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Background. Glomerular hypertrophy has been described in several populations at high risk of chronic kidney disease. Total nephron (and thereby glomerular) number (Nglom) varies widely in normal adult human kidneys and is generally inversely correlated with mean glomerular volume (Vglom). However, little is known about the range of individual glomerular volumes (IVglom) within single human kidneys and the association with Nglom. The aim of the present study was to estimate IVglom in Caucasian and African Americans and identify any associations between heterogeneity in IVglom and nephron number.
Methods. Using unbiased stereological techniques, IVglom 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’ Nglom (>1.2 million nephrons per kidney) and the other half had ‘low’ Nglom (<600 000).
Results. Caucasians with high Nglom had a relatively homogeneous distribution of IVglom as well as a relatively low mean value, while those with low Nglom had much greater heterogeneity of IVglom, as well as a larger IVglom (P < 0.0001) compared with those with high Nglom. This disparity was not apparent in African Americans, however, where subjects with both high and low Nglom showed substantial heterogeneity in IVglom and larger mean values (P = 0.95).
Conclusions. High Nglom appeared to protect against glomerular enlargement and volume heterogeneity in Caucasians. However, substantial variation in IVglom and net enlargement in glomerular size in African Americans with high nephron numbers suggest that additional forces, independent of low Nglom, are driving glomerular enlargement and heterogeneity.
Several studies have reported a large range of total nephron number (Nglom) in normal human kidneys. In 37 Danish kidneys examined at autopsy, Nglom ranged from 331000 to 1424000 ; in a German cohort of 20 subjects, Nglom ranged from 531140 to 1959914  and in our study of 189 Caucasian and African American subjects, Nglom ranged from 210332 to 2026541 .
In most studies, Nglom has been shown to be inversely correlated with mean glomerular volume (Vglom) [4–6], although Nyengaard et al.  did not observe this relationship. In studies that did observe this inverse relationship, it was proposed that compensatory hypertrophy of individual glomeruli acted to ensure adequate filtration in subjects with low Nglom . Glomerular hypertrophy (an increase in the size of glomeruli) can occur as the result of an increase in the number of cells (resident and infiltrating) within a glomerulus and/or an increase in the size of individual cells and/or accumulation of the extracellular matrix . Glomerular hypertrophy is observed in a number of normal and pathological states. For instance, glomeruli undergo hypertrophy during body growth [8,9], with the development of primary renal pathology such as focal and segmental glomerular sclerosis , secondary to systemic abnormalities such as hypertension or diabetes , with obesity , and following single kidney transplant . Focal segmental glomerular sclerosis can result from low nephron number or following nephron loss. Reduced nephron endowment leads to increased demands on remaining nephrons, increases in glomerular capillary pressure and subsequent hyperperfusion leading to glomerular hypertrophy . In turn, glomerular hypertrophy is known to lead to premature sclerosis and ultimately to obsolescence of glomeruli  and is therefore a risk factor for kidney disease and renal failure [6,10].
Previous studies have estimated Vglom indirectly during Nglom estimation, in which the average size of all glomeruli in a kidney is derived. However, this single estimate of glomerular volume for a kidney does not reveal the range of individual glomerular volumes (IVglom) within the kidney. Previous studies have estimated glomerular volumes in paraffin sections, which are well known to be strongly affected by shrinkage, and often by using potentially biased techniques [15–18]. In a recent study by Samuel et al. , IVglom was estimated in different zones of the normal adult human kidney in glycolmethacrylate-embedded tissue using unbiased stereological techniques, similar to the present study. Samuel et al.  showed that the range of IVglom within subjects varied from 2- to 8-fold with an overall 12-fold range among subjects.
The aim of the present study was to evaluate the distribution of IVglom in the kidneys of adults with high and with low Nglom to better understand the process of compensatory hypertrophy at the individual glomerular level and the association between nephron number and glomerular size. Caucasian and African American males were studied whose Nglom was known to be either <600000 or >1.2 million.
Right kidneys were obtained at coronial autopsy from Caucasian and African American adult males who died unexpectedly (of non-renal causes). Autopsies were conducted at the University of Mississippi Medical Center, Jackson,MS, USA. Subjects were excluded from the study if the right and left kidneys were considerably unequal in size or if there was histologic evidence of glomerular or tubulointerstitial disease. Ethical approval from the Institutional Review Board of the University of Mississippi Medical Center and the Monash University Ethics Committee and informed consent from the next of kin were obtained prior to clinical research.
The right kidney was perfusion fixed with 10% buffered formalin and sent to Monash University, Melbourne, Australia, where stereological estimation of Nglom and IVglom was performed. Nglom was estimated using the physical disector/fractionator combination prior to this study as previously described .
In the present study, 24 adult male subjects (12 American Caucasians and 12 African Americans) with either high (>1.2 million) or low Nglom (<600000) (Tables (Tables33 and and4)4) were selected from a total of 187 kidneys collected at the University of Mississippi Medical Centre at the beginning of the study. Of those subjects 167 were adults. Twenty-four subjects had nephron endowment in excess of 1.2 million, of which 19 were male, 8 Caucasian and 11 African American. Of the 37 subjects who had nephron numbers <600000 per kidney, 21 were male, 9 Caucasian and 12 African American. In total, six Caucasian and six African American adult male subjects with high or low nephron numbers were selected for the analysis of IVglom. The subjects were chosen so that group aggregates were matched as best as possible for age and for body surface area (BSA). The characteristics of adult male subjects used in this study are shown in Tables Tables11 and and22.
The stereological estimation of IVglom has been described previously [19,20]. Briefly, kidneys were bisected in the coronal plane and one-half was randomly chosen for sampling. From the mid-hilar region of the kidney a 1 cm × 1 cm × 1 mm sample was obtained through the full thickness of the cortex (including some of the underlying medulla), dehydrated and embedded in glycolmethacrylate (Technovit 7100; Heraeus Kulzer, Germany). The blocks were serially sectioned at 10 μm using a Leica DM2165 Supercut rotary microtome, with every second section collected and stained with periodic acid Schiff (PAS).
Using an Olympus BH-2 microscope equipped with a projection arm, a ‘map’ was drawn of the image projected onto a white surface at low magnification (×60). The thickness of the renal cortex was measured and divided into three regions of equal thickness: superficial, middle and juxtamedullary. Those glomeruli present in a section, which were not present in the previous section, were sampled according to disector sampling criteria . At a final magnification of ×320, all glomerular profiles from sampled glomeruli were projected onto a white surface with a superimposed orthogonal grid (1 cm2). Using the Cavalieri technique and stereological point counting, IVglom was obtained for 10 glomeruli from each of the three zones of the kidney of each subject (n = 30 glomeruli per subject). Thus, IVglom was estimated using a disector/Cavalieri combination. Sclerosed glomeruli or glomeruli falling within one glomerular diameter from an artificial section edge were excluded. All kidneys were analysed in a blind fashion.
Data were analysed using STATA (version 9.1) statistical software. Comparisons between groups were performed using a clustered linear regression analysis to consider the correlation between IVglom and Nglom, taking into account both the variation in IVglom within and between subjects. Linear regression analyses were graphed and analysed using GraphPad Prism (version 4.00) for Windows, (GraphPad Software, San Diego CA, USA). Values are mean ± standard deviation. The statistical significance was accepted at P < 0.05.
Within the aggregate group of adult males in the broader study, the previously described inverse relationship between Nglom and Vglom was confirmed in both Caucasians (n = 54, P < 0.0001; Figure Figure1)1) and African Americans (n = 56, P = 0.0013; Figure Figure2).2). This significance was applied for unadjusted values as well as for values adjusted for age and current body size (BSA). However, race was a significant determinant of Vglom even after accounting for Nglom, age and BSA, with higher Vglom estimates observed in African Americans than for Caucasians (P = 0.004).
The current study estimated IVglom for 720 glomeruli from the superficial, middle and juxtamedullary renal cortices of 24 adult male Caucasian and African Americans. Tables Tables33 and and44 show the results for the 24 subjects.
The mean IVglom for the 720 glomeruli was 5.16 ± 2.25 μm3 × 106 (median value was 4.87 μm3 × 106). The range of IVglom within subjects ranged from 1.71- to 6.03-fold, with a 12.85-fold range between the smallest (1.17 μm3 × 106) and the largest (15.04 × μm3 × 106) glomerulus observed in the study (Tables (Tables33 and and44).
Caucasians with high Nglom had significantly smaller mean IVglom and a more homogeneous distribution (lower variance) of IVglom than those with low Nglom (P < 0.0001 for IVglom comparisons between the two Caucasian groups). However, African Americans with high Nglom had mean IVglom almost twice as large as those of Caucasians with high Nglom, with marked heterogeneity, and with values very similar to Caucasian and African American subjects with low Nglom (P = 0.95 for IVglom compared between the two African American groups). These phenomena are shown graphically in Figure Figure33.
There were no significant differences in the mean or the variance of IVglom by zone (10 glomeruli were analysed per subject per zone) either in Caucasians with high or low Nglom (P = 0.96 and P = 0.79, respectively), or in African Americans with high or low Nglom (P = 0.79 and P = 0.95, respectively).
In previous human studies, the association between low nephron number and large glomerular size has been demonstrated mostly through average glomerular volume estimates for whole kidneys [1,4,5,22]. Such studies provide no information about the range of glomerular size within kidneys. Due to shrinkage associated with paraffin embedding, previous studies in which absolute glomerular volumes were estimated in paraffin blocks cannot be compared to the values obtained in the present study in which tissue was embedded in glycolmethacrylate. Plastic-embedded sections reveal less tissue shrinkage and fewer artefacts  and therefore were used in the present study. Other studies have however, using an unbiased stereological technique similar to that used in the present study, shown a modest range of individual glomerular volumes within single kidneys. For example, MacLeod et al.  estimated the volumes of 10 individual glomeruli within each of 20 subjects using resin-embedded tissue. They obtained a mean IVglom of 4.21 ± 1.23 μm3 × 106 that compares well with the present value of 4.64 ± 2.39 μm3 × 106. IVglom within subjects in their study ranged from 1.24 to 7.40 μm3 × 106, a 5.99-fold range, whilst in the present study the range was from 1.17 to 15.04 × μm3 × 106, a 12.85-fold range overall in Caucasian subjects. Importantly, the present data for IVglom confirm the large range in glomerular size, both within and between individuals, noted by Samuel et al. .
In the present study, we have shown in white adult males that this process of compensatory glomerular hypertrophy in the face of low nephron number is associated not only with the enlargement of remaining glomeruli on average, but also with a marked heterogeneity in volume of different glomeruli within a kidney and between individuals. This heterogeneity in glomerular size suggests differential hypertrophic responses and/or mechanisms within the glomerular population within a single kidney. The mechanisms that drive this process of apparent compensatory hypertrophy appear to be differentially experienced and expressed at any particular point in time by different glomeruli. At some critical point of enlargement, glomeruli are thought to be at accentuated risk for injury and sclerosis as hypothesized by Brenner et al. . In people with lower nephron numbers, it is probable that the largest glomeruli among the great splay of individual glomerular volumes are at the greatest risk of undergoing sclerosis. As substantial numbers of glomeruli become obsolescent, the ever-diminishing number of nephrons only compound the hypertrophic stimulus for those that remain, and they in turn enter this cycle of progressive enlargement and subsequent loss. In this study, we were unable to demonstrate that this hypertrophic process was preferentially expressed in any particular zone of the cortex. More targeted research remains to be done in this area.
In Caucasian American males, robust nephron numbers (>1.2 million) were strongly protective against glomerular enlargement and volume heterogeneity. This finding implies that the risk for hyperperfusion injury and sclerosis is minimized in such subjects, and presumably their risk for rising blood pressures and loss of renal function is also minimized [2,4]. It should be noted that glomeruli with evidence of glomerular sclerosis were not included in this study.
Overall, African Americans had larger glomeruli with a more heterogeneous volume distribution than Caucasians. In addition, African Americans with high Nglom were not perceptibly protected from glomerular enlargement or heterogeneity that characterized Caucasian and African Americans with low Nglom. This implies that additional forces are driving glomerular enlargement and heterogeneity in African Americans beyond those associated with compensatory hypertrophy in a setting of low nephron number. Focal dysregulation of preglomerular afferent arteriolar blood flow is one strong candidate for this phenomenon. Indeed, Tracey et al.  have noted that vascular wall hyaline changes and luminal dilatation of individual arterioles are linked to the enlargement of the glomeruli they perfuse. This phenomenon is almost certainly linked to the excessive rates of hypertension in African Americans .
The glomerular hypertrophy seen in the African American subjects might expose them to accelerated loss of nephrons and might indicate increased susceptibility to chronic kidney disease. Furthermore, the persistence of these characteristics in those with robust Nglom indicates that additional factors, as well as Nglom, make important, perhaps critical contributions to their risk for renal disease and hypertension. Therein probably lies the greater susceptibility of African Americans to these conditions than Caucasians. These observations prompt us to further consider pathophysiology, as well as to expand considerations of modalities for prevention and for treatment.
The heterogeneity of individual glomerular volumes associated with compensatory hypertrophy in Caucasians suggests that renal protective treatments, which reduce glomerular perfusion, might both minimize the damage to the largest glomeruli and help to preserve the many nephrons whose volumes still remain relatively small. Such treatment should also be effective in African Americans, but, depending on the nature of the additional stimuli driving hyperperfusion and glomerular enlargement, might need to be supplemented by other interventions.
This research was funded by grants from the National Institutes of Health NIH 1 R01 DK065970-01, NIH Center of Excellence in Minority Health 5P20M000534-02 and the Colonial Foundation of Australia. The results of this study were presented as a free communication at the American Society of Nephrology Conference, November 2006, San Diego.
Conflict of interest statement. None declared.