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. 2015 Dec;26(12):3162-78.
doi: 10.1681/ASN.2014080752. Epub 2015 Jun 2.

Glomerular Aging and Focal Global Glomerulosclerosis: A Podometric Perspective

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Free PMC article

Glomerular Aging and Focal Global Glomerulosclerosis: A Podometric Perspective

Jeffrey B Hodgin et al. J Am Soc Nephrol. .
Free PMC article

Abstract

Kidney aging is associated with an increasing proportion of globally scarred glomeruli, decreasing renal function, and exponentially increasing ESRD prevalence. In model systems, podocyte depletion causes glomerulosclerosis, suggesting age-associated glomerulosclerosis could be caused by a similar mechanism. We measured podocyte number, size, density, and glomerular volume in 89 normal kidney samples from living and deceased kidney donors and normal poles of nephrectomies. Podocyte nuclear density decreased with age due to a combination of decreased podocyte number per glomerulus and increased glomerular volume. Compensatory podocyte cell hypertrophy prevented a change in the proportion of tuft volume occupied by podocytes. Young kidneys had high podocyte reserve (podocyte density >300 per 10(6) µm(3)), but by 70-80 years of age, average podocyte nuclear density decreased to, <100 per 10(6) µm(3), with corresponding podocyte hypertrophy. In older age podocyte detachment rate (urine podocin mRNA-to-creatinine ratio) was higher than at younger ages and podocytes were stressed (increased urine podocin-to-nephrin mRNA ratio). Moreover, in older kidneys, proteinaceous material accumulated in the Bowman space of glomeruli with low podocyte density. In a subset of these glomeruli, mass podocyte detachment events occurred in association with podocytes becoming binucleate (mitotic podocyte catastrophe) and subsequent wrinkling of glomerular capillaries, tuft collapse, and periglomerular fibrosis. In kidneys of young patients with underlying glomerular diseases similar pathologic events were identified in association with focal global glomerulosclerosis. Podocyte density reduction with age may therefore directly lead to focal global glomerulosclerosis, and all progressive glomerular diseases can be considered superimposed accelerators of this underlying process.

Keywords: aging; glomerulosclerosis; podocyte.

Figures

Figure 1.
Figure 1.
Podometric methodology. The upper panels show TLE4-red fluorescent podocyte nuclei (red) in glomerular profiles from young (A), middle-aged (B), and older (C) people. The lower panels show the same sections for which the coverslip has been removed and peroxidase immunocytochemistry performed to delineate the Glepp1 peroxidase–positive area (brown) occupied by podocytes. Calibrated photomicrographs of the TLE4 immunofluorescent sections are used to measure podocyte nuclear mean caliper diameter. Photomicrographs of the Glepp1-stained sections are used to measure glomerular tuft area and the percentage of the tuft area occupied by podocytes. These primary data measured in the histologic section are then used to estimate podocyte nuclear density, glomerular volume, number of podocyte nuclei per tuft, Glepp1-positive tuft volume, Glepp1-negative tuft volume, and podocyte cell volume. The bar shows 100 μm.
Figure 2.
Figure 2.
Aging-associated changes in podocyte density resulting from changes in podocyte nuclear number per tuft and glomerular volume. (A) Podocyte nuclear density (number per tuft volume). (B) Reciprocal glomerular volume per podocyte. The underlying causes of the density change are a reduction in podocyte number per glomerular tuft (C) and an increase in glomerular volume (D). Table 1 provides slopes and statistical information for these data using bivariate linear regression models.
Figure 3.
Figure 3.
Podocyte cell hypertrophy compensates for reduction in podocyte density with age. Glepp1 area density does not change with successful aging (A). However, the volume occupied by podocytes (Glepp1 percentage area×glomerular volume) does increase with aging (B). The average podocyte cell volume (Glepp1 volume/podocyte nuclear number per tuft) increases with age (C). Average podocyte nuclear volume also increases with aging (D). Podocyte cell volume and nuclear volume are highly correlated (E). In contrast, average glomerular nonpodocyte nuclear volumes do not change with aging (F). Table 1 provides slopes and statistical information for these data. The data shown in Figures 2 and 3 are for male and female patients combined, using linear regression modeling. Analysis by sex showed no statistical difference between sexes (or trends toward differences) for any of the preceding variables (data not shown).
Figure 4.
Figure 4.
Podocyte density/size plot providing a mechanistic explanation for podocyte hypertrophic failure associated with aging. A plot of podocyte density against cell size with the mean and 95% confidence limits demonstrates how a reduction in podocyte density with aging necessitates an exponential (inversely proportional) increase in cell size to maintain the filtration barrier. At high podocyte densities (younger age), a density decrease is easily accommodated by small increases in podocyte size. However, at a density <100 per 106 μm (by older age), small decrements in podocyte density require large and ever-increasing compensatory changes in cell size, thereby driving hypertrophic stress and detachment. Best fit curve estimation revealed a “power curve” with R2=0.93.
Figure 5.
Figure 5.
Urine pellet podocyte mRNA markers by age showing accelerated detachment and podocyte stress in the group older than age 60 years. (A) The urine podocin mRNA-to-creatinine ratio is a measure of the rate of podocyte detachment relative to urine creatinine excretion analogous to the urine protein-to-creatinine ratio. The urine podocin mRNA-to-creatinine ratio in the >60-year-old group is increased 3.3-fold above the mean of younger people (<60 years; P<0.001). (B) The urine podocin-to-aquaporin2 mRNA ratio is a measure of the rate of podocyte detachment relative to detachment of another kidney cell type (distal tubule and collecting duct cells that express aquaporin2 mRNA) demonstrating that podocytes are lost preferentially to other kidney cells in the >60-year-old age group. (C) The urine pellet podocin-to-nephrin mRNA ratio is a ratio of two podocyte-specific markers that we have previously reported as a measure of podocyte stress. Distribution of male and female patients did not significantly differ in the various age groups. The box plots show median and interquartile ranges, and the corresponding means were compared using ANOVA with Bonferroni correction. Outliers outside the mean±2SD range are shown as circles and those outside the mean±3SD range are shown as asterisks.
Figure 6.
Figure 6.
Old kidneys show stages of podocyte stress and detachment by Glepp1 and podocalyxin immunoperoxidase histochemistry. Images from >60-year-old kidneys. (A) A subset of old glomeruli develop Glepp1-negative proteinaceous material in the Bowman space (arrowhead). (B) Glepp1-positive subcellular cell particles detach from podocytes and are present within the proteinaceous matrix in the Bowman space (arrowhead). (C) The proteinaceous matrix in the Bowman space becomes Glepp1 positive, suggesting loss of glycocalyx material from podocytes. (D–F) Glepp1-positive podocytes detach from the glomerulus and appear within proteinaceous matrix material in the Bowman space. (G) Glomeruli become globally scarred but still contain remnants of Glepp1. (H) Similar staining is present for podocalyxin, as confirmation that two independent podocyte antibodies show the same patterns. (I) Control immunoperoxidase staining showing that the observed peroxidase product was specific.
Figure 7.
Figure 7.
Detaching podocytes are multinucleate and associated with wrinkling of the GBM, glomerular collapse, and periglomerular fibrosis. (A) Normal PAS-stained old glomerulus with peroxidase-stained WT1-positive podocyte nuclei. (B) Proteinaceous material in the Bowman space is PAS positive (arrowhead) and the glomerulus shows a wrinkled appearance with early periglomerular layering. (C) WT1-positive nuclei present within the PAS-positive proteinaceous material in the Bowman space (arrowhead). (D and E) WT1-positive podocyte nuclei within the Bowman space (arrowhead). Podocyte nuclei of cells remaining attached to the glomerular tuft (arrowheads) are shown at higher power to be binucleate (E, arrowheads). (F) Detached podocytyes are binucleate (arrowheads). (G) Glomerulus with binucleate detached WT1-positive cells are present in the Bowman space (G, arrowhead) and remain attached to the glomerular tuft (H, arrowheads). (H) High-power view of panel G to illustrate binucleate cells (arrowheads) and wrinkled glomerular capillary loops (arrow). (I) Glomerulus with detached podocytes in the Bowman space (shown by arrowheads) that is wrinkled and collapsed (arrow). (J) PAS-stained section showing a glomerulus at an earlier stage with detached podocytes in the Bowman space (arrowheads) at left and a glomerulus with major tuft collapse (arrow) at right. (K) PAS-stained section with WT1 peroxidase again showing three glomeruli. The upper glomerulus appears normal. The left glomerulus shows detached podocytes (arrowhead), proteinaceous material in the Bowman space, and periglomerular layering. The right glomerulus shows major glomerular tuft collapse and global glomerulosclerosis. The average glomerular tuft diameter is 137 micrometers.
Figure 8.
Figure 8.
Glomeruli with proteinaceous material in the Bowman space have reduced podocyte density and larger podocyte nuclei. (A) Podocyte density. Kidney sections in which a large proportion of glomeruli had PMBS (n=5) (PMBS >30%) had significantly lower podocyte density than glomeruli that did not have a large proportion of glomerular tufts with protein in the Bowman space (n=15). (B) Podocyte nuclear volume as a surrogate for podocyte cell volume: WT1-positive podocyte nuclear volume was measured in glomeruli that contained Bowman space proteinaceous cast material (PMBS) versus neighboring glomeruli in the same kidney section that did not have PMBS. The average podocyte nuclear volume in glomeruli containing PMBS was larger (582±137 μm; n=609) than in neighboring glomeruli in the same kidney sections that did not contain PMBS (372±72 μm; n=554) (P<0.01), compatible with greater podocyte hypertrophy in the subset of glomeruli associated with PMBS.
Figure 9.
Figure 9.
Glomeruli from diverse forms of glomerular injury not associated with old age also show mass podocyte detachment events, multinucleate podocytes in the Bowman space, wrinkled GBM, glomerular collapse, and FGGS in the absence of arteriosclerosis. See Tables 5 and 6 for clinical information. (A) PAS-stained kidney biopsy from a 52-year-old kidney showing two normal glomeruli, one glomerulus with proteinaceous material and cells in the Bowman space, and one glomerulus with a collapsed tuft, global sclerosis, and periglomerular fibrosis. (B–D) Higher-power views of glomeruli from panel A. Note a normal glomerulus (panel A), proteinaceous material (arrow) and detached cells (arrowhead) in the Bowman space of a glomerulus with wrinkled GBM (panel C) and a collapsed glomerulus with periglomerular fibrosis (arrowhead) in panel D. (E) Glepp1-peroxidase showing that both cells (arrowhead) space and proteionaceous material (arrow) in Bowman space contain Glepp1. (F–H) Immunofluorescent images using TLE4 to identify podocyte nuclei (red in panel F) and DAPI to identify nuclei (blue with overlap between TLE4 and DAPI showing as purple). Note that detached cells contain nuclei staining for TLE4 and some nuclei are binucleate (gray arrowheads). Parietal podocytes are present. (I–K) PAS-stained sections showing detached cells in the Bowman space in biopsy specimens from 9-, 16-, and 31-year old kidneys, respectively (arrowheads). (L) Masson trichrome–stained section from a 38-year-old showing detached podocytes (arrowhead) and globally sclerosed glomerulus at right (arrow). (M) PAS staining of the same glomerulus as shown in panel L showing an apparently patent arteriole feeding the collapsed glomerulus (arrow). (N–P) Sections from a 9-year-old kidney. (N) Three glomeruli, including one glomerulus with PMBS, one glomerulus with numerous detached cells in the Bowman space (arrowhead), and one collapsed globally sclerotic glomerulus (arrow) (silver stain). (O) Collapsing glomerulus (arrow) associated with cells in the Bowman space (arrowhead). (P) Normal artery in this biopsy specimen (PAS). The average glomerular tuft diameter is 137 micrometers.
Figure 10.
Figure 10.
Multivariate regression analysis shows that ischemic-like glomeruli correlate with podocyte detachment events but not arteriosclerosis. PAS-stained sections from 44 nephrectomies (average age, 71.1±6.9 years) containing 8570 glomerular profiles were scored for glomeruli with FGGS (average, 9.5%±7.9%), PMBS (average, 1.0%±1.3%), MPDEs containing at least three cells in the Bowman space (0.2%±0.6%), ischemic-like glomerular tufts (average, 2.8%±3.7%), and arteriosclerosis score (average, 2.4±1.0; range, 0–5). Partial correlation coefficients with the corresponding P values are shown where the net relationship between two variables is a product of multivariate linear regression model after adjustment with the effect of other covariates. Note that podocyte stress/detachment events (PMBS+MPDE) correlated with ischemic-like lesions (P=0.02) while arteriosclerosis did not (P=0.21). This finding suggests that the ischemic-like lesions are not directly related to arteriosclerosis. However, all three variables (arteriosclerosis, ischemic-like lesions, and podocyte stress/detachment) independently correlated with FGGS. Bivariate comparison correlating arteriosclerosis or podocyte detachment events with FGGS showed independent correlations of approximately equal weight (R=0.4; P<0.01). This result suggests that arteriosclerosis-associated processes can independently amplify the rate of development of FGGS being caused by other mechanisms (e.g., podocyte depletion) or cause FGGS by different de novo mechanisms.
Figure 11.
Figure 11.
Glomerular life cycle hypothesis. The data suggest that a glomerulus can have a life cycle analogous to that of a star. During normal life span, podocyte density decreases. At some point critical podocyte depletion is reached when podocytes are forced into attempting to divide. Mitosis is incompatible with podocytes remaining attached to the GBM, so they detach en masse (catastrophic mitotic detachment) in a manner analogous to a supernova. Rapidly following mass detachment the glomerular tuft vasoconstriction/collapse (possibly to minimize protein loss) periglomerular fibsosis is activated, leading to global glomerulosclerosis and loss of function (analogous to a dwarf star). Under normal conditions a small proportion of glomeruli reach criticality so that the typical mass podocyte detachment events are rarely observed, but under conditions of accelerated reduction in podocyte density (due to podocyte loss and/or glomerular enlargement), this mechanism can become a major driver of FGGS.

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