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Am J Physiol Renal Physiol 294: F758-F767, 2008. First published January 16, 2008; doi:10.1152/ajprenal.00457.2007
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Glomerular hypertrophy precedes albuminuria and segmental loss of podoplanin in podocytes in Munich-Wistar-Frömter rats

D. H. T. IJpelaar,1 A. Schulz,2 K. Koop,1 M. Schlesener,2 J. A. Bruijn,1 D. Kerjaschki,3 R. Kreutz,2 and E. de Heer1

1Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands; 2Department of Clinical Pharmacology and Toxicology, Charité Universitätsmedizin, Berlin, Germany; and 3Department of Clinical Pathology, University of Vienna, Vienna, Austria

Submitted 4 October 2007 ; accepted in final form 15 January 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Focal segmental glomerulosclerosis (FSGS) is a common cause of end-stage renal disease. Albuminuria is a risk factor for FSGS and is influenced by environmental, genetic, and sex-specific factors. Podocytes play a central role in the development of albuminuria, but the precise relationship between early glomerular and podocyte-associated damage and albuminuria is unclear. Furthermore, experimental findings demonstrate a sex difference in development of albuminuria and FSGS. We investigated the early glomerular changes in male Munich-Wistar-Frömter (MWF) rats, which spontaneously develop albuminuria, and male albuminuria-resistant spontaneously hypertensive rats (SHR). In addition, since female MWF rats are protected from overt proteinuria and progressive renal disease, we compared the phenotypic changes in podocytes during early development of albuminuria in male and female MWF rats. In male MWF rats, glomerular hypertrophy preceded the onset of albuminuria and was greater than in male SHR. Albuminuria developed starting at 6 wk of age and coincided with focal and segmental loss of podoplanin, increased expression of desmin, entrapment of albumin in affected podocytes, and focal and segmental foot process effacement at the ultrastructural level. Other podocyte-associated molecules, such as nephrin and zonula occludens 1, were unaffected. Early glomerular hypertrophy and podocyte damage did not differ between male and female MWF rats. Our data show for the first time that albuminuria in male and female MWF rats is preceded by glomerular hypertrophy and accompanied by focal and segmental loss of podoplanin when FSGS was not yet present.

FOCAL SEGMENTAL GLOMERULOSCLEROSIS (FSGS) is one of the most frequent causes of end-stage renal disease (ESRD) (17). It is clinically characterized by proteinuria, usually in the nephrotic range, and development of glomerulosclerosis (39). The severity of proteinuria and the rate of progression to ESRD are influenced by sex, environmental, and genetic factors (12, 23, 33, 44). FSGS can be divided into two etiological categories: idiopathic (or primary) and secondary. The causes of idiopathic FSGS are not known and are outside the scope of this study. Secondary FSGS has multiple causes, including genetic mutations in glomerular epithelial cells, metabolic disease, glomerular immunologic injury, and hemodynamic factors (5, 6). Irrespective of the root cause, secondary FSGS is thought to result from nephron loss and subsequent hyperperfusion (4).

In recent years, podocyte damage has been demonstrated to play a central role in the development of secondary FSGS (20, 22). However, the exact relationship between damage to the podocyte and development of FSGS is unclear. It is well established that podocyte damage contributes to the development of albuminuria in human renal disease and animal models (15, 47). Albuminuria is typically accompanied by podocyte damage, loss, or reorganization of podocyte-associated molecules and flattening of the foot processes at the ultrastructural level (16). Also, damaged podocytes may detach from the glomerular basement membrane, and a reduction in the podocyte number has been linked to progression of renal disease (22, 51, 55).

Albuminuria itself is an independent predictor of the development of ESRD, and, in the microalbuminuric and even high normal normoalbuminuric range, it is an independent risk factor for cardiovascular events (7, 38, 50). Greater insight into the molecular events occurring during the early stages of microalbuminuria may lead to better understanding of the pathogenesis of proteinuria and progressive glomerulosclerosis and eventually improved therapeutic options.

However, the sequence of events in the development of microalbuminuria, podocyte-associated changes, overt proteinuria, and glomerulosclerosis is difficult to investigate in humans due to a lack of repetitive biopsies and human genetic heterogeneity. An ideal tool for investigating the time course of early changes in development of albuminuria and FSGS is the Munich-Wistar-Frömter (MWF) rat (36, 37). Male MWF rats have an inherited deficit in the number of nephrons and spontaneously develop albuminuria and mild hypertension followed at an older age by overt proteinuria, FSGS, and renal failure (9, 37). In contrast, although female MWF rats demonstrate a similar reduction in the total number of nephrons (9), they do not develop overt proteinuria or FSGS, although they exhibit mild albuminuria and mild hypertension (19, 37). Consistent with this finding, sex is known to influence the severity of proteinuria and the development of FSGS in humans and experimental models (33).

Previous studies have demonstrated a depletion of podocytes and changes in the podocyte-associated molecules zonula occludens 1 (ZO-1) and nephrin in male MWF rats with overt albuminuria and when obvious light microscopic changes are present; these studies have suggested that podocyte damage is a consequence of albuminuria and may lead to FSGS in this rat model (25, 26). In this study, we investigated the early glomerular changes that precede the development of microalbuminuria and light microscopic changes in MWF rats. In particular, we examined whether these glomerular changes involve podocyte-associated molecular changes. In addition, we compared early glomerular molecular changes between male and female MWF rats.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Study Design

Fifteen female MWF rats, 15 male MWF rats, and 15 male spontaneously hypertensive rats (SHR) were obtained from the colony at the Charite-Universitatsmedizin, Campus Benjamin Franklin (Berlin, Germany). All rats were fed a normal pellet diet and had free access to food and water. All animal experiments were previously approved by the ethical committee for animal experimentation of Leiden University and conducted in accordance with Dutch legislation on animal experiments.

Each strain was divided into three groups (n = 5) and was studied at 4, 6, and 8 wk of age. For the determination of urinary albumin excretion, rats were placed in metabolic cages for 2 days, and urine was collected over the last 24-h period. Urinary albumin excretion was determined using a sensitive and rat-specific ELISA (19).

Subsequently, rats were anesthetized by intraperitoneal injection of ketamine and xylazine. Kidneys were perfused with PBS and removed. Small pieces of the cortex were fixed in 1.5% glutaraldehyde and 1% paraformaldehyde for 24 h and stored in cacodylate buffer for electron microscopy. One kidney was snap-frozen and stored at –80°C. The other was fixed in formalin for 24 h, transferred to 70% ethanol, and embedded in paraffin for histology and immunohistochemistry.

Immunohistochemistry

Periodic acid Schiff staining was performed on 4-µm sections of paraffin-embedded samples to determine changes in morphology.

In addition to staining for 11 podocyte-associated molecules (Table 1), expression of albumin and Jg12 proteins was assessed. Jg12 is a bradykinin-degrading membrane peptidase that is expressed on glomerular and tubulointerstitial endothelial cells (30). Four-micrometer paraffin sections were dewaxed, and endogenous peroxidase was blocked by incubation with 0.12% H2O2 in PBS for 20 min. Sections stained for Wilms tumor 1 (WT-1) and Jg12 were additionally blocked with normal goat serum for 1 h. Sections were then incubated with primary antibodies diluted in 1% BSA in PBS for 2 h. Binding was detected by incubation with peroxidase-labeled secondary antibodies. Peroxidase activity was visualized using 3,3'-diaminobenzidine tetrahydrochloride (DakoCytomation, Glostrup, Denmark), and sections were counterstained with hematoxylin and mounted. Staining for nephrin and {alpha}-actinin 4 were performed on 3-µm-thick frozen sections (Table 1).


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  		Table 1. Antibodies and protocol immunohistochemistry

 
The specificity of the anti-podoplanin antibody has been described before (29, 32).

Quantification

Staining for desmin and podoplanin was analyzed by counting the percentage of glomeruli with loss of podocyte staining for podoplanin or desmin. At least 30 glomeruli per section were scored.

Slides stained for WT-1 were used for morphometric analysis of the size of the glomerulus and the number of podocytes. WT-1 is a podocyte-specific transcription factor previously used to identify and count podocytes in tissue sections (40). The number of WT-1-positive nuclei per glomerular cross-section (4 µm) was counted. Ten randomly chosen regions of the outer glomerular cortex were photographed at x200 magnification with a Zeiss Axioplan microscope equipped with a Sony DXC-950P 3CCD color camera (Sony, Tokyo, Japan). The surface area of all glomeruli in the photographs was measured using ImageJ 1.34 software (National Institutes of Health, http://rsb.info.nih.gov/ij). From these measurements, the mean glomerular volume, the number of podocytes per glomerulus, and the glomerular volume per podocyte were calculated, as described previously (49).

Electron Microscopy

Small pieces of cortex were fixed in 1.5% glutaraldehyde and 1% paraformaldehyde for 24 h and stored in 0.1 M cacodylate buffer with 6% sucrose. After postfixation in 1% reduced osmium in 0.1 M cacodylate buffer, pieces were dehydrated and embedded in Epon. Ultrathin sections were cut using a Leica Ultracut microtome and mounted on uncoated copper grids. Sections were contrasted with uranyl acetate and lead citrate and evaluated with a JEOL JEM-1011 electron microscope equipped with a digital camera.

Statistical Analysis

Results are expressed as means ± SD. ANOVA with least significant difference post hoc correction was used to test for differences between groups. Correlation coefficients were calculated using the Pearson correlation test. Statistical significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Time Course Analysis in Male MWF Rats vs. Male SHR

Albuminuria. At 4 wk of age, no appreciable amounts of albumin were detected in the urine of MWF rats or SHR. Starting at 6 wk of age, MWF rats, but not SHR, developed increased albuminuria (Fig. 1, P < 0.05).


Figure 1
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  		Fig. 1. Urinary albumin excretion (mg/24 h) in male Munich-Wistar-Frömter (MWF), female MWF, and male spontaneously hypertensive rats (SHR). Male and female MWF rats, but not SHR, developed albuminuria at 6 and 8 wk of age. At 8 wk of age, albuminuria was significantly greater in male vs. female MWF rats. **P < 0.01 vs. SHR at same age; #P < 0.05 vs. other sex of MWF rats at same age.

 
Glomerular volume and number of podocytes. The mean volume per glomerulus increased with age in both MWF rats and SHR (Fig. 2A). However, the glomerular volume was significantly higher in MWF rats vs. SHR at all time points (P = 0.001). Glomerular hypertrophy was already present at 4 wk of age in male MWF rats and preceded the development of albuminuria.


Figure 2
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  		Fig. 2. Quantification of glomerular volume (A), number of podocytes (B), and glomerular volume per podocyte (C) in male MWF rats (solid bars) and male SHR (shaded bars) at 4, 6, and 8 wk of age. A: at all time points, glomerular volume was significantly greater in MWF vs. SHR rats. B: at 8 wk, there were fewer Wilms tumor 1 (WT-1)-positive podocytes in MWF vs. SHR rats. C: at all time points, the glomerular volume per podocyte was significantly greater in MWF vs. SHR rats. **P < 0.01 vs. SHR at same age.

 
The number of WT-1-positive podocytes did not differ between MWF rats and SHR at 4 and 6 wk of age (Fig. 2B). However, at 8 wk, glomeruli from male MWF rats contained significantly fewer podocytes than did glomeruli from male SHR (P = 0.008). The glomerular volume per podocyte (Fig. 2C) was significantly greater in male MWF rats compared with male SHR at 4, 6, and 8 wk of age (P < 0.001).

Glomerular morphology and protein expression. Evaluation of periodic acid-Schiff staining revealed no obvious changes in the glomerular and tubulointerstitial compartments within the first 8 wk.

Since quantitative Western blotting of glomerular protein extracts would not reveal subtle segmental changes in the expression of glomerular proteins, we performed immunohistochemistry during the course of disease. Desmin, an intermediate-sized filament expressed by mesangial cells in normal glomeruli (13), is a well-known marker of damaged or stressed podocytes (10). At 4 wk of age, desmin protein was found only in mesangial cells in MWF rats and SHR. At 6 and 8 wk of age, desmin was focally and segmentally expressed in podocytes of male MWF rats, whereas podocytes of SHR did not express desmin at any time point (Fig. 3, a–f).


Figure 3
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  		Fig. 3. Expression of desmin and podoplanin proteins in male MWF and male SHR. Representative photographs are of desmin staining in SHR (a–c) and MWF rats (d–f) at 4 wk (a and d), 6 wk (b and e), and 8 wk (c and f) of age. At 6 and 8 wk of age, MWF rats exhibited focal and segmental expression of desmin protein in podocytes. Protein expression in SHR did not change. Representative photographs also show podoplanin staining in SHR (g–i) and MWF rats (j–l) at 4 wk (g and j), 6 wk (h and k), and 8 wk (i and l) of age. At 6 and 8 wk of age, MWF rats exhibited focal and segmental loss of podoplanin protein expression in podocytes. Protein expression in SHR did not change.

 
Consistent with the pattern of desmin expression, there was focal and segmental loss of podoplanin protein in MWF rats at weeks 6 and 8 (Fig. 3, g–l). Sequential sections of glomeruli stained for podoplanin and desmin showed that desmin was expressed in podocytes in which podoplanin expression had been lost (Fig. 4).


Figure 4
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  		Fig. 4. Sequential kidney sections from an 8-wk-old male MWF rat showing staining for periodic acid-Schiff (a), podoplanin (b), desmin (c), and albumin (d).

 
Albumin protein reabsorption droplets in proximal tubular cells were present in all groups at all ages, but were more pronounced in MWF rats at weeks 6 and 8, when albuminuria was present (Fig. 5). Concomitant with the development of albuminuria, albumin-positive granules were found in podocytes in male MWF rats. Staining of sequential sections for albumin, podoplanin, and desmin revealed that albumin droplets were present predominantly in desmin-positive, podoplanin-negative podocytes (Fig. 4).


Figure 5
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  		Fig. 5. Expression of albumin protein in male and female MWF rats and male SHR. a–f: Representative photographs of albumin expression in MWF (a–c) and SHR (d–f) at 4 wk (a and d), 6 wk (b and e), and 8 wk (c and f) of age. Albumin droplets were present in podocytes of 6- and 8-wk-old male and female MWF rats. g: Higher magnification of a glomerulus of a male MWF rat showing albumin droplets in podocytes.

 
Immunohistochemical experiments revealed no change in expression of the podocyte-associated molecules {alpha}-actinin-4, {alpha}-dystroglycan, ezrin, podocalyxin, podocin, nephrin, ZO-1, and synaptopodin at any time and in either rat strain (data not shown). To investigate changes in glomerular endothelial cells, we performed staining with the monoclonal antibody Jg12 (30). There were no changes in the endothelial distribution over time and no differences between MWF rats and SHR (data not shown).

Electron microscopy. Electron microscopy at 8 wk of age revealed focal and segmental foot process effacement in close proximity to morphologically normal capillaries with unaffected podocytes. Some podocytes with foot process effacement contained electron-dense granules, suggestive of albumin-containing absorption droplets (Fig. 6).


Figure 6
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  		Fig. 6. Electron microscope image of a glomerulus of an 8-wk-old male MWF rat. a: A normal capillary with fenestrated endothelium and normal foot processes. b: The same glomerulus as in a. This capillary is surrounded by podocytes with foot process effacement (arrow) and droplets suggestive of albumin (asterisks). Bar = 5 µm.

 
Comparison of Male and Female MWF Rats

Female MWF rats, like male MWF rats, developed albuminuria starting at 6 wk of age; levels were significantly higher in males vs. females at 8 wk of age (Fig. 1). There were no significant differences between males and females in the number of podocytes, glomerular volume, and glomerular volume per podocyte (Table 2). Immunohistochemistry on renal tissue of female MWF rats also showed focal and segmental loss of podoplanin protein expression and increased expression of desmin in the same podocytes. There were no differences between males and females in the percentage of glomeruli exhibiting loss of podoplanin (Fig. 7A). However, the percentage of glomeruli with desmin-positive podocytes was significantly higher in males vs. females at 8 wk of age (Fig. 7B). As in males, there were no changes in the expression of other podocyte-associated molecules.


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  		Table 2. Clinical and morphological parameters for female and male Munich-Wistar-Frömter rats

 

Figure 7
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  		Fig. 7. Percentage of glomeruli exhibiting podocyte loss of podoplanin and desmin in male (open bars) and female (solid bars) MWF rats at 4, 6, and 8 wk of age. a: The percentage of glomeruli exhibiting podocyte loss of podoplanin. b: The percentage of glomeruli with podocyte expression of desmin protein. *P < 0.05 vs. other sex at the same age.

 
Correlations

To identify statistically significant relationships between variables, Pearson correlations were calculated between albuminuria and podocyte-associated characteristics in the total group of MWF rats (male and female). There was a significant, positive correlation between the level of albuminuria and the percentage of glomeruli exhibiting loss of podoplanin, the increase in desmin expression, glomerular volume, and volume per podocyte in MWF (Table 3). The percentage of glomeruli with desmin in podocytes correlated positively with the percentage of glomeruli exhibiting loss of podoplanin (Table 3). Separate correlation analyses for males and females produced the same results (data not shown).


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  		Table 3. Correlations between podocyte morphology and protein expression in male and female Munich-Wistar-Frömter rats

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To our knowledge, this is the first demonstration that glomerular hypertrophy precedes the development of albuminuria in male and female MWF rats. Most interestingly, we observed that development of albuminuria at 6 wk of age is accompanied by focal and segmental loss of podoplanin protein expression in podocytes and de novo expression of desmin in the same podocytes. This occurred concomitantly with entrapment of albumin in these podocytes and focal and segmental foot process effacement at the electron microscopic level. There were no changes in other podocyte-associated molecules (Table 1).

Glomerular hypertrophy is present in biopsies of patients with secondary FSGS, and it is linked to development of proteinuria and glomerulosclerosis in humans and experimental animals (reviewed by Fogo in Ref. 11). It was hypothesized that the expansion of the glomerular tuft requires adaptation of the glomerular epithelial cells to cover a wider area of the glomerular capillary wall, a process that necessitates reorganization of the actin cytoskeleton and increased podocyte workload (21). This increased workload may lead to podocyte damage and eventually podocyte loss. The glomerular hypertrophy observed in the present study is most likely the result of the previously described inborn nephron deficit in MWF rats (9). The resulting increased workload of podocytes was followed by loss of podoplanin protein in podocytes and development of albuminuria. This strongly suggests that glomerular hypertrophy is involved in development of podocyte damage and albuminuria in MWF rats.

Loss of podoplanin protein and de novo expression of desmin in podocytes occurred simultaneously. These changes seem to be causally related, as podocytes exhibiting loss of podoplanin specifically expressed desmin. However, we do not know which event occurred first; therefore, we cannot speculate about the mechanisms preceding this very early event in FSGS. There were no changes in other podocyte-associated molecules such as nephrin and ZO-1 during the development of microalbuminuria. This suggests that segmental loss of podoplanin is the primary molecular event in the development of albuminuria and that the previously described changes in nephrin and ZO-1 protein (25, 26) are secondary to albuminuria.

Breiteneder-Geleff (3) and Matsui et al. (29) described a role for podoplanin in the development of proteinuria. This 43-kDa glycoprotein is expressed by lymphatic endothelial cells in the kidney and is also localized on cell membranes of podocytes, predominantly at the urinary surface, but also at the base of foot processes (3, 28). Podoplanin was downregulated in podocytes in puromycin aminonucleoside nephrosis, a rat model of specific podocyte damage and proteinuria (3). Furthermore, treatment with divalent IgG anti-podoplanin antibodies has been shown to induce rapidly reversible proteinuria, concomitant with extensive foot process effacement (29). Podoplanin is upregulated in the invasive fronts of several human carcinomas, and ectopic expression of podoplanin results in the formation of cell extensions and reorganization of the cytoskeleton. These observations suggest a role for podoplanin in tumor cell migration and invasion (27, 41, 43, 52, 53). Data obtained in experimental models of proteinuric renal disease (3, 29) indicate that podoplanin in podocytes may also be involved in maintaining the highly specialized structure of the podocyte and its foot processes during hypertrophy, which is essential to normal functioning of the glomerular filtration barrier (3, 29). Thus far it has been impossible to investigate this hypothesis in more detail because podoplanin-deficient mice exhibit lethal lymphatic abnormalities (41). In addition, podocytes in mice do not highly express podoplanin. Thus podocyte-specific downregulation of podoplanin in rats is more suitable to investigation of podoplanin function in the glomerulus.

We hypothesize that segmental loss of podoplanin protein leads to structural changes in podocytes and to a dysfunctional glomerular filtration barrier within the affected segments. This is supported by our observation that albumin accumulates in podocytes in which podoplanin protein has been lost. Furthermore, at the electron microscopic level, albumin droplets were specifically found in podocytes with foot process effacement. This may be the result of increased albumin filtration at these sites or an indication of increased reabsorption of albumin by these podocytes. We conclude from these results that increased albumin filtration occurs in glomerular segments exhibiting podoplanin loss. However, we did not find a correlation between the loss of podoplanin and mean area positive for albumin per glomerulus (data not shown). One explanation for this finding is that the staining for albumin is not as specific as the staining for podoplanin or desmin. Furthermore, the presence of albumin could be transient. The loss of podoplanin and increase of desmin seem more permanent, whereas albumin reabsorption and degradation is a dynamic process.

The present study showed loss of podoplanin on protein level. Podoplanin has been investigated on mRNA level in acquired human renal diseases before (18, 42). These two studies describe an increase of podoplanin mRNA level in glomeruli of patients with human renal disease, such as minimal change disease and membranous nephropathy. Although podoplanin was not investigated at protein level in these studies, in the study of Koop et al. (18), other podocyte-associated molecules were downregulated at protein level, while the corresponding mRNA expression levels were increased. This discrepancy between mRNA and protein was suggested to represent a compensatory upregulation of mRNA when protein expression was decreased. Although we have not investigated podoplanin in MWF rats at mRNA level, a compensatory upregulation of podoplanin mRNA could also be present in MWF rats.

Our results raise the question of why there are only segmental changes in the expression of podoplanin and desmin in the glomeruli of MWF rats. Although there has been thorough investigation of the role of hyperfiltration and hypertrophy in the development of podocyte damage (54), no clear explanation has been found for the segmental pattern of glomerulosclerosis. Is it a stochastic process, or is there a propensity for glomerulosclerosis at weak spots in the glomerular wall? Possibly, intraglomerular differences in capillary pressure or podocyte-associated adaptation processes play a role. As vascular damage is present in predictable locations and structures, such as branches and bifurcations in systemic hypertension, intraglomerular differences may also render some segments of the glomerulus more prone to hyperfiltration-induced damage. In MWF rats, we did not observe podocyte damage at a single preferred location in the glomerulus. Further study of intraglomerular adaptation processes is necessary to account for the segmental changes.

Segmental glomerular changes are also present in patients with FSGS (6). Although the exact mechanism underlying the development of segmental glomerulosclerosis is unknown, podocyte damage is thought to play a role. Because male MWF rats develop FSGS late in life (36), it is tempting to speculate that glomerulosclerosis will develop in glomeruli with segmental loss of podoplanin.

Genetic factors are known to determine albuminuria, renal lesions, and the inborn deficit of nephrons in MWF rats. Linkage analysis of male MWF rats and contrasting reference strains with low albuminuria have revealed multiple quantitative trait loci that are involved in the development of albuminuria but are independent of blood pressure (44, 45). Transfer of chromosome 6 of SHR into male MWF rats normalizes the nephron deficit and produces a marked suppression of albuminuria and glomerulosclerosis in the resulting consomic strain (46). Therefore, genes located within the quantitative trait loci on chromosome 6 may play a causal role in the inherited deficit in nephron number and the subsequent changes in glomeruli and podocytes reported here.

In addition to genetic factors, sex influences the severity and progression of renal disease in humans and experimental models (33). In MWF rats, there is sexual dimorphism in the development of overt proteinuria and FSGS. Males develop overt proteinuria and FSGS later in life, whereas females are protected from proteinuria and FSGS (37). Glomerular hypertrophy and loss of podoplanin are apparently not sufficient for the development of overt proteinuria and FSGS in female MWF rats. Additional factors are required for the aggravation of albuminuria in male MWF rats. The increased percentage of glomeruli with desmin-positive podocytes in males suggests an increased podocyte workload, which could contribute to the development of FSGS. It is not yet clear what sex-specific factor contributes to the increased workload in male MWF rats late in life. However, one possibility is the relatively low number of nephrons in males vs. females. Although the absolute number of nephrons is similar in male and female MWF rats, the number of nephrons corrected for body weight is lower in males (9). This may cause male MWF rats to have a less favorable ratio between the number of nephrons and metabolic demand, which may lead to aggravation of hypertrophy, albuminuria, and progressive glomerulosclerosis in males (9).

Other possibilities are specific sex-related differences in renal hemodynamics, alterations in the renin-angiotensin system, and direct effects of sex hormones (14, 31, 34). The presence of testosterone worsens the outcome of renal disease, whereas estrogens seem to be protective in several experimental models (1, 2, 8, 24, 48). Estrogen and androgen receptors within the glomerulus are thought to mediate the induction or prevention of glomerulosclerosis (8, 35). The exact influence of sex hormones on the severity of renal disease and progression to ESRD needs further investigation.

In conclusion, our data show, for the first time, that development of albuminuria in male and female MWF rats is preceded by glomerular hypertrophy and accompanied by focal and segmental loss of podoplanin protein and podocyte stress before histological signs of FSGS are present.


   ACKNOWLEDGMENTS
 
We thank Kirsten van Beelen for assistance with the immunohistochemistry. We also thank Dr. Kawachi for the anti-nephrin antibody, Drs. Schweikert and Beggs for the anti-{alpha}-actinin-4 antibody, Dr. Antignac for the anti-podocin antibody, and Dr. Miettinen for the anti-podocalyxin antibody. We thank Peter Neeskens and Frans Prins for help with the electron microscopy studies.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. H. T. IJpelaar, Leiden Univ. Medical Center, Dept. of Pathology Bldg. 1, L1Q, P.O. Box 9600, 2300 RC Leiden, The Netherlands (e-mail: d.ijpelaar{at}lumc.nl)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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K. Koop, M. Eikmans, M. Wehland, H. Baelde, D. Ijpelaar, R. Kreutz, H. Kawachi, D. Kerjaschki, E. de Heer, and J. A. Bruijn
Selective Loss of Podoplanin Protein Expression Accompanies Proteinuria and Precedes Alterations in Podocyte Morphology in a Spontaneous Proteinuric Rat Model
Am. J. Pathol., August 1, 2008; 173(2): 315 - 326.
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