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

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Clin Exp Nephrol 2008; 13: Received for publication: ; Accepted in revised form: Nephron number and individual glomerular volumes in male Caucasian and African American subjects Monika A. Zimanyi 1, Wendy E. Hoy 2, Rebecca N. Douglas-Denton 1, Michael D. Hughson 3, Libby M. Holden 2 and John F. Bertram 1 1 Department of Anatomy and Developmental Biology, School of Biomedical Sciences, Monash University, Victoria, 2 Centre for Chronic Diseases, The University of Queensland, Brisbane, Australia and 3 Department of Pathology, University of Mississippi Medical Center, Jackson, MS, USA Correspondence and offprint requests to: Monika A. Zimanyi; monika.zimanyi@med.monash.edu.au Abstract 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 (< ). 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 < ) compared with those with high N glom. This disparity was not apparent in African Americans, C The Author [2009]. 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2 Nephron number and individual glomerular volume 2429 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. Keywords: African American; Caucasian; glomerular volume; nephron number; stereology IV glom 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. [19] showed that the range of IV glom 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 IV glom in the kidneys of adults with high and with low N glom 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 N glom was known to be either < or >1.2 million. Introduction Several studies have reported a large range of total nephron number (N glom ) in normal human kidneys. In 37 Danish kidneys examined at autopsy, N glom ranged from to [1]; in a German cohort of 20 subjects, N glom ranged from to [2] and in our study of 189 Caucasian and African American subjects, N glom ranged from to [3]. In most studies, N glom has been shown to be inversely correlated with mean glomerular volume (V glom) [4 6], although Nyengaard et al. [1] 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 N glom [6]. 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 [7]. 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 [10], secondary to systemic abnormalities such as hypertension or diabetes [11], with obesity [12], and following single kidney transplant [13]. 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 [12]. In turn, glomerular hypertrophy is known to lead to premature sclerosis and ultimately to obsolescence of glomeruli [14] and is therefore a risk factor for kidney disease and renal failure [6,10]. Previous studies have estimated V glom indirectly during N glom 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 (IV glom ) 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. [19], Subjects and methods Subject selection 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 N glom and IV glom was performed. N glom was estimated using the physical disector/fractionator combination prior to this study as previously described [4]. In the present study, 24 adult male subjects (12 American Caucasians and 12 African Americans) with either high (>1.2 million) or low N glom (< ) (Tables 3 and 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 < 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 IV glom. 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 1 and 2. Estimation of individual glomerular volume (IV glom ) The stereological estimation of IV glom 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 a1cm 1cm 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 [21]. At a final magnification of 320, all glomerular profiles from sampled glomeruli were projected onto a white surface with a superimposed orthogonal grid (1 cm 2 ). Using the Cavalieri technique and stereological point counting, IV glom was obtained for 10 glomeruli from each of the three zones of the kidney of each subject (n = 30 glomeruli

3 2430 M. A. Zimanyi et al. Table 1. Age, height, body weight, body surface area (BSA) and kidney weight of the 12 adult male Caucasian subjects Caucasian Age Height Body Kidney subjects (year) (cm) weight (kg) BSA (m 2 ) weight (g) Mean Mean Table 2. Age, height, body weight, body surface area (BSA) and kidney weight of the 12 adult male African American subjects African American Age Height Body Kidney subjects (year) (cm) weight (kg) BSA (m 2 ) weight (g) Mean Mean per subject). Thus, IV glom 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. Statistical analyses 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 IV glom and N glom, taking into account both the variation in IV glom 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 < Results Within the aggregate group of adult males in the broader study, the previously described inverse relationship between N glom and V glom was confirmed in both Caucasians (n = 54, P < ; Figure 1) and African Americans (n = 56, P = ; Figure 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 V glom even after accounting for N glom, age and BSA, with higher V glom estimates observed in African Americans than for Caucasians (P = 0.004). The current study estimated IV glom for 720 glomeruli from the superficial, middle and juxtamedullary renal cortices of 24 adult male Caucasian and African Americans. Tables 3 and 4 show the results for the 24 subjects. The mean IV glom for the 720 glomeruli was 5.16 ± 2.25 µm (median value was 4.87 µm ). The range of IV glom within subjects ranged from to 6.03-fold, with a fold range between the smallest (1.17 µm ) and the largest (15.04 µm ) glomerulus observed in the study (Tables 3 and 4). Caucasians with high N glom had significantly smaller mean IV glom and a more homogeneous distribution (lower variance) of IV glom than those with low N glom (P < for IV glom comparisons between the two Caucasian groups). However, African Americans with high N glom had mean IV glom almost twice as large as those of Caucasians with high N glom, with marked heterogeneity, and with values very similar to Caucasian and African American subjects with low N glom (P = 0.95 for IV glom compared between the two African American groups). These phenomena are shown graphically in Figure 3. There were no significant differences in the mean or the variance of IV glom by zone (10 glomeruli were analysed per subject per zone) either in Caucasians with high or low N glom (P = 0.96 and P = 0.79, respectively), or in African Americans with high or low N glom (P = 0.79 and P = 0.95, respectively). Discussion 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 [23] 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. [22] estimated the volumes of 10 individual glomeruli within each of 20 subjects using resin-embedded tissue. They obtained a mean IV glom of 4.21 ± 1.23 µm that compares well with the present value of 4.64 ± 2.39 µm IV glom within subjects in their study ranged from 1.24 to 7.40 µm , a 5.99-fold range, whilst in the present study the range was from 1.17 to µm , a fold range overall in Caucasian subjects. Importantly, the present data for IV glom confirm

4 Nephron number and individual glomerular volume 2431 Table 3. Total nephron number (N glom ), smallest individual glomerular volume (IV glom ), largest IV glom, mean IV glom, standard deviation of IV glom and fold range of IV glom of 12 adult male Caucasians with high (> ) or low (< ) total N glom Caucasian Smallest IV glom Largest IV glom Mean IV glom SD of IV glom subjects N glom (µm ) (µm ) (µm ) (µm ) Fold range Mean Mean Table 4. Total nephron number (N glom ), smallest individual glomerular volume (IV glom ), largest IV glom, mean IV glom, standard deviation of IV glom and fold range of IV glom of 12 adult male African Americans with high (> ) or low (< ) total N glom African American Smallest IV glom Largest IV glom Mean IV glom SD of IV glom subjects N glom (µm ) (µm ) (µm ) (µm ) Fold range Mean Mean Mean glomerular volume (µm 3 x 10 6 ) r 2 = 0.26 p < Total Nephron Number Fig. 1. Graph showing an inverse correlation between total nephron number and mean glomerular volume (V glom ) in Caucasian Americans (n = 54). Mean glomerular volume (µm 3 x 10 6 ) r 2 = 0.18 p = Total Nephron Number Fig. 2. Graph showing an inverse correlation between total nephron number and mean glomerular volume (V glom ) in African Americans (n = 56). the large range in glomerular size, both within and between individuals, noted by Samuel et al. [19]. 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

5 2432 M. A. Zimanyi et al. Fig. 3. Individual glomerular volumes (IV glom ) of Caucasian and African Americans with high or low total nephron number (N glom ). Subjects within each group are ranked by mean IV glom. Each dot represents a glomerulus, with values for 30 glomeruli shown for each subject (column). 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. [24]. 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 N glom were not perceptibly protected from glomerular enlargement or heterogeneity that characterized Caucasian and African Americans with low N glom. 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. [25] 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 [4]. 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 N glom indicates that additional factors, as well as N glom, 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. Acknowledgements. This research was funded by grants from the National Institutes of Health NIH 1 R01 DK , NIH Center of Excellence in Minority Health 5P20M 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.

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Nephrol Dial Transplant 1999; 14: Received for publication: ; Accepted in revised form: The value of post-biopsy ultrasound in predicting complications after percutaneous renal biopsy of native kidneys Bryan Waldo 1, Stephen M. Korbet 1, Maija G. Freimanis 2 and Edmund J. Lewis 1 1 Section of Nephrology, Department of Medicine and 2 Department of Radiology, Rush University Medical Center, 1653 West Congress Pkwy, Chicago, IL 60612, USA Correspondence and offprint requests to: Stephen M. Korbet; skorbet@aol.com Abstract Background. Clinically significant bleeding complications occur in >30% of patients undergoing percutaneous renal biopsy (PRB) of native kidneys and can be severe in up to 10% of patients. A noninvasive measure that would reliably predict which patients will do well with an uncomplicated post-biopsy course or which patients may be at risk of developing a clinically significant complication is in great demand. Methods. PRB of native kidneys was performed in 162 adult patients from February 2002 through February 2007 using real-time ultrasound and automated needle. Renal C The Author [2009]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org