1 Division of Nephrology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 2 Clinical Research Center, Samsung Biomedical Research Institute, Seoul, Korea and 3 Institute of Nephrology, Niigata University School of Medicine, Japan
Correspondence and offprint request to: Dae Joong Kim, MD, Division of Nephrology, Samsung Medical Center, 50 Ilwondong, Kangnamgoo, Seoul, Korea 135-710. E-mail: kimdjmed{at}hanmail.net
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods. Twenty rats were divided into three experimental groups (n = 16 total) and a control group (n = 4). Rats in the experimental groups received a single intravenous injection of puromycin aminonucleoside (PAN), and were sacrificed at 1 (n = 4), 2 (n = 6) and 3 weeks (n = 6) post-injection. Nephrin expression was assessed by immunoelectron microscopy using a polyclonal antibody against nephrin and gold particles. It was quantified by counting the gold particles and the slit diaphragms and by measuring the average foot process width in microphotographs.
Results. The average foot process width in the 1 week group (5924.5±1523.9 nm) was far greater than that of controls (1112.9±79.8 nm), but decreased thereafter. The average number of total gold particles per unit length (10 000 nm) of the glomerular basement membrane (GBM) underlying the foot processes was reduced at 1 week (26.0±9.5), compared with controls (335.3±125.5), but increased thereafter. Also, the average number of junctional gold particles per unit length of the GBM was lower than controls (208.4±1.7) at 1 week (10.1±3.5), but increased thereafter. There were no significant differences between the numbers of junctional gold particles per slit diaphragm among the groups, but significant differences were observed in the distributions of gold particles among the groups. Gold particles were more frequently seen in cytoplasm at 1 week.
Conclusions. The present ultrastructural studies showed that nephrin expression and its distribution were altered in PAN-treated rats, and this occurred in parallel with foot process effacement. Nephrin expression returned to normal with improved resolution of the effacement. Nephrin expression was found to be rather preserved in areas without foot process effacement, even in PAN-treated rats. The significance of the above findings in terms of proteinuria and foot process effacement needs further clarification.
Keywords: nephrin; proteinuria; puromycin aminonucleoside
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Puromycin aminonucleoside (PAN) nephrosis, a model of human minimal change disease, can be induced in experimental rats by injection of PAN [8]. Proteinuria develops from day 4 after a single injection and increases to a maximum at around day 10. Morphological changes in PAN nephrosis include pronounced effacement and fusion of foot processes, which is consistent with the general morphological features of CNF. In this study, experimental animals were sacrificed at 1, 2 and 3 weeks after injection of PAN. We investigated the distribution of nephrin on podocytes at the ultrastructural level.
![]() |
Subjects and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cortical renal tissues from each animal were immediately isolated for immunoelectron microscopic studies. The tissues were fixed in 3.5% paraformaldehyde (Sigma) and 0.01% glutaraldehyde (Electron Microscopy Science, Fort Washington, PA), in a 0.1 M phosphate buffer.
Antibodies
We used polyclonal rabbit antibodies against the intracellular domain of nephrin. Preparation of the antibodies is described in detail elsewhere [9]. Briefly, we chose a peptide of 21 amino acids, DRDTR SSTVS TAEVD M, which is located in the cytoplasmic region of the denuded amino acid sequence, as an immunogen. The rabbits were immunized with 0.1 mg of the peptide conjugated with keyhole limpet haemocyanin as a carrier protein, and boosted twice with 0.5 mg of antigen. Rabbits were sacrificed and bled 2 weeks after the last immunization.
Immunoelectron microscopy
Immunoelectron microscopy was performed by using the post-embedding immunogold technique. Samples were fixed for 2 h at 4°C, rinsed in 0.1 M phosphate buffer at pH 7.4 for 12 h and dehydrated in a graded ethanol series. They were then infiltrated initially with 50% LR white resin (London Resin, Basingstroke, UK) for 2 h in 100% ethanol and then with LR white resin for 12 h. Tissues were embedded in gelatine capsules (Agar, Essex, UK) filled with LR white resin. After UV polymerization, specimens were cut into ultrathin sections with a Leichert ultracut S (Wein, Austria) and mounted on carbon/formvar nickel grids. To minimize non-specific labelling, ultrathin sections were incubated twice with a protein blocking agent (Immunotech, Glostrup, Denmark) in PGB (1% bovine serum albumin and 0.1 M glycine in 0.1 M phosphate-buffered saline) at room temperature for 10 min. Grids were incubated for 1 h at 37°C with primary antibody (1:100 dilution for rabbit anti-nephrin antibody in PGB). After rinsing, the grids were incubated with a secondary antibody, immunogold conjugate EM goat anti-rabbit IgG (British BioCell International, Cardiff, UK), in PGB at 37°C for 30 min. The grids were then rinsed in PGB and post-fixed in 0.1 M phosphate buffer containing 1% glutaraldehyde for 10 min. The specimens were first stained with uranyl acetate, then with lead citrate, and were then examined under an electron microscope (Hitachi-7100, Hitachi, Osaka, Japan) at 75 kV.
Morphometric evaluation
Ten micrographs were taken from each sample. Printed copies with a final magnification of x 15 000 were examined and gold particles in the foot processes were counted. To quantify the amount of nephrin in and around the slits, a circle of 0.5 cm diameter on transparent film was placed centrally on each slit. Gold particles in the defined circles were counted and expressed as gold particles located at the junction. The gold particles other than junctional particles were classified as located at the basal membrane if they were located within 0.5 cm from the basal cytoplasmic membrane. The gold particles were classified as located at the apical membrane if they were located within
0.5 cm from a cytoplasmic membrane other than the basal membrane. The gold particles were classified as located within the cytoplasm if they were located within cytoplasm and the distances from the cytoplasmic membrane were > 0.5 cm.
On each photograph, the curved length of the peripheral capillary basement membrane was measured using a flat scanner and a computer-based morphometric system. The number of slit diaphragms overlying the capillary basement membrane was counted and the average foot process width (Wp) was calculated using the following formula [10]:
![]() |
The above equation was used to determine the total number of gold particles/10 000 nm Wp, the number of junctional gold particles/10 000 nm Wp, and the number of junctional gold particles/slit diaphragm.
Statistical analysis
Results are expressed as means ± SD. Between-group comparisons were analysed using the KruskalWallis test. Pairwise comparisons were performed using Scheffe's test. Fisher's exact test and multinomial logistic analysis were also used to determine the gold particle distributions. A P-value of < 0.05 was considered statistically significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunoelectron microscopy
Most of the gold particles, corresponding to nephrin, were characteristically located on the podocyte foot processes at the level of the slit diaphragms in the control kidneys (Figure 1). However, some immunogold particles were also seen in the cytoplasm and at the apical plasma membrane and basal portion of podocytes.
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Since the slit diaphragm is believed to be the ultimate barrier for the ultrafiltration of proteins such as albumin, and since proteinuria is the hallmark of most if not all glomerular diseases such as glomrulonephritis, diabetic nephropathy and hypertensive glomerulosclerosis, nephrin has been suggested to play a role in acquired proteinuric glomerular disease [5,6].
Because of this, many studies have investigated nephrin expression in acquired human glomerular disease and in experimental animal models. However, the human studies have not been consistent. Several authors found reduced nephrin expression, whereas others reported contradictory findings [6,1214]. Studies in experimental animal models, especially in puromycin-induced nephrosis models, have more consistently found that nephrin expression is decreased [5,15]. The discrepancies between these studies are difficult to explain, but may be due to different methodologies to detect nephrin expression. Morphological changes in podocytes such as foot process effacement during glomerular disease are not always consistent in the same category of disease and can change even in samples from the same disease. The foot processes may be severely effaced in certain areas of the samples, whereas they may be preserved in other areas. Therefore, when studying nephrin expression, which is localized in the slit diaphragm of the podocyte foot process, ultrastructural methods such as immunoelectron microscopy are better able to detect changes in nephrin expression in association with foot process effacement than methods such as immunohistochemistry or immunofluorescence.
Only a small number of studies have attempted a quantitative ultrastructural analysis of nephrin expression in acquired human glomerular disease or in disease models. In previous quantitative analysis based on immunoelectron microscopy, we found that nephrin expression was reduced in glomerulonephritis patients, but not in areas where the podocyte foot processes were well preserved [13]. Wernerson et al. [12] recently reported similar results. By employing a similar quantitative immunoelectron microscopy method, they found that nephrin expression was decreased in minimal change nephrotic syndrome patients, whereas it was preserved in the intact slit area [12].
The present study was designed to examine ultrastructural nephrin expression. PAN nephrosis is a well-established animal model which closely resembles human minimal change nephropathy. After administration of a single PAN injection, heavy proteinuria reaches its peak around day 10 and this is accompanied by podocyte foot process effacement. Thereafter, the proteinuria decreases with resolution of foot process effacement [8]. PAN nephrosis is an excellent model to study nephrin expression and its significance in kidney pathology since it induces reversible heavy proteinuria accompanied by reversible foot process effacement. These are the only pathologic changes and there are no changes in mesangial or capillary cells, or in inflammatory cell infiltration or immune complex deposits. Using PAN and immunoelectron microscopy, we observed overall decreases in nephrin expression where there was heavy proteinuria and foot process effacement. However, we also observed normal nephrin expression where the slit area was preserved. These findings are in good agreement with previous studies examining nephrin expression using quantitative ultrastructural analysis [8,12,13]. We found that the distribution of nephrin appeared to be altered at 1 week after PAN administration, which agrees with the report of Doublier et al. [14]. These alterations in subcellular localization of nephrin may be secondary to cytoskeletal alterations caused by puromycin [14]. Wernerson et al. [12] suggested that local reductions in nephrin below a certain level may dissolve the slit membranes causing nephrin to recirculate intracellularly [12]. We observed a slight increase in nephrin expression per slit which was not statistically significant at 1 week after PAN injection. Increased expression of nephrin has been found in the early phase of experimental diabetes, and the authors suggested a role for activated protein kinase C as a mediator of the nephrin increase [16]. Furthermore, our study showed that the altered expression of nephrin was reversed when the proteinuria decreased and foot process effacement resolved.
It is possible that the altered nephrin expression in our PAN-treated rats played a pathogenetic role in proteinuria and foot process effacement. As occurs in CNF, an altered expression of nephrin by PAN can lead to foot process effacement and slit membrane disruption that ultimately results in proteinuria. However, it is also possible that the altered nephrin expression in our PAN-treated rats occurred as a consequence of cytoskeletal alterations and podocyte foot process effacement. Indeed, several examples of this have been reported in the literature. Disruption of a single component of the cytoskeletal structure interconnecting the actin cytoskeleton, the molecules around the slit membrane such as nephrin, or cell surface molecules such as podocalyxin can lead to foot process effacement and proteinuria. Mutations of podocin [17] or -actinin-4 [18], or disruption of CD2-AP [19] or podocalyxin [20] are well-described examples of this mechanism, although the degree of proteinuria and podocyte effacement and the pace of these changes can be variable. In addition, the podocyte depletion that was reported in PAN nephrosis [21] may be linked to cytoskeletal alterations and podocyte foot process effacement, leading to the altered nephrin expression that we observed in the current experiment. Morphological studies such as ours do not allow detailed mechanistic studies of how PAN causes altered nephrin expression, nor of the pathogenetic role of this altered expression in foot process effacement and proteinuria.
In summary, our quantitative ultrastructural studies showed that nephrin expression in PAN-treated rats was altered in the areas that showed foot process effacement. However, nephrin expression was comparable with that in control animals in areas where the foot process interspaces were well preserved. The altered nephrin expression was reversed when proteinuria was reduced and foot process effacement was resolved. The significance and pathogenetic mechanism of these observations warrant further investigation.
![]() |
Acknowledgments |
---|
Conflict of interest statement. None declared.
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|