Urinary Podocyte Loss Is a More Specific Marker of Ongoing Glomerular Damage than Proteinuria

Experimental Design

All animal experiments were approved by the local review boards.

In Vivo Studies

Bladder Urine Collection and Cell Culture

To collect sterile bladder urine, animals were anesthetized by ether inhalation and an intramuscular injection of a mixture containing 80% of 2% Rompun (Bayer Vital GmbH, Leverkusen, Germany) and 20% of 10% Ketamine (CEVA Tiergesundheit GmbH, Duesseldorf, Germany). A butterfly needle was then inserted into the tail vein and a bolus of 5 to 6 ml/200 g body weight of normal saline was injected over 2 to 5 min to induce a forced diuresis as described previously (10). Next, the abdomen was opened through a longitudinal incision. The urinary bladder was punctured with a 1-ml syringe, and urine (0.8 to 1 ml) was removed and rapidly transferred into a sterile 15-ml tube. Urine pH was partially neutralized by adding 11 ml of prewarmed PBS (Life Technologies, Paisley, UK). After centrifugation at 1200 × g at room temperature for 5 min, the supernatant was removed and discarded, and the cell pellet resuspended in 12 ml of culture medium for rat podocytes (20). The medium was made up of 338 ml DMEM medium (Biochrom AG; Berlin; Germany), 113 ml Ham’s F-12 (Biochrom), 25 ml 10% FBS, 5 ml penicillin/streptomycin, 5 ml glutamine (2 mmol/L; all from Life Technologies), 5 ml HEPES (Sigma, Taufkirchen, Germany), 5 ml sodium bicarbonate (Sigma), and 5 ml trace element mix (BioSource, Solingen, Germany).

The resuspended pellet was divided into 2-ml aliquots and seeded into six-well tissue culture plates (Becton Dickinson Labware, Franklin Lakes, NJ) coated with collagen I (Bovine Collagen I; Collaborative Biomedical Products, Bedford, MA). Tissue culture plates were incubated at 37°C in 95% air/5% CO₂. Twenty-four h after seeding, red blood cells and nonattached cells were removed by washing with PBS. Adherent cells were subsequently grown at 37°C in DMEM medium containing 5% FBS, which was replaced every 3 d.

After 24 h of cell culture, the number of individual adherent cells was counted and morphologic changes were documented. Cultivated cells were further characterized by immunofluorescent staining with podocyte-specific antibodies as described previously (10).

Immunostaining

To assess podocyte cell-cycle activity in the different disease models, kidney samples from rats with PAN, anti-Thy 1.1 nephritis, 5/6-nephrectomy, and aging rats were fixed in formalin, embedded in paraffin, and cut into 2-μm thick sections. Indirect immunoperoxidase staining was performed as reported previously (7) using the following primary antibodies and overnight incubation at 4°C: cyclin D1 (mouse monoclonal; Neomarkers, Fremont, CA), cdc2 (mouse monoclonal; Neomarkers), Wilm’s tumor (WT-1) (mouse monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA), and proliferating cell nuclear antigen (PCNA) (mouse monoclonal; Oncogene, Schwalbach, Germany). Controls included omitting the primary antibody and substituting the primary antibody with irrelevant mouse IgG.

Tissue sections were boiled in citric acid (10 mmol; pH 6.0) for 10 min to unmask antigens. Nonspecific background staining was reduced with the avidin/biotin blocking kit (Vector Laboratories Inc, Burlingame, CA) and Background Buster (Innovex Biosciences, Fichmond, CA). A biotinylated horse anti-mouse secondary antibody (Vector) was then added and detected using the ABC-Kit (Vector). Antigen was finally visualized using diaminobenzidine (Sigma) with nickel chloride enhancement. To recognize binucleated or multinucleated podocytes, WT-1 staining was followed with PAS-staining.

To detect cell-cycle active podocytes in situ, double immunostaining was performed. Tissue sections were boiled in citric acid and nonspecific background staining was reduced with the avidin/biotin blocking kit as described. Staining was performed with a polyclonal rabbit anti-podocin antibody (a kind gift of Peter Mundel, Department of Medicine and Anatomy, Albert Einstein College, New York, NY) overnight at 4°C, followed by a biotinylated horse anti-rabbit secondary antibody (Vector) and the ABC-Kit. Antigen was finally visualized using diaminobenzidine (Sigma) with nickel chloride enhancement for approximately 6 min. Nonspecific background was then reduced by Background Buster followed by an overnight staining with an antibody either against cyclin D1 or PCNA at 4°C. A biotinylated horse anti-mouse secondary antibody was then added and detected using the AEC staining kit (Sigma) as described (21).

Quantitation of Immunostaining

The glomerular expression of each cell-cycle protein was evaluated in a blinded fashion in control and diseased animals at each time point. For this, 30 consecutive glomerular cross-sections were evaluated in each study and control animal by counting the number of cells staining positively for each antigen. Because all glomerular cells can express cdc2 and cyclin D1, and because this study was specifically targeted to podocytes, we counted only those PCNA and cyclin D1 cells that also expressed podocin as a podocyte-specific cell marker. Because we were not able to establish convincing double immunostaining for cdc2 and different podocyte-specific proteins, we counted those cdc2 cells that located to typical podocyte positions, i.e., to the edge of the glomerular tuft. In the case of WT-1, all positive glomerular cells were counted, because in the renal glomerulus WT-1 is specifically expressed by podocytes (22).

The number of WT-1–positive cells per glomerular cell volume was calculated using the thin and thick section method for estimation of podocyte number, glomerular volume, and glomerular volume per podocyte as described (23). In brief, mean glomerular volume in a given tissue was calculated by measuring 50 glomerular diameters and calculating the mean glomerular diameter. From these data, the maximal glomerular diameter can be derived using the Weibel formula (maximum diameter = 4/π × mean measured diameter). Because the glomerulus is seen as a sphere, mean glomerular volume can be derived from these data. To calculate the podocyte number per glomerulus, sections of different thickness (3 and 9 μm) were cut from the same tissue and WT-1–positive nuclei counted in each. The difference in podocyte counts between thin and thick sections is directly proportional to the difference in slice thickness. For both thin and thick sections, the mean podocyte number per glomerular tuft area is calculated. Differences in podocyte number are related to the difference in section thickness (6 μm). By dividing the known difference in section thickness by the podocyte number per glomerular area, one can derive the glomerular volume per podocyte. The mean podocyte number per tuft is equal to the mean glomerular volume divided by glomerular volume per podocyte.

Mean values per time point were calculated and the results were expressed as the number of cells staining positively per 30 glomerular cross-sections in the case of cdc2, cyclin D1, and PCNA or as the number of cells per 50 glomerular volumes in the case of WT-1.

Statistical Analysis

All data are expressed as mean ± SD. Statistical significance (defined as P < 0.05) was evaluated using the Bonferroni t test and Spearman test.

Viable Podocytes Can Be Cultivated from the Urine of Study Rats in Different Disease Models

Similar to recent data obtained in the rat model of passive Heymann nephritis (10), the induction of other glomerular diseases in rats was paralleled by the appearance of podocytes in the urine. The cells were viable and attached to the culture dishes. After 24 h of culture, colonies of cells with similar morphologic features were visible (Figure 1A). During the following days, they showed an increase in cell size and other characteristic changes in cell morphology (Figure 1B) as described recently in urinary cells cultivated from rats with passive Heymann nephritis (10). Approximately one-third of the podocytes cultured ex vivo exhibited polyploidy, again confirming earlier observations (10). By immunofluorescent staining with antibodies against nephrin and podocin, all cells in culture stained positively for these specific podocyte proteins from day 1 of cell culture onwards (data not shown). However, in this study we failed to note any increase in cell numbers over time, which is in contrast to our recent data obtained in passive Heymann nephritis (10). Rather, despite changing the culture medium every third day, cells rounded up and finally detached over a time course of approximately 15 d.

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Figure 1.

Morphology of cultivated urinary cells ex vivo. The characteristic morphology is shown after (A) 24 h (magnification ×400) and (B) 10 d of cell culture (magnification ×200). Note the increase in cell size, characteristic changes in morphology as described in detail previously (10), as well as the appearance of binucleated or multinucleated cells (*).

Although an intravenous fluid bolus was used before harvesting the cells from the bladder to standardize experimental conditions, we noted that a similar amount of cells also appeared in the urine without fluid loading (data not shown). More importantly, loss of these cells into the urine was disease-specific, because with or without fluid loading, such cells were noted in normal control rats.

Urinary Podocyte Loss Exhibits a Characteristic Time Course in Each Disease Model and Can Be Dissociated from Proteinuria

In healthy control rats as well as in aging rats of up to 1 yr of age, we consistently failed to detect any podocytes in the urine (data not shown) despite the slow development of age-dependent proteinuria (1-mo-old rats: 26 ± 2 mg/24 h versus 12-mo-old rats: 84 ± 11 mg/24 h).

After the induction of PAN in rats, low numbers of urinary podocytes first appeared in some animals on days 3 and 5 (Figure 2A). On day 10 after disease induction, podocyturia increased massively in all animals and reached 8000 or more cells after 24 h of culture (Figure 2A). These changes closely paralleled the onset of marked proteinuria (Figure 2A). However, whereas proteinuria remained high on day 15, podocyturia declined sharply to near baseline (Figure 2A).

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Figure 2.

Time course of podocyturia and proteinuria in the different disease models. (A) puromycin aminonucleoside-induced nephrosis (PAN) model. (B) Anti-Thy 1.1 nephritis. (C) 5/6 nephrectomy. Data are means ± SD; n = 6 per time point. *P < 0.05 versus d or wk 0. **P < 0.01 versus d or wk 0.

In the anti-Thy 1.1 model of mesangioproliferative nephritis, low-grade podocyturia (approximately 30-fold lower than that observed in PAN) was transiently detected on days 4 and 5 but at no other time points (Figure 2B). Days 4 and 5 of the disease are characterized by the onset of massive mesangial cell proliferation and matrix expansion, which subsides at later time points such as days 21 and 30 (24,25). As in rats with PAN, proteinuria increased in parallel with the occurrence of podocyturia but persisted on day 21 when podocyturia had already subsided (Figure 2B).

Podocyturia in rats with 5/6-nephrectomy, in which glomerular injury in contrast to PAN and anti-Thy 1.1 nephritis is continuous, followed a completely different time course. It was first detected in some animals at week 1 and 2 after disease induction and markedly increased toward the end of the observation period, i.e., week 12 (Figure 2C). Also, in 5/6-nephrectomized rats, podocyturia did not dissociate from proteinuria and rather exhibited a significant correlation with proteinuria (Figure 3A).

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Figure 3.

Relationship between podocyturia and proteinuria (A), mean arterial BP and podocyturia (B), and the number of Wilm’s tumor (WT-1)–positive nuclei in podocyte locations (C) at the different time points after 5/6-nephrectomy in rats. **P < 0.01.

In contrast to PAN and anti-Thy 1.1 nephritis, the 5/6-nephrectomy model is characterized by systemic hypertension (Table 1). We therefore investigated a potential relationship between podocyturia and mean arterial BP. As shown in Figure 3B, BP and the extent of podocyturia were positively correlated.

Urinary Podocyte Loss Is Not Associated with a Significant Decrease in Glomerular WT-1–Positive Cells

WT-1 is a nuclear protein specific for podocytes and parietal glomerular epithelial cells in the adult kidney (22). WT-1–positive cells within the glomerular tuft excluding the parietal epithelium were counted at each time point in aging rats as well as in rats with PAN, anti-Thy 1.1 nephritis, and 5/6-nephrectomy, and the number per glomerular cell volume was calculated as described (23) to determine whether podocyte loss into the urine led to a reduction of glomerular podocyte numbers.

Despite a trend toward lower podocytes per glomerulus in both the PAN and anti-Thy 1.1 nephritis model (Figure 4, A and B), none of the changes reached statistical significance.

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Figure 4.

Quantification of WT-1–positive podocytes in glomeruli during the course of PAN (A), anti-Thy 1.1 nephritis (B), or after 5/6-nephrectomy including age-matched normal controls (C). Data are means ± SD; n = 6 per time point. *P < 0.05 versus d or wk 0. **P < 0.01 versus d or wk 0. (D) Example of multinucleated podocytes staining positively for WT-1 on day 15 of PAN.

Assessment of glomerular WT-1 expression was notable for the appearance of WT-1–positive binucleated or multinucleated cells in all disease models examined (Figure 4D) but not in normal controls or aging rats.

Increase in Cell-Cycle Markers Precedes Podocyturia

In our experiments, PCNA was used as a marker of cell-cycle activity, cyclin D1 as a marker of the G1 phase, and cdc2 as a marker of the G2/M-phase (26,27). In the case of PCNA and cyclin D1, double immunostaining with podocin was performed to prove the podocyte character of cell-cycle active cells. In healthy control animals, rare glomerular cells expressed either of these cell-cycle markers (Figure 5, C and D). Rarely, expression of PCNA or cyclin D1 was noted in normal podocytes (Figure 5, C and D). Also, no increase in cell-cycle activity above this low baseline level occurred during normal aging (data not shown). Immunohistological control stains, in which the primary antibody was replaced by irrelevant mouse IgG, resulted in the complete loss of immunoreactivity (data not shown).

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Figure 5.

Immunohistological detection of proliferating cell nuclear antigen (PCNA) (A, B) and cyclin D1 (C, D) by AEC staining in normal control rats (C, D) and rats at day 10 after induction of PAN (A, B). Rare intraglomerular cells express PCNA (C) or cyclin D1 in normal rats (D) (red color). In PAN, PCNA is detectable in both cells within the glomerular tuft and at the edge in podocyte locations (A). Expression of cyclin D1 in PAN can be demonstrated in podocytes as well (B). Podocytes can be differentiated from nonpodocytes by double immunostaining with an antibody against podocin (brown color). Podocin expression appears similar in control and disease animals. Immunohistological detection of cdc2 in the same tissue samples was performed using diaminobenzidine in control rats (E) and PAN rats (F). Immunoperoxidase staining, original magnification ×400. *Cell-cycle–positive podocytes.

In rats with PAN, PCNA cyclin D1 and cdc2 expression was noted in cells within the glomerular tuft (Figure 5, A and B) and in podocytes. When podocytes were assessed separately, the number of PCNA or cyclin D1-positive podocytes increased significantly on day 2 after disease induction and thereby preceded the increase of the G2/M-phase marker cdc2 by 1 d (Figure 5E). All three markers showed maximum expression on day 10 and decreased thereafter (Figure 6A), thus mirroring the course of podocyturia (Figure 2A).

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Figure 6.

Time courses of the expression of cell-cycle markers PCNA, cyclin D1, and cdc2 in podocytes of rats with PAN (A), anti-Thy 1.1 nephritis (B), and 5/6-nephrectomy (C). *P < 0.05 versus day or week 0. **P < 0.01 versus day or week 0.

In the anti-Thy 1.1 nephritis model, expression of all three cell-cycle markers in podocytes increased on day 2 and reached a maximum on day 4 to 5 (Figure 6B), i.e., paralleling the podocyte loss in urine. Whereas cell-cycle markers subsequently remained elevated (Figure 6B), podocyturia returned to baseline (Figure 2B).

After 5/6-nephrectomy, we failed to detect any significant changes in cell-cycle activity, i.e., PCNA, cyclin D1, or cdc2 expression, over the whole period of observation (Figure 6C).