Abstract
Enlarged glomerular size is a feature of focal segmental glomerulosclerosis, obesity-related glomerulopathy, diabetic nephropathy, and hypertension. The distribution of glomerular volumes within different cortical zones and glomerular volume alterations with age and obesity may contribute to understanding the evolution of these diseases. We analyzed the distributions of volumes of individual glomeruli in the superficial, middle, and juxtamedullary cortex of normal human kidneys using the disector/Cavalieri method. Volumes (Vglom) of 720 nonsclerotic glomeruli (30 per kidney, 10 per zone) were estimated in autopsy kidneys of 24 American men, 12 aged 20 to 30 yr and 12 aged 51 to 69 yr. Black and white individuals were represented equally. The range of individual Vglom within subjects varied from two- to eight-fold. There were no significant zonal differences in Vglom in the young or those with body surface area (BSA) ≤ 2.11 m2. In contrast, superficial glomeruli in the older age group, in those with BSA > 2.11 m2, and in white subjects were significantly larger than juxtamedullary glomeruli. Black subjects tended toward larger Vglom than white subjects, and this difference was significant and most marked in the juxtamedullary zone and independent of age, BSA, and glomerular number. There is a wide range in individual Vglom in adults. BSA, race, and age independently influence Vglom in different zones of the renal cortex. These findings might reflect processes of aging and susceptibility factors to renal disease.
Mean glomerular volume (Vglom) and the total number of nephrons in adult human kidneys vary widely. In our recent study of 78 kidneys, estimates obtained using unbiased stereologic methods showed a 5.5-fold range in mean Vglom and a range of 247,652 to 1,825,380 nephrons per kidney (1).
The significance of Vglom has become increasingly appreciated. Enlarged glomeruli are a feature of diabetic nephropathy and hypertension, the disorders that cause the major burden of ESRD in North America, Australia, and Europe (2–6). Glomerulomegaly distinguishes minimal-change disease from focal segmental glomerulosclerosis and marks a risk for glomerulosclerosis in obesity-related glomerulopathy (7–11).
Glomerular enlargement is a surrogate microanatomic marker of low nephron number in primary hypertension and in people of lower birth weight (6,12). However, estimates of the mean Vglom of the kidney are often derived indirectly using stereologic techniques that do not provide volumes of individual glomeruli. Accordingly, the distribution of Vglom within the normal adult human kidney is unknown. Diversity in the distribution of Vglom within the same kidney or in kidneys of different individuals could reflect divergent pathways of nephron maturation and glomerular obsolescence and reflect differences in the incidence and severity of progressive renal disease among different populations The aims of this study were to define the distribution of volumes of individual glomeruli in different zones of the normal adult kidney using an unbiased stereologic method and to identify the determinants of individual Vglom that may be related to age, race, body size, and glomerulosclerosis.
Materials and Methods
We estimated volumes of individual glomeruli in kidneys of 24 North American men who were previously healthy and died unexpectedly of nonrenal causes. Kidneys were obtained at coroner autopsy at the University of Mississippi Medical Center (Jackson, MS). Ethical approval for the use of autopsy tissue for clinical research was obtained by informed consent from the first of kin and approved by the Internal Review Board of the University of Mississippi Medical Center. We studied 12 people (six blacks and six whites) aged 20 to 30 yr and 12 (six blacks and six whites) aged 51 to 69 yr. Kidneys were excluded from the study when the right and left kidneys were substantially unequal in size or there was histologic evidence of glomerular or tubulointerstitial disease.
Tissue Processing and Estimation of Individual Vglom
The right kidney was perfusion-fixed with 10% buffered formalin and bisected in a midsagittal plane. A single block of approximately 10 × 10 × 1 mm of tissue that contained full-thickness cortex and underlying medulla was cut from the mid-hilar region of one random half of the kidney and embedded in glycol methacrylate (Technovit 7100; Heraeus Kulzer Gmbh, Wehrheim, Germany). Glycol methacrylate was preferred to paraffin to minimize the effects of shrinkage during tissue processing and embedding on glomerular dimensions (13). Blocks were sectioned exhaustively at 10 μm using a Leica DM2165 Supercut rotary microtome. The same microtomist cut all sections to minimize section thickness variation.
The renal cortex in the 10-μm glycol methacrylate sections was divided into outer (superficial), middle, and inner (juxtamedullary) zones using the standard classification of nephrons into these zones by the location of their glomerulus within the renal cortex (14). Outer cortical glomeruli were defined as the most superficial, located within three glomerular diameters of the capsule (15). Juxtamedullary glomeruli were the deepest, located within three glomerular diameters above the arcuate arteries. Glomeruli between these two zones were considered midcortical. Compressed glomeruli at the edge of sections were excluded.
Slides stained with periodic acid-Schiff (PAS) were projected onto a white surface using an Olympus BH-2 microscope at a magnification of ×320. Thirty glomeruli (10 per zone) in each kidney were sampled using disectors (16), and their volumes were estimated using the Cavalieri method (13,17). This disector/Cavalieri technique provides unbiased estimates of Vglom and requires no knowledge or assumptions of glomerular size or shape. Each glomerulus having its volume estimated was completely (exhaustively) sectioned at a known section thickness (t) or distance between sections (10 μm). The glomerular profile tuft area of every second section was measured by point counting using an orthogonal grid system (area per point = 1 cm2). Glomerular tuft volume (Vglom) was estimated using the formula Vglom = sum of glomerular profile areas × 2t (2 × 10 μm). On average, 12 sections (range nine to 15 sections) were measured from each glomerulus to estimate Vglom.
Estimation of Total Nephron Number
Tissue blocks from the other half of each kidney were sampled using a systematic uniform random-sampling scheme and used to estimate total glomerular number (Nglom) using the physical disector/fractionator combination. Details of this approach have been published previously (1,18). Briefly, this technique involves a series of subsampling events. Glomeruli are counted in a known fraction of the kidney. One random half of the kidney was cut into 4-mm-thick slices, and one in four of these slices was sampled using a random number between 1 and 4. The sampled slices were cut into small blocks approximately 10 × 10 × 1 mm. These blocks were randomly aligned, and one in 25 was sampled. The sampled blocks were embedded in glycol methacrylate and sectioned exhaustively at 20 μm, and every 10th and 11th section pair was stained with PAS. Slides with complete kidney sections (and their corresponding pair) were projected at ×320 magnification with Olympus BH-2 light microscopes onto an orthogonal grid (6 × 6 cm). Grid intersections were used to estimate the fraction of the section area used for glomerular counting (Pf). Q− was the actual number of glomeruli counted using the physical disector. With this technique, glomeruli are counted only when they are present in one section but absent from the next in the pair. The total Nglom in the kidney was estimated using the formula total Nglom = 4 × 25 × 10 × Ps/2Pf× Q−, where 4 was the inverse of the first sampling fraction, 25 was the inverse of the second sampling fraction, 10 was the inverse of the third sampling fraction, and Ps was the total area of kidney sections.
Paraffin Histology
Remaining portions of the kidney were processed for embedding in paraffin, and 4-μm sections were stained with PAS, hematoxylin and eosin, Masson’s trichome, and periodic acid-methenamine silver stains. All kidneys were examined carefully by light microscopy.
Glomerulosclerosis
Glomerulosclerosis was assessed on the thicker (20 μm) sections used to estimate total Nglom. The first 210 glomeruli on random sections from each kidney (70 per zone) were examined for sclerosis. The number of sclerotic glomeruli was expressed as a percentage of the total number of glomeruli examined.
Statistical Analyses
Data were analyzed using STATA statistical software (19). Univariate correlations were determined by Pearson parametric method for normally distributed variables and the Kendall Tau nonparametric method for skewed data. Multivariate linear regression was used to predict significant variations of one variable on another. Comparisons between two groups were performed using unpaired t test. P < 0.05 was considered significant.
Results
The volumes of 720 nonsclerotic glomeruli (30 per kidney, 10 from each zone) were estimated in 24 subjects. The mean (SD) volume of glomeruli in the 24 subjects was 4.48 (1.81) μm3× 106. Surprising, within individuals, Vglom varied two- to eight-fold (Tables 1 and 2). There was a low percentage of global glomerulosclerosis, and no segmental glomerulosclerosis was observed. The heterogeneity of Vglom in each of the three zones of the 24 kidneys analyzed is shown in Tables 3 and 4. The coefficient of variation of mean Vglom for the 24 subjects was 25%. The variance was approximately the same in the three cortical zones in the younger and older age groups, among black and white individuals and was not influenced by body surface area (BSA).
Characteristics of 12 male subjects aged 20 to 30 yr and the total Nglom, volumes of sampled glomeruli, and the extent of sclerosis in each kidney (unadjusted data)a
Characteristics of 12 male subjects aged 51 to 69 yr and the total Nglom, volumes of sampled glomeruli, and the extent of sclerosis in each kidney (unadjusted data)
Distribution of Vglom and extent of global sclerosis in different zones of the kidney in 12 men aged 20 to 30 yr (unadjusted data)a
Distribution of Vglom and extent of global sclerosis in different zones of the kidney in 12 men aged 51 to 69 yr (unadjusted data)
Cortical zone, BSA, age, and race all were shown to influence Vglom. The independent effect of each of these factors is described below using adjusted data to overcome the influence of confounding variables.
Vglom and BSA
BSA strongly correlated with mean Vglom for the 24 subjects (R = 0.48, P < 0.01), and this applied to every cortical zone (superficial R = 0.48, P = 0.017; middle R = 0.46, P = 0.025; juxtamedullary R = 0.48, P = 0.018). In multivariate analysis using age, race, BSA, total Nglom, and glomerulosclerosis as independent variables, BSA significantly predicted mean Vglom for a subject (BSA r2 = 0.458, P = 0.011; age P = 0.176; race P = 0.051; total Nglom P = 0.451; glomerulosclerosis, P = 0.356; α = 0.05:0.965). Multivariate linear regression revealed an enlargement in Vglom of 2.08 μm3× 106 for each m2 increase in BSA.
In Figure 1, mean Vglom per person in the three cortical zones is compared in individuals with BSA above and below the 50th percentile (2.11 m2) after adjustment for age, race, and glomerulosclerosis. In people with BSA ≤ 2.11 m2, there were no zonal differences in Vglom. In contrast, in people with BSA > 2.11 m2, there was progressive increase in Vglom from juxtamedullary to superficial cortical zones with glomeruli at each cortical level being larger than for people with BSA ≤ 2.11 m2. Mean Vglom in the superficial cortex of people with BSA > 2.11 m2 was significantly larger than that in the superficial cortex of people with BSA ≤ 2.11 m2.
Comparison of glomerular volume (Vglom) by body surface area (BSA) and cortical zone in 24 American men divided into two groups with BSA ≤ the median of 2.11 m2 (n = 12) and > 2.11 m2 (n = 12). Vglom of superficial cortex (SC) for individuals with BSA > 2.11 m2 is larger than those with BSA ≤ 2.11 m2 (*P = 0.047). Values for Vglom are adjusted for age, race, and glomerulosclerosis. JM, juxtamedullary cortex; MC, midcortex.
Vglom and Age
In Figure 2, mean Vglom per person in the three cortical zones is compared in the younger and older age groups after adjustment for race, BSA, glomerulosclerosis, and total Nglom. There were no zonal differences in Vglom in the younger group. In contrast, the older group showed a gradient of glomerular enlargement between the juxtamedullary, middle, and superficial cortex, with the largest glomeruli being found in the superficial cortex. In multivariate analysis with age, race, BSA, total Nglom, and glomerulosclerosis as independent variables, both age (P = 0.028) and BSA (P = 0.01) predicted superficial cortical Vglom.
Comparison of Vglom by age and cortical zone in young and older American men (n = 12 in each category). Vglom of SC is larger than that of JM in older subjects (51 to 69 yr; *P = 0.008). Vglom of SC for the 51- to 69-yr age group is larger than that for the 20- to 30-yr age group (**P = 0.022). Values for Vglom are adjusted for race, BSA, glomerulosclerosis, and total glomerular number.
Vglom and Race
Figure 3 shows the zonal distribution of Vglom in black and white individuals after adjustment for age, BSA, glomerulosclerosis, and total Nglom. There was a zonal difference in Vglom among white individuals, unlike black individuals, who had similarly sized glomeruli in all three cortical zones. Black individuals tended toward larger glomeruli than white individuals in all cortical zones. The adjusted mean (SD) Vglom for black individuals of all ages was 5.01 (1.92) μm3× 106 compared with 3.98 (1.52) μm3× 106 for white individuals (P = 0.05). The zonal difference was most marked in the juxtamedullary glomeruli, where Vglom for black individuals was 4.87 (1.42) μm3× 106 compared with 3.44 (0.91) μm3× 106 for white individuals (P = 0.023). In multivariate analysis with age, race, BSA, total Nglom, and glomerulosclerosis as independent variables, Vglom of juxtamedullary glomeruli was predicted by race (P = 0.031) and BSA (P = 0.039). Multivariate linear regression showed that black race was associated with a 1.15 μm3× 106 enlargement of juxtamedullary Vglom over white individuals (α = 0.05:0.938). There was no significant racial difference in age, BSA, total Nglom, or extent of glomerulosclerosis.
Comparison of Vglom by race and cortical zone in American men (n = 12 in each category). Vglom of SC is larger than that of JM among white individuals (*P = 0.023). Vglom of JM of black individuals is larger than of white individuals (**P = 0.028). Values for Vglom are adjusted for age, BSA, glomerulosclerosis, and total glomerular number.
Glomerulosclerosis
Glomerulosclerosis significantly and directly correlated with age (r = 0.768, P < 0.001) and inversely correlated with total Nglom (r = −0.681, P < 0.001). Total Nglom was also inversely correlated with age (r = −0.642, P < 0.001). In stepwise regression using age, race, BSA, Vglom, and total Nglom as independent variables, only age significantly predicted the severity of glomerulosclerosis (age P < 0.001; total Nglom P = 0.079). In the older group, global sclerosis affected 0 to 19% (mean 6.3%) of glomeruli in the superficial cortex compared with 0 to 7% (mean 2.7%) of juxtamedullary glomeruli. Glomerulosclerosis did not correlate significantly with Vglom.
Discussion
The novelty of this study lies in the use of unbiased stereologic techniques to sample and estimate Vglom in different zones of the kidney in both young and older adults of different races. We identified a notable degree of heterogeneity in the distribution of Vglom in the “normal” human adult kidney. Significant relationships were found between the zonal distributions of Vglom and BSA, race, and age.
During kidney development, glomeruli develop at the tips of the branching ureteric bud in a centrifugal pattern. Glomeruli that will be located in the juxtamedullary region develop first and are larger than superficial glomeruli at birth and during early postnatal life (20). In humans, all glomeruli are formed by 36 wk gestation (21). Earlier studies documented significant glomerular hypertrophy from birth to adolescence (22–24), but alterations in glomerular size in later life remain uncertain. There are known anatomic and functional differences in glomeruli. This relates to their location within the cortex and whether the glomerulus is superficial and belongs to a nephron with a short loop of Henle or juxtamedullary and belongs to a nephron with a long loop of Henle that descends into the inner medulla (25).
Textbooks on the anatomy of the kidney state that the juxtamedullary glomeruli are 50% or more larger than superficial glomeruli (26,27). This seems to be well documented and to persist after maturity in rats and rabbits. However, for humans, any zonal variations in Vglom that may exist in early postnatal life seem to be lost in childhood. Fetterman et al. (24) used a maceration technique to study the kidneys of 23 individuals aged term to 18 yr. They found no differences in mean glomerular diameter between the inner and outer cortical glomeruli at any age. Oliver and McDowell (28) also used the maceration method to examine a composite collection of glomeruli from the normal kidneys of three adults with an average age of 35 yr. They found the average diameter of outer and juxtamedullary glomeruli to be essentially the same, although they observed more heterogeneity in size and larger glomeruli in the juxtamedullary cortex. Although we found a large variance in Vglom, the variance was distributed approximately equally in all cortical zones and did not change significantly with age, race, BSA, or Nglom.
Newbold et al. (29) measured single cross-sectional areas of outer cortical and juxtamedullary glomeruli in 41 adults aged 22 to 92 yr (mean 61 yr) and counted the proportion of obsolete, globally sclerotic glomeruli in these cortical zones. In these predominantly elderly subjects, global glomerulosclerosis was most severe in the outer cortex, and the glomerular area of juxtamedullary glomeruli was significantly greater than that of outer cortical glomeruli. The enlargement of juxtamedullary glomeruli was positively correlated with glomerulosclerosis. However, the correlation was significant only for glomerulosclerosis in the outer and not juxtamedullary cortex. The authors suggested that the juxtamedullary glomeruli were enlarged in compensation for the loss of glomeruli in the outer cortex. They proposed that the compensatory hypertrophy may be due to hyperfiltration and place the juxtamedullary glomeruli at risk for hyperfiltration injury.
We found a considerable variation in Vglom within each kidney in 12 young male adults aged 20 to 30 yr but no significant differences between the juxtamedullary and superficial glomeruli. In 12 older male adults aged 51 to 69 yr, the mean Vglom in the superficial cortex was 20% larger than in the juxtamedullary zone. Global glomerulosclerosis increased with age and was most severe in the superficial cortex. Unlike the findings of Newbold et al. (29), the age-related glomerular enlargement occurred in superficial glomeruli and occurred with global glomerulosclerosis in the same cortical region.
We could not establish any statistical correlation between the proportion of sclerotic glomeruli and the degree of enlargement of nonsclerotic glomeruli. However, analysis of a single glomerular profile without apparent sclerosis does not preclude the existence of segmental sclerosis in another section of that glomerulus. Thus, analysis of one section per glomerulus could have led to underestimation of the number of glomeruli with segmental lesions.
The proportion of globally sclerotic glomeruli was inversely correlated with total Nglom. This may be the result of age and hypertension-related glomerular loss that has been shown to be most severe in kidneys with low nephron number (6). In this and other studies, nephron number correlated inversely with age, suggesting that nephrons are possibly lost with age (1,30,31). This could also be the result of generational differences in kidney size and nephron endowment, with the older generation subjects being born with fewer nephrons. The proportion of identifiable obsolete glomeruli increased with age, although the overall incidence was low and we did not identify significant tubulointerstitial fibrosis on qualitative inspection. Experimental evidence to determine whether such scarred glomeruli are permanent or eventually disappear without leaving recognizable scars is lacking (32,33).
Loss of glomeruli in the superficial cortex may shift perfusion to adjacent functional glomeruli within the peripheral vascular supply of the superficial cortex and promote their enlargement. The change in Vglom is in all likelihood a compensation for reduced glomerular number.
Black individuals had larger glomeruli in every zone than white individuals, although the difference was statistically significant only in the juxtamedullary zone. This did not seem to be a function of BSA or age because there were no significant racial differences in these variables.
People with a BSA > 2.11 m2 were above the median body size and, on average, had larger glomeruli in every zone than those of smaller body size, although only the difference between the superficial zones was statistically significant. In addition, the differences in Vglom by zone were largely limited to people whose BSA was above the group median. Kasiske and Umen (34) previously identified BSA as a major determinant of glomerular area in normal humans. Our study establishes a direct correlation between body size and Vglom that has a distinctly zonal distribution. This relationship follows the recognized associations of obesity with glomerulomegaly and glomerulosclerosis (9–11).
An unexpected finding in this study was the lack of correlation between total Nglom of the 24 subjects and mean Vglom based on 30 glomeruli per kidney. We previously reported an inverse correlation between total Nglom and mean Vglom in a larger study of 78 individuals (1). In this study, individual Vglom measurements were confined to a small portion of the kidney from the mid-hilar region, whereas in the preceding study, samples of tissue were distributed uniformly throughout the full three-dimensional anatomy of the kidney and estimation of mean Vglom was based on grid points overlying hundreds of glomerular profiles per kidney. Thus, the number of subjects, the size of the tissue sample per kidney, and the distribution of the sample through the kidney were very different in the two studies. These factors almost certainly account for the different findings in these studies. It is interesting that a landmark investigation of total Nglom and mean Vglom using unbiased stereologic techniques by Nyengaard and Bendtsen (30) found no correlation between these parameters among 37 subjects. The relationship between total Nglom and mean Vglom is complex and influenced by other factors, including BSA. This is demonstrated by the following two interesting subjects in our study. One white man aged 30 yr had the highest BSA of 2.66 m2, 1,208,151 glomeruli, and mean Vglom of 6.03 μm3× 106. In contrast, another white man aged 29 yr had the lowest BSA of 1.60 m2, the lowest number of glomeruli (247,652), and mean Vglom of only 2.27 μm3× 106. When the latter individual was excluded, there was an inverse relationship between total Nglom and mean Vglom for the remaining 23 subjects, although this did not reach statistical significance (r = −0.2; P = 0.36).
The main limitation of our study is that volumetric analysis was based on a small number of glomeruli from samples restricted to a mid-hilar slice of the kidney. The renal papillae of the poles of the kidney are convex and provide the drainage of fused lobes that are different than the largely conical and unilobular papillae in the middle of the kidney (26). These differences may be reflected by variations in Vglom in the respective renal cortices. In a preliminary study, we found mean Vglom in the poles of two kidneys to be very similar to that in the mid-hilar region. In a third kidney, mean Vglom in the pole was 45% larger than in the mid-hilum.
A defined region of each kidney was analyzed, because the preparation and analysis of serial sections are technically laborious and require a significant amount of time. Nevertheless, with the 720 glomeruli measured using unbiased stereologic techniques, we have documented a wide range of Vglom within these zones of renal cortex and established that BSA, race, and age are independent determinants of Vglom.
The well-known risk factors for renal disease such as obesity and older age are characterized by enlargement of superficial glomeruli, whereas black race is attended by enlargement of juxtamedullary glomeruli. Future studies will attempt to define Vglom distribution in individuals with known BP, both hypertensive and nonhypertensive, and in people with low nephron number.
It is interesting that Toyota et al. (35) recently used x-ray microcomputed tomography scan to estimate Vglom distribution in rats with early diabetic nephropathy and found global heterogeneity, which they suggested might be a sensitive marker of early disease. A future attraction is the potential clinical application of x-ray microcomputed tomography scan or similar noninvasive techniques that can be used for the accurate volumetric analysis of large numbers of glomeruli in healthy and diseased human kidneys.
Acknowledgments
This research was funded by grants from Janssen-Cilag Australia Pty. Ltd., the American Heart Association (Southeastern affiliate), and National Institutes of Health grants 1 RO1 DK065970-01 and 5 P20M000534-02 (“Project Export,” National Institutes of Health, Center of Excellence in Minority Health). T.S. received a Monash PhD scholarship.
The content of this paper was presented as a poster at the American Society of Nephrology Conference, Renal Week; October 2004; St. Louis, MO.
We thank our dedicated microtomist, Sue O’Connell, and Jenny Nicol for statistical support.
Footnotes
Published online ahead of print. Publication date available at www.jasn.org.
- © 2005 American Society of Nephrology
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