Podocyte Number in Children and Adults: Associations with Glomerular Size and Numbers of Other Glomerular Resident Cells.

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.

Glomerular hypertrophy in subjects with low nephron number: contributions of sex, body size and race.

BACKGROUND: We have shown that low nephron number (Nglom) is a strong determinant of individual glomerular volume (IGV) in male Americans. However, whether the same pattern is present in female Americans remains unclear. The contributions of body surface area (BSA) and race to IGV in the context of Nglom also require further evaluation. METHODS: Kidneys without overt renal disease were collected at autopsy in Mississippi, USA. The extremes of female Nglom were used to define high and low Nglom for both sexes. Nglom and IGV were estimated by design-based stereology. A total of 24 African and Caucasian American females (n = 12 per race; 6 per Nglom extreme) were included. These subjects were subsequently matched to 24 comparable males by age and Nglom and to 18 additional males by age, Nglom and BSA. RESULTS: IGV average and variance were very similar in female African and Caucasian Americans with high and low Nglom. Males with low Nglom from both races showed greater IGV average and variance than comparable females matched by age and Nglom. These differences in IGV between sexes were not observed in Caucasian Americans with low Nglom that were matched by age, Nglom and BSA. In contrast, glomeruli from African Americans were larger than those from Caucasian Americans, especially in subjects with high Nglom. CONCLUSIONS: While female Americans with low Nglom did not show glomerular hypertrophy, comparable males with low Nglom showed marked glomerular hypertrophy that was closely associated with high BSA. Glomerular size in African Americans may be confounded by multiple additional factors.

Hypertension, glomerular hypertrophy and nephrosclerosis: the effect of race.

BACKGROUND: African Americans have more severe hypertensive nephrosclerosis than white Americans, possibly at similar levels of blood pressure. Glomerular volume is increased in African Americans relative to whites, but it is uncertain how this relates to nephrosclerosis and whether it contributes to or compensates for glomerulosclerosis. METHODS: Stereological disector/fractionator estimates of glomerular number (N(glom)) and average glomerular volume (V(glom)) were obtained on autopsy kidneys of 171 African Americans and 131 whites. Eighty-eight African Americans and 49 whites were identified as hypertensive. Nephrosclerosis was measured morphometrically as the percentage of glomerulosclerosis, proportion of cortical fibrosis and interlobular artery intimal thickness, and analyzed with V(glom) by age, race, gender, body mass index (BMI) and blood pressure. RESULTS: African Americans were more frequently hypertensive (58.5%) than whites (35.8%) and when hypertensive had higher levels of blood pressure (P = 0.02). N(glom) was significantly lower in hypertensive compared with non-hypertensive subjects among white women (P = 0.02) but not white males (P = 0.34) or African American females (P = 0.10) or males (P = 0.41). For each race and gender, glomerulosclerosis, cortical fibrosis and arterial intimal thickening were statistically correlated with age (P < 0.001) and hypertension (P < 0.001) and increased V(glom) with hypertension (P < 0.001) and BMI (P < 0.001). In multivariate analysis, African American race was associated with increased V(glom) (P = 0.01) and arterial intimal thickening (P < 0.01), while interactions between race and blood pressure indicated that the severity of nephrosclerosis including increased V(glom) was linked most directly to hypertension without significant contributions from race. The hypertension-associated enlargement of V(glom) was present with mild degrees of glomerulosclerosis and changed little as the severity of glomerulosclerosis increased. CONCLUSIONS: Glomerular hypertrophy was identified as an integral feature of hypertensive nephropathy and appeared to precede rather than compensate for glomerulosclerosis. An effect of race on V(glom) and arterial intimal thickening seemed to be related to the more frequent and more severe hypertension among African Americans.

Design-based stereological methods for estimating numbers of glomerular podocytes.

The podocyte depletion hypothesis has emerged as a unifying concept in glomerular pathology. According to this hypothesis podocyte depletion may be absolute (decrease in number of healthy mature podocytes), relative (fewer podocytes per unit of glomerular volume) or involve alterations to the specialized podocyte architecture (such as foot process effacement). To study and understand podocyte depletion it is important to be able to accurately and precisely count these cells. Here we present new design-based stereological methods for estimating podocyte number in individual glomeruli of known volume, and in average glomeruli. Both methods involve serial histological sectioning, triple label immunohistochemistry, laser confocal microscopy and cell counting with the optical disector/fractionator.

Discovery and evolution of phenoxypiperidine hydroxyamide dual CCR3/H1 antagonists. Part I.

The discovery of potent small molecule dual antagonists of the human CCR3 and H(1) receptors is described for the treatment of allergic diseases, for example, asthma and allergic rhinitis. Optimizing in vitro potency and metabolic stability, starting from a CCR1 lead compound, led to compound 20 with potent dual CCR3/H(1) activity and in vitro metabolic stability.

Discovery and evolution of phenoxypiperidine hydroxyamide dual CCR3/H1 antagonists. Part II: optimising in vivo clearance.

The second part of this communication focuses on the resolution of issues surrounding the series of hydroxyamide phenoxypiperidine CCR3/H(1) dual antagonists described in Part I. This involved further structural exploration directed at reducing metabolism and leading to the identification of compound 60 with a greatly improved in vivo pharmacokinetic profile.

Scaffold-hopping with zwitterionic CCR3 antagonists: identification and optimisation of a series with good potency and pharmacokinetics leading to the discovery of AZ12436092.

The discovery and optimisation of a series of zwitterionic CCR3 antagonists is described. Optimisation of the structure led to AZ12436092, a compound with excellent selectivity over activity at hERG and outstanding pharmacokinetics in preclinical species.

Renal biopsy findings among Indigenous Australians: a nationwide review.

Australia's Indigenous people have high rates of chronic kidney disease and kidney failure. To define renal disease among these people, we reviewed 643 renal biopsies on Indigenous people across Australia, and compared them with 249 biopsies of non-Indigenous patients. The intent was to reach a consensus on pathological findings and terminology, quantify glomerular size, and establish and compare regional biopsy profiles. The relative population-adjusted biopsy frequencies were 16.9, 6.6, and 1, respectively, for Aboriginal people living remotely/very remotely, for Torres Strait Islander people, and for non-remote-living Aboriginal people. Indigenous people more often had heavy proteinuria and renal failure at biopsy. No single condition defined the Indigenous biopsies and, where biopsy rates were high, all common conditions were in absolute excess. Indigenous people were more often diabetic than non-Indigenous people, but diabetic changes were still present in fewer than half their biopsies. Their biopsies also had higher rates of segmental sclerosis, post-infectious glomerulonephritis, and mixed morphologies. Among the great excess of biopsies in remote/very remote Aborigines, females predominated, with younger age at biopsy and larger mean glomerular volumes. Glomerulomegaly characterized biopsies with mesangiopathic changes only, with IgA deposition, or with diabetic change, and with focal segmental glomerulosclerosis (FSGS). This review reveals great variations in biopsy rates and findings among Indigenous Australians, and findings refute the prevailing dogma that most indigenous renal disease is due to diabetes. Glomerulomegaly in remote/very remote Aboriginal people is probably due to nephron deficiency, in part related to low birth weight, and probably contributes to the increased susceptibility to kidney disease and the predisposition to FSGS.

Estimating total nephron number in the adult kidney using the physical disector/fractionator combination.

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.

Estimating individual glomerular volume in the human kidney: clinical perspectives.

BACKGROUND: Measurement of individual glomerular volumes (IGV) has allowed the identification of drivers of glomerular hypertrophy in subjects without overt renal pathology. This study aims to highlight the relevance of IGV measurements with possible clinical implications and determine how many profiles must be measured in order to achieve stable size distribution estimates. METHODS: We re-analysed 2250 IGV estimates obtained using the disector/Cavalieri method in 41 African and 34 Caucasian Americans. Pooled IGV analysis of mean and variance was conducted. Monte-Carlo (Jackknife) simulations determined the effect of the number of sampled glomeruli on mean IGV. Lin's concordance coefficient (R(C)), coefficient of variation (CV) and coefficient of error (CE) measured reliability. RESULTS: IGV mean and variance increased with overweight and hypertensive status. Superficial glomeruli were significantly smaller than juxtamedullary glomeruli in all subjects (P < 0.01), by race (P < 0.05) and in obese individuals (P < 0.01). Subjects with multiple chronic kidney disease (CKD) comorbidities showed significant increases in IGV mean and variability. Overall, mean IGV was particularly reliable with nine or more sampled glomeruli (R(C) > 0.95, <5% difference in CV and CE). These observations were not affected by a reduced sample size and did not disrupt the inverse linear correlation between mean IGV and estimated total glomerular number. CONCLUSIONS: Multiple comorbidities for CKD are associated with increased IGV mean and variance within subjects, including overweight, obesity and hypertension. Zonal selection and the number of sampled glomeruli do not represent drawbacks for future longitudinal biopsy-based studies of glomerular size and distribution.

Distribution of volumes of individual glomeruli in kidneys at autopsy: association with physical and clinical characteristics and with ethnic group.

We have demonstrated considerable variability in the volumes of different glomeruli in given individuals (individual glomerular volume: IGV) in a stereologic study of kidneys at forensic autopsy performed to investigate sudden or unexpected death in people without manifest kidney disease. We review some important associations of IGV by subject characteristics and by ethnic groups. IGVs were measured by the Cavalieri method in 30 glomeruli in each of 111 adult males who belonged to 4 ethnic groups, i.e. US Whites, African-Americans, Africans from Senegal, and Australian Aborigines. Correlations of pooled IGV values with certain subject characteristics were evaluated in the US Whites. Pooled IGV data were compared in subjects across the 4 ethnic groups. In US Whites, mean IGV and its variance were greater with higher age, lower nephron number, lower birth weight, and with gross obesity, hypertension and cardiovascular death. In comparisons by ethnic group, mean IGV and IGV ranges were higher in African-Americans and Australian Aborigines than in US Whites and African Senegalese subjects. We conclude that glomerular enlargement with volume heterogeneity marks more advanced age, relative nephron deficiency, lower birth weight, obesity, hypertension, and advanced cardiovascular disease. The findings in African-Americans and Australian Aborigines suggest that larger IGVs and volume heterogeneity might mark populations with accentuated susceptibility to hypertension and kidney disease, but the data need to be further examined in the context of the determining characteristics defined in the US Whites.

Human nephron number: implications for health and disease.

Several studies have shown that total nephron (glomerular) number varies widely in normal human kidneys. Whereas the studies agree that average nephron number is approximately 900,000 to 1 million per kidney, numbers for individual kidneys range from approximately 200,000 to >2.5 million. Several studies have shown loss of glomeruli due to age-related glomerulosclerosis. The rates of loss vary among individuals depending upon blood pressure, diseases affecting the kidney, and other attributes of health, but most of the variation in nephron number is present at birth and is therefore developmentally determined. For example, in a relatively small study of nephron number in 15 children <3 months of age, we found that nephron number ranged from approximately 250,000 to 1.1 million. Given that no new nephrons are formed in human kidneys after approximately 36 weeks' gestation, much interest has focused on renal function and health in individuals born with relatively low nephron endowment. Several studies have reported a direct correlation between birth weight and nephron number and an indirect association between nephron number and blood pressure. Associations between low birth weight and cardiovascular disease, including hypertension, have also been widely reported. This report provides an update on our current knowledge of human nephron number and the associations with adult health and disease.

Prediction of human renal clearance from preclinical species for a diverse set of drugs that exhibit both active secretion and net reabsorption.

Identifying any extrahepatic excretion phenomenon in preclinical species is crucial for an accurate prediction of the pharmacokinetics in man. This understanding is particularly key for drugs with a small volume of distribution, because they require an especially low total clearance to be suitable for a once-a-day dosing regimen in man. In this study, three animal scaling techniques were applied for the prediction of the human renal clearance of 36 diverse drugs that show active secretion or net reabsorption: 1) direct correlations between renal clearance in man and each of the two main preclinical species (rat and dog); 2) simple allometry; and 3) Mahmood's renal clearance scaling method. The results show clearly that the predictions to man for the methods are improved significantly when corrections are made for species differences in plasma protein binding. Overall, the most accurate predictions were obtained by using a direct correlation with the dog renal clearance after correcting for differences in plasma protein binding and kidney blood flow (r² = 0.84), where predictions, on average, were within 2-fold of the observed renal clearance values in human.

Towards a definition of glomerulomegaly: clinical-pathological and methodological considerations.

BACKGROUND: Glomerulomegaly, the abnormal enlargement of glomeruli, has been related to an increased risk of glomerulosclerosis, but the degree of enlargement that constitutes glomerulomegaly has not been defined. METHODS: The principal stereological methods for estimating glomerular volume are [1] the disector/Cavalieri method that is considered the 'gold standard' for measuring individual glomerular volume (IV(glom)) and [2] the disector/fractionator technique that estimates average glomerular volume (V(glom)) together with total glomerular number (N(glom)) for the entire kidney. The two methods produce different estimates with V(glom) consistently exceeding IV(glom). This study compares glomerular volumes obtained by the two methods in autopsy kidneys of 39 African American and 34 US white adult males, and correlates the values with N(glom), body mass index (BMI), hypertension, glomerulosclerosis and race, factors known or thought to influence glomerular volume. RESULTS: For the smallest glomeruli, V(glom) was 25% larger than IV(glom) with the difference increasing to over 50% for kidneys with the largest glomeruli. Both V(glom) and IV(glom) showed significant inverse correlations with N(glom) and significant direct correlations with BMI and hypertension. African Americans had larger IV(glom) and V(glom) than whites, but only IV(glom) was significant. The 90th percentile for IV(glom) was 6.81 µm(3) × 10(6) and 13.10 µm(3) × 10(6) for V(glom), but larger glomerular size did not separate hypertensive from non-hypertensive subjects nor did it show any significant relationship to glomerulosclerosis. While V(glom) overestimated glomerular size compared with IV(glom), both measurements demonstrated similar relationships to factors influencing glomerular volume. CONCLUSIONS: With neither method could glomerulomegaly, the abnormal enlargement of glomerular size predisposing to glomerulosclerosis, be determined.

Glomerular number and size variability and risk for kidney disease.

PURPOSE OF REVIEW: This review discusses current understandings of variability in glomerular number and size, and the implications for renal health. RECENT FINDINGS: The quantitative microanatomy of the normal human kidney varies widely. Of greatest significance, total nephron number varies at least 13-fold, and several genes and environmental factors that regulate human nephron endowment have been identified. Full or partial deletion of more than 25 genes in mice has been shown to result in renal hypoplasia and, when measured, reduced nephron endowment. Many more will likely be identified. As would be expected, some gene abnormalities increase nephron endowment above that found in control mice. Glomerular volume also varies widely, both between and within kidneys, and increased heterogeneity of glomerular volume within kidneys is associated with risk factors for kidney disease, including birth weight, age, race, body size and hypertension. SUMMARY: Data from several human populations indicate that the quantitative microanatomy of the human kidney varies considerably: total glomerular number varies at least 13-fold, mean glomerular volume varies up to seven-fold and the volumes of individual glomeruli within single kidneys can vary as much as eight-fold. Human glomerular number, size and size distribution are being found to correlate with risk factors for kidney disease. The genetic and fetal environmental regulators of nephrogenesis, and thereby nephron endowment, are being rapidly identified and will provide the bases for future clinical interventions. In contrast, the molecular regulation of glomerular size remains unclear.

A rapid computational filter for predicting the rate of human renal clearance.

In silico models that predict the rate of human renal clearance for a diverse set of drugs, that exhibit both active secretion and net re-absorption, have been produced using three statistical approaches. Partial Least Squares (PLS) and Random Forests (RF) have been used to produce continuous models whereas Classification And Regression Trees (CART) has only been used for a classification model. The best models generated from either PLS or RF produce significant models that can predict acids/zwitterions, bases and neutrals with approximate average fold errors of 3, 3 and 4, respectively, for an independent test set that covers oral drug-like property space. These models contain additional information on top of any influence arising from plasma protein binding on the rate of renal clearance. Classification And Regression Trees (CART) has been used to generate a classification tree leading to a simple set of Renal Clearance Rules (RCR) that can be applied to man. The rules are influenced by lipophilicity and ion class and can correctly predict 60% of an independent test set. These percentages increase to 71% and 79% for drugs with renal clearances of < 0.1 ml/min/kg and > 1 ml/min/kg, respectively. As far as the authors are aware these are the first set of models to appear in the literature that predict the rate of human renal clearance and can be used to manipulate molecular properties leading to new drugs that are less likely to fail due to renal clearance.

CKD in Aboriginal Australians.

Chronic kidney disease (CKD) is one component of a spectrum of chronic disease in Aboriginal Australians. CKD is marked by albuminuria, which predicts renal failure and nonrenal natural death. Rates vary greatly by community and region and are much higher in remote areas. This reflects the heterogeneous characteristics and circumstances of Aboriginal people. CKD is multideterminant, and early-life influences (notably low birth weight), infections (including poststreptococcal glomerulonephritis), metabolic/hemodynamic parameters, and epigenetic/genetic factors probably contribute. CKD is associated intimately with cardiovascular risk. Albuminuria progresses over time, with a high incidence of new onset of pathologic levels of albuminuria in all age groups. All the usual morphologic findings are found in renal biopsy specimens. However, glomerular enlargement is notable in individuals from remote regions, but not those living closer to population centers. Glomerulomegaly probably represents compensatory hypertrophy caused by low nephron number, which probably underlies the accentuated susceptibility to renal disease. In the last decade, health care services have been transformed to accommodate systematic chronic disease surveillance and management. After a relentless increase for 3 decades, rates of Aboriginal people starting renal replacement therapy, as well as chronic disease deaths, appear to be stabilizing in some regions. Official endorsement of these system changes, plus ongoing reductions in the incidence of low birth weight and infections, hold promise for continued better outcomes.

A comparison of nephron number, glomerular volume and kidney weight in Senegalese Africans and African Americans.

BACKGROUND: Low nephron number is determined in utero and is a proposed risk for essential hypertension. Glomerular volume is inversely correlated with nephron number, and genetic and environmental factors that determine nephron number are thought to determine glomerular volume. This study compared total glomerular (nephron) number (N(glom)), mean glomerular volume (V(glom)) and kidney weight in two geographically separated black populations with significant common genetic ancestry. METHODS: Unbiased stereology was used to determine N(glom) and V(glom) in kidneys collected at coronial autopsy in an age- and sex-matched sample of 39 adult Africans from Dakar in Senegal, West Africa and 39 African Americans from Mississippi in the USA. RESULTS: African Americans were taller and heavier than their Senegalese counterparts. N(glom) was remarkably similar-with a geometric mean of 937 967 in Senegalese and 904 412 in African Americans (P = 0.62). V(glom) was correlated inversely with N(glom) and directly with body surface area in both groups, but V(glom) was 54% greater in African Americans than in Senegalese Africans [8.30 +/- 2.92 (SD) and 5.38 +/- 1.25 microm(3) x 10(6), respectively] and remained significantly larger (38%) after adjustment for body size. V(glom) increased with age in African Americans, but not in the Senegalese. Kidney weight was larger in African Americans (P < 0.0001), but kidney-to-body weight ratio was not different between groups. CONCLUSIONS: Despite similar nephron numbers, a common genetic constitution, and even in relation to current body size, African Americans have larger V(glom) than Senegalese subjects. This may mark exposure to environmental stressors or hereditary traits concentrated in the population's relocation to North America.

Nephron number and individual glomerular volumes in male Caucasian and African American subjects.

BACKGROUND: Glomerular hypertrophy has been described in several populations at high risk of chronic kidney disease. Total nephron (and thereby glomerular) number (N(glom)) varies widely in normal adult human kidneys and is generally inversely correlated with mean glomerular volume (V(glom)). However, little is known about the range of individual glomerular volumes (IV(glom)) within single human kidneys and the association with N(glom). The aim of the present study was to estimate IV(glom) in Caucasian and African Americans and identify any associations between heterogeneity in IV(glom) and nephron number. METHODS: Using unbiased stereological techniques, IV(glom) was determined for 30 glomeruli in each of 24 adult male kidneys from Jackson, MS, USA (12 Caucasian and 12 African American). Half of each group had 'high' N(glom) (>1.2 million nephrons per kidney) and the other half had 'low' N(glom) (<600 000). RESULTS: Caucasians with high N(glom) had a relatively homogeneous distribution of IV(glom) as well as a relatively low mean value, while those with low N(glom) had much greater heterogeneity of IV(glom), as well as a larger IV(glom) (P < 0.0001) compared with those with high N(glom). This disparity was not apparent in African Americans, however, where subjects with both high and low N(glom) showed substantial heterogeneity in IV(glom) and larger mean values (P = 0.95). CONCLUSIONS: High N(glom) appeared to protect against glomerular enlargement and volume heterogeneity in Caucasians. However, substantial variation in IV(glom) and net enlargement in glomerular size in African Americans with high nephron numbers suggest that additional forces, independent of low N(glom), are driving glomerular enlargement and heterogeneity.

Indomethacin, ibuprofen and gentamicin administered during late stages of glomerulogenesis do not reduce glomerular number at 14 days of age in the neonatal rat.

Premature neonates are frequently administered indomethacin, ibuprofen and gentamicin during the period of active glomerulogenesis. These drugs are known to have nephrotoxic effects, but the morphological effect of these drugs is unknown. The purpose of this study was to determine whether administration of these drugs during the late stages of glomerulogenesis in the rat has an effect on glomerular endowment. Rat pups were given, intraperitoneally, indomethacin, ibuprofen or indomethacin and gentamicin for the first 5 days of their postnatal life. The pups were killed at 14 days of age at completion of glomerulogenesis. The total number of glomeruli in the left kidney was determined by the physical disector/fractionator stereological technique. There was no difference between treatment groups in total number of glomeruli per kidney (P = 0.45). There were significantly fewer glomeruli per gram of kidney in those rat pups that had received indomethacin or ibuprofen (P < 0.0001). The reduction in the number of glomeruli per gram of kidney may indicate augmented growth of nephron tubules and/or collecting ducts, and/or be a consequence of oedema secondary to drug exposure. Further study is required to determine whether reduced glomerular number is seen in older animals or following exposure to these drugs at different time-points in kidney development.