method has been extended to analysis of the same ureteric tree over time (4D). The results obtained provide accurate and precise quantitation of ureteric tree growth in the developing mouse or rat kidney.

There is strong evidence from human and animal models that exposure to maternal hyperglycemia during in utero development can detrimentally affect fetal kidney development. Notwithstanding this knowledge, the precise effects of diabetic pregnancy on the key processes of kidney development are unclear due to a paucity of studies and limitations in previously used methodologies. The purpose of the present study was to elucidate the effects of hyperglycemia on ureteric branching morphogenesis and nephrogenesis using unbiased techniques. Diabetes was induced in pregnant C57Bl/6J mice using multiple doses of streptozotocin (STZ) on embryonic days (E) 6.5-8.5. Branching morphogenesis was quantified ex vivo using Optical Projection Tomography, and nephrons were counted using unbiased stereology. Maternal hyperglycemia was recognised from E12.5. At E14.5, offspring of diabetic mice demonstrated fetal growth restriction and a marked deficit in ureteric tip number (control 283.7 ± 23.3 vs. STZ 153.2 ± 24.6, mean ± SEM, p<0.01) and ureteric tree length (control 33.1 ± 2.6 mm vs. STZ 17.6 ± 2.7 mm, p = 0.001) vs. controls. At E18.5, fetal growth restriction was still present in offspring of STZ dams and a deficit in nephron endowment was observed (control 1246.2 ± 64.9 vs. STZ 822.4 ± 74.0, p<0.001). Kidney malformations in the form of duplex ureter and hydroureter were a common observation (26%) in embryos of diabetic pregnancy compared with controls (0%). Maternal insulin treatment from E13.5 normalised maternal glycaemia but did not normalise fetal weight nor prevent the nephron deficit. The detrimental effect of hyperglycemia on ureteric branching morphogenesis and, in turn, nephron endowment in the growth-restricted fetus highlights the importance of glycemic control in early gestation and during the initial stages of renal development.

1. Prenatal alcohol exposure impairs kidney development, resulting in a reduced nephron number. However, the mechanism through which alcohol acts to disrupt renal development is largely unknown. Retinoic acid (RA) is critically involved in kidney development and it has been proposed that a diminished concentration of RA is a contributing factor to fetal alcohol syndrome. 2. In the present study we proposed that the ethanol-induced inhibition of ureteric branching morphogenesis and glomerular development in the cultured rat kidney would be ameliorated by coculture with exogenous RA and that examining the expression profile of key genes involved in the development of the kidney would provide insights into the potential molecular pathways involved. 3. Whole rat metanephroi cultured in the presence of exogenous RA (10-20 nmol/L) without ethanol appeared larger and had significantly more ureteric branch points, tips and glomeruli than metanephroi cultured in control media. Those cultured in the presence of ethanol alone (0.2%) had 20% fewer ureteric branch points, tips and glomeruli, which was ameliorated by coculture with retinoic acid. 4. Gene expression analysis identified changes in the expression of enzymes involved in the metabolism of alcohol in conjunction with changes in key regulators of kidney development, including cRET. 5. These results demonstrate that the teratogenic effects of alcohol in vitro on kidney development resulting in reduced ureteric branching morphogenesis and glomerular development can be ameliorated through coculture with RA. These results provide the foundation for future research into the mechanism through which alcohol acts to disrupt kidney development., (Clinical and Experimental Pharmacology and Physiology © 2012 Blackwell Publishing Asia Pty Ltd.)

Globally 30% of adults are overweight or obese. The white adipocyte is a major component of adipose tissue, and as the obesity epidemic increases it is critically important to understand the factors determining adipocyte development and function. Adipogenesis has two distinct phases; determination of the adipocyte from a multipotent stem cell, and terminal differentiation of a pre-adipocyte into a mature adipocyte. The environment encountered in early life can alter adipocyte number and size and potentially impact upon adipocyte endocrine function in adulthood. These alterations may contribute to the pathophysiology of chronic diseases and thus targeted therapy of the adipocyte has great potential for treating the current obesity epidemic., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Intrauterine growth restriction caused by uteroplacental insufficiency increases risk of cardiovascular and metabolic disease in offspring. Cardio-renal and metabolic responses to pregnancy are critical determinants of immediate and long-term maternal health. However, no studies to date have investigated the renal and metabolic adaptations in growth restricted offspring when they in turn become pregnant. We hypothesised that the physiological challenge of pregnancy in growth restricted females exacerbates disease outcome and compromises next generation fetal growth. Uteroplacental insufficiency was induced by bilateral uterine vessel ligation (Restricted) or sham surgery (Control) on day 18 of gestation in WKY rats and F1 female offspring birth and postnatal body weights were recorded. F1 Control and Restricted females were mated at 4 months and blood pressure, renal and metabolic parameters were measured in late pregnancy and F2 fetal and placental weights recorded. Age-matched non-pregnant Control and Restricted F1 females were also studied. F1 Restricted females were born 10-15% lighter than Controls. Basal insulin secretion and pancreatic β-cell mass were reduced in non-pregnant Restricted females but restored in pregnancy. Pregnant Restricted females, however, showed impaired glucose tolerance and compensatory glomerular hypertrophy, with a nephron deficit but normal renal function and blood pressure. F2 fetuses from Restricted mothers exposed to physiological measures during pregnancy were lighter than Controls highlighting additive adverse effects when mothers born small experience stress during pregnancy. Female rats born small exhibit mostly normal cardio-renal adaptations but altered glucose control during late pregnancy making them vulnerable to lifestyle challenges.

Nephron number has emerged as a useful parameter for assessing the roles of specific genes and feto-maternal environmental factors in kidney development. Nephron number is also of clinical interest due to increasing evidence suggesting that low nephron number is associated with increased risk for developing chronic adult disease, including cardiovascular and renal disease. The physical disector/fractionator combination is considered the gold standard method for estimating total nephron number in kidneys. Here we describe the use of this method to estimate total nephron number in mouse and rat kidneys, and variations to the method required to estimate nephron number in larger species, including human.

Design-based stereology is considered the gold-standard method for estimating the total number of glomeruli, and thereby nephrons, in the adult kidney. However, until recently, a design-based method for estimating nephron number in the developing kidney was not available. For such a method to provide accurate and precise estimates, unambiguous identification of developing nephrons is essential. Here, we describe a combined histochemical/stereological technique for estimating total nephron number in the developing mouse and rat kidney. The method can be modified for use in other species.

Low glomerular (nephron) endowment has been associated with an increased risk of cardiovascular and renal disease in adulthood. Nephron endowment in humans is determined by 36 wk of gestation, while in rats and mice nephrogenesis ends several days after birth. Specific genes and environmental perturbations have been shown to regulate nephron endowment. Until now, design-based method for estimating nephron number in developing kidneys was unavailable. This was due in part to the difficulty associated with unambiguously identifying developing glomeruli in histological sections. Here, we describe a method that uses lectin histochemistry to identify developing glomeruli and the physical disector/fractionator principle to provide unbiased estimates of total glomerular number (N(glom)). We have characterized N(glom) throughout development in kidneys from 76 rats and model this development with a 5-parameter logistic equation to predict N(glom) from embryonic day 17.25 to adulthood (r(2) = 0.98). This approach represents the first design-based method with which to estimate N(glom) in the developing kidney.

We examined whether deficits in glomerular capillary surface area associated with a congenital nephron deficit could be corrected by glomerular hypertrophy. Using unbiased stereological techniques, we examined the time course and mode of glomerular hypertrophy in mice lacking one allele for glial cell line-derived neurotrophic factor (GDNF). These GDNF heterozygous (Het) mice are born with approximately 30% less nephrons than wild-type (WT) littermates. An additional group of GDNF Het mice received the angiotensin type 1 (AT1)-receptor antagonist candesartan (Cand; 10 mg x kg(-1) x day(-1)) from 5 wk of age to determine the role of AT1 receptors in the compensatory hypertrophy. At 10 wk of age, the total volume of renal corpuscles, glomerular capillary surface area, and length of glomerular capillaries in the kidneys of GDNF Het mice were all markedly (approximately 45%) less than that of WT mice (P < 0.001). However, by 30 wk, and persisting at 60 wk of age, GDNF Het and WT mice did not significantly differ in any of these parameters. Furthermore, conscious 24-h mean arterial pressure (MAP) did not differ between GDNF Het and WT mice at any time point. MAP of GDNF Het-Cand mice was 20-30 mmHg less than that of GDNF Het-vehicle mice at all three ages, but Cand treatment did not significantly alter glomerular capillary dimensions. In conclusion, we have demonstrated that the deficit in glomerular capillary surface area associated with a congenital nephron deficit can be corrected for in adulthood by an increase in the total length of glomerular capillaries. This process does not require AT1 receptor activation.

Maternal administration of dexamethasone (DEX) for 48 h early in rat kidney development results in offspring with a reduced nephron endowment. However, the mechanism through which DEX inhibits nephrogenesis is unknown. In this study, we hypothesized that DEX may indirectly inhibit nephrogenesis by inhibiting ureteric branching morphogenesis. Whole metanephroi from embryonic day 14.5 (E14.5) rat embryos were cultured in the presence of DEX. DEX (10(-5) M) exposure for 2 days significantly inhibited ureteric branching compared with metanephroi grown in control media or DEX (10(-7) M). Culturing metanephroi for a further 3 days (in control media only) reduced total glomerular number in metanephroi previously exposed to DEX (10(-5) M) or (10(-7) M) compared with control cultures. Expression of genes known to regulate ureteric branching morphogenesis was determined by real-time PCR in metanephroi after 2 days in culture. DEX exposure in vitro decreased expression of glial cell line-derived neurotrophic factor (GDNF) and increased expression of bone morphogenetic protein-4 (BMP-4) and transforming growth factor-beta1 (TGF-beta1). Similar gene expression changes were found in E16.5 metanephroi in which the dam had been exposed to 2 days of DEX (0.2 mg.kg(-1).day(-1)) at E14.5/15.5 in vivo. However, in kidneys collected at E20.5 after in vivo exposure for 2 days, GDNF expression was increased and BMP-4 and TGF-beta1 expression decreased suggesting a biphasic response in gene expression to DEX exposure. These results show for the first time that inhibition of ureteric branching morphogenesis may be a key mechanism through which DEX exposure results in a reduced nephron endowment.

Maternal treatment with the synthetic glucocorticoid, dexamethasone has been reported to result in a nephron deficit and development of hypertension in the offspring of rats. However, it is not known whether elevated maternal corticosterone (CORT), the natural glucocorticoid, has similar effects on blood pressure and nephron endowment. The present study investigated the effects of CORT (0.8 mg kg(-1) day(-1)) administration on embryonic day 14 (E14) and E15 of pregnancy on: (1) nephron number at postnatal day 30 (PN30); (2) blood pressure at PN120; and (3) receptors of the renal renin-angiotensin system (RRAS) (AT(1)Ra, AT(1)Rb and AT(2)Ra) during both embryonic (E16, E20) and adolescent (PN30) life. Plasma CORT concentrations were approximately doubled 30 min after injection. Unbiased stereological analysis revealed that maternal CORT treatment resulted in a nephron deficit of 21 and 19% in male and female offspring, respectively. Mean arterial pressures were significantly elevated in offspring of both sexes from the CORT group. Real-time PCR revealed that CORT treatment increased expression of AT(1)Ra and AT(2)R at E16, and at PN30. Expression of AT(1)Rb was downregulated in embryonic life but upregulated at PN30. We believe that these results are the first to demonstrate that maternal CORT treatment results in a nephron deficit and development of hypertension in the rat offspring. Changes in the RRAS may be contributing to these phenotypes. Critically, this study suggests that increased but physiological levels of the natural glucocorticoid can programme similar changes to those seen with pharmacological doses of the synthetic glucocorticoid. This may have important implications for women experiencing significant stress during pregnancy.

The effects of prenatal protein restriction on adult renal and cardiovascular function have been studied in considerable detail. However, little is known about the effects of life-long protein restriction, a common condition in the developing world. Therefore, we determined in rats the effects of combined pre- and postnatal protein restriction on adult arterial pressure and renal function and responses to increased dietary sodium. Nephron number was also determined. Male Sprague-Dawley rats were born to mothers fed a low [8% (wt/wt), LP] or normal [20% (wt/wt), NP] isocaloric protein diet throughout pregnancy and maintained on these diets after birth. At postnatal day 135, nephron number, mean arterial pressure (MAP), and renal function were determined. A high-NaCl [8.0% (wt/wt), high-salt] diet was fed to a subset of rats from weaning. MAP was less in LP than in NP rats (120 +/- 2 vs. 128 +/- 2 mmHg, P < 0.05) and was not significantly altered by increased salt intake. Nephron number was 31% less in LP than in NP rats (P < 0.001). The volume of individual glomeruli was also less in LP than in NP rats, as were calculated effective renal plasma flow and glomerular filtration rate. Glomerular filtration rate, but not effective renal plasma flow, appeared to be increased by high salt intake, particularly in LP rats. In conclusion, protein restriction induced a severe nephron deficit, but MAP was lower, rather than higher, in protein-restricted than in control rats in adulthood. These findings indicate that the postnatal environment plays a key role in determining the outcomes of developmental programming.

In recent years, a great deal has been learnt about the molecular regulation of kidney development. While most research has focused on the molecular regulation of ureteric branching morphogenesis and nephron formation, significant insights into the definition and functions of the renal stroma have emerged. Many molecules expressed in the developing renal stroma are now known to play significant regulatory roles in kidney development. However, the term 'renal stroma' continues to have different meanings to different researchers. This review clarifies this situation and defines the derivation, location and functions of the stroma in the developing metanephros., (Copyright (c) 2005 S. Karger AG, Basel.)

The loss of one allele for glial cell line-derived neurotrophic factor (GDNF) results in approximately 30% fewer but normal sized glomeruli in young mice. Low nephron number, inherited or acquired, has been linked to increased risk of development of hypertension and renal failure. This study examines whether GDNF heterozygous mice, with an inherent reduction in nephron number, demonstrate a deterioration in renal structure and function and rise in arterial pressure in later life. Fourteen-month-old male GDNF heterozygous (n=7) and wild-type (n=6) mice were anesthetized and prepared for measurement of mean arterial pressure, glomerular filtration rate (GFR), and renal blood flow. After measurement of renal function, kidneys were fixed for stereological determination of total glomerular number and mean glomerular volume. Mean arterial pressure was, on average, 18 mm Hg higher in GDNF heterozygous (98+/-4 mm Hg) than wild-type mice (80+/-2 mm Hg; P<0.01). However, GFR (0.656+/-0.054 versus 0.688+/-0.076 mL/min per g kidney wt) and renal blood flow (5.29+/-0.42 versus 4.70+/-0.34 mL/min per g kidney wt) were not different between groups. Fourteen-month-old GDNF heterozygous mice had approximately 30% fewer glomeruli than wild-type mice (9206+/-934 versus 13440+/-1275; P<0.01) and significantly larger glomeruli (4.51+/-0.39 versus 3.72+/-0.63x10(-4)mm(3); P<0.01). Thus, aged GDNF heterozygous mice maintained a normal GFR and renal blood flow despite reduced nephron numbers. The elevated arterial pressure, glomerular hypertrophy, and hyperfiltration demonstrated in the GDNF heterozygous mice at this age may indicate a compensatory mechanism whereby GFR is maintained in the presence of a reduced nephron endowment.

The growth and branching of the epithelial ureteric tree is critical for development of the permanent kidney (metanephros). Current methods of analysis of ureteric branching are mostly qualitative. We have developed a method for measuring the length of individual branches, and thereby the total length of the ureteric tree in 3 dimensions (3D). The method involves confocal microscopy of whole-mount immunostained metanephroi and computer-based image segmentation, skeletonisation and measurement. The algorithm performs semi-automatic segmentation of a set of confocal images and skeletonisation of the resulting binary object. Length measurements and number of branch points are automatically obtained. The final representation can be reconstructed providing a fully rotating 3D perspective of the skeletonised tree. After 36 h culture of E12 mouse metanephroi, the total length of the ureteric tree was 6103 +/- 291 microm (mean +/- SD), a four-fold increase compared with metanephroi cultured for just 6 h (1522 +/- 149 microm). Ureteric duct length increased at a rate of 153 microm/h over the first 30 h period and was maximal between 18 and 24 h at 325 microm/h. The distribution of branch lengths at the six time points studied was similar, suggesting tight control of ureteric lengthening and branching. This method will be of use in analysing ureteric growth in kidneys cultured in the presence of specific molecules suspected of regulating ureteric growth. The method can also be used to analyse in vivo kidneys and to quantify branching morphogenesis in other developing organs.

Background: The exact molecular mechanisms that regulate ureteric branching morphogenesis in the developing metanephros have not been fully elucidated. However, in vivo and in vitro evidence indicates that glial cell line-derived neurotrophic factor (GDNF) is a key regulator of the initiation of ureteric branching. GDNF knockout mice show renal agenesis or severe dysgenesis and die 24 hours after birth from renal failure. Inhibition of GDNF activity in metanephric organ culture inhibits ureteric branching. Since nephron initiation only occurs at the tips of ureteric branches, the aim of the present study was to determine whether nephron number in GDNF heterozygous mice is reduced., Methods: Male GDNF heterozygous mice of hybrid 129/Sv and C57/BL genetic background were mated with C57BL/6 females. Offspring were genotyped at postnatal day 30 (PN30) by polymerase chain reaction. Left kidneys were used for estimating kidney volume and total nephron number. We also estimated absolute and relative volumes of ureteric duct epithelium. Unbiased stereological methods were used throughout (Cavalieri method, physical disector/fractionator combination)., Results: GDNF wild-type and heterozygous mice had similar body weights at PN30. However, heterozygous kidneys were 25% smaller than wild-type kidneys (wild-type, 114.75 +/- 16.46 mm3; heterozygous, 87.11 +/- 21.84 mm3, P < 0.001) and contained approximately 30% fewer nephrons (wild-type, 11886 +/- 1277; heterozygous, 8573 +/- 2240, P < 0.01). In addition, the absolute ureteric duct volume was significantly reduced in heterozygous mice (P < 0.001)., Conclusions: : These results indicate that the loss of one GDNF allele results in reduced nephron endowment in the adult kidney, presumably as the result of reduced branching morphogenesis of the ureteric bud.