Expression of human nephrin mrna in diabetic nephropathy

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1 Nephrol Dial Transplant (2004) 19: DOI: /ndt/gfg545 Original Article Expression of human nephrin mrna in diabetic nephropathy Masao Toyoda, Daisuke Suzuki, Tomoya Umezono, Goro Uehara, Mayumi Maruyama, Masashi Honma, Takako Sakai and Hideto Sakai Division of Nephrology and Metabolism, Department of Internal Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan Abstract Background. Diabetic nephropathy (DN) is associated with functional changes in the filtration barrier, and microalbuminuria is a strong predictor of the development of overt DN. Nephrin is a novel podocytespecific protein which localizes at the slit diaphragm. This study examines the expression of nephrin mrna in the kidneys of type 2 diabetics with DN. Methods. Renal tissues were obtained from 13 type 2 diabetics with DN. We also examined samples from five patients with minimal change nephrotic syndrome (MCNS) and five normal kidneys (normals) as control. The severity of DN was classified into two grades based on histopathological findings. DN grade 1 (DN1 ¼ seven patients) presented mild mesangial expansion, and DN grade 2 (DN2 ¼ six patients) moderate mesangial expansion. Nephrin mrna was quantitated and localized by in situ hybridization. Results. Cells positive for nephrin mrna were detected exclusively in glomerular epithelial cells. The percentage of cells positive for nephrin mrna in DN2 was significantly lower than in MCNS and normal kidneys. Furthermore, there was an inverse correlation between the percentage of cells positive for nephrin mrna and extent of proteinuria. Conclusion. The low expression of nephrin mrna may be closely linked to development and/or progression of proteinuria in human diabetic nephropathy. Keywords: diabetic nephropathy; in situ hybridization; nephrin; proteinuria Introduction Many kidney diseases entail renal failure or proteinuria. Proteinuria is a sign of serious renal problems, and Correspondence and offprint requests to: Daisuke Suzuki, MD, PhD, Division of Nephrology and Metabolism, Department of Internal Medicine, Tokai University School of Medicine, Isehara, Kanagawa , Japan. daisuke@is.icc.u-tokai.ac.jp frequently indicates a poor prognosis. Proteinuria has also been considered an important risk factor for the progression of diabetic and non-diabetic renal disease. The dysfunction of the glomerular filter, leading to extensive leakage of plasma proteins and diffuse alteration of podocyte foot processes (detected by electron microscopy), is observed in various forms of glomerulonephritis or nephropathy. Heavy proteinuria results in the nephrotic syndrome. The molecular processes underlying glomerular diseases with proteinuria are not fully understood, although increasing evidence suggests a key role for podocytes in permeability changes at the glomerular filtration barrier. Congenital nephrotic syndrome of the Finnish type (NPHS1) is an autosomal recessive disorder characterized by massive proteinuria beginning in utero. Recently, mutations in the NPHS1 gene were identified as responsible for NPHS1 [1]. NPHS1 is a recently discovered 29-exon gene that codes for nephrin, a cell adhesion protein that is expressed on podocytes and localized at the slit diaphragm [2]. Experimental animal models of glomerulonephritis have shown a correlation between the alteration of nephrin expression and proteinuria [3 5]. Therefore, the expression of nephrin has been studied in certain acquired kidney diseases [6 10]. The aim of this study was to investigate the expression of nephrin mrna in different severities of human diabetic nephropathy (DN) using nonradioactive in situ hybridization. Subjects and methods Patients Open renal biopsy tissues were obtained from 13 patients with type 2 diabetes mellitus and DN. The presence of DN was confirmed by histologic evaluation of renal biopsy specimens using light microscopy, immunofluorescence staining and electron microscopy. After resection, kidney samples were embedded in compound OCT (Tissue Tek; Miles, Elkhart, IN) and stored for future use. The severity of Nephrol Dial Transplant Vol. 19 No. 2 ß ERA EDTA 2004; all rights reserved

2 Nephrin in diabetic nephropathy 381 Fig. 1. Light microscopic findings in glomeruli from representative patients with DN prepared by periodic acid-schiff staining. The severity of DN was classified based on the degree of mesangial expansion. (a) DN1. Note the mild degree of mesangial expansion (original magnification 100). (b) DN2. Note the moderate degree of mesangial expansion (100). Table 1. Clinical characteristics of patients at the time of open renal biopsy DN was classified into two grades based on examination of the biopsy specimens: DN grade 1 (DN1 ¼ seven patients) presented mild mesangial expansion while DN grade 2 (DN2 ¼ six patients) had moderate mesangial expansion [11] (Figure 1). We also examined control samples from five patients with minimal change nephrotic syndrome (MCNS) and five others from whom we used uninvolved portions of surgically removed kidneys afflicted with malignancies. The patients with MCNS fulfilled the criteria for MCNS clinically and histopathologically. The Human Research Committee of Tokai University School of Medicine approved the study, and informed consent for open renal biopsy and in situ hybridization studies was obtained from each patient prior to the commencement of the study [12]. The following clinical parameters were noted or tested at the time of open renal biopsy: gender, age, serum creatinine, total protein, HbA1c, urinary protein and creatinine clearance (Table 1). In situ hybridization The nucleotide sequence of human nephrin cdna was used as the probe. The probe for nephrin corresponded to DN1 (n ¼ 7) DN2 (n ¼ 6) MCNS (n ¼ 5) Normals (n ¼ 5) Gender (M/F) 6/1 5/1 4/1 2/3 Age (years) 37.4 ± ± 14.0 a 26.4 ± ± 7.8 b Serum creatinine (mg/dl) 0.73 ± ± 1.04 c 0.86 ± ± 0.14 Total protein (g/dl) 6.97 ± ± ± 0.90 d 6.48 ± 0.41 HbA1c (%) 11.0 ± ± 2.8 N.D. N.D. Urinary protein (g/day) 0.25 ± 0.27 e 1.32 ± 1.34 f 4.71 ± 3.13 f 0.02 ± 0.06 Creatinine clearance (ml/min) 91.5 ± ± ± ± 2.4 Data are presented as mean ± SD. a P < 0.05 vs MCNS. b P < 0.05 vs DN1, DN2 and MCNS. c P < 0.05 vs DN1, Normals and MCNS. d P < 0.05 vs DN1, DN2 and Normals. e P < 0.05 vs Normals. f P < 0.01 vs DN1 and Normals. N.D., not done. sequence nos of human nephrin cdna. Of the oligonucleotide probe, 100 pm was labelled, using a digoxigenin (DIG) oligonucleotide tailing kit according to the standard protocol (Boehringer Mannheim, Mannheim, Germany). The free DIG was removed by ethanol precipitation, and dissolved in diethylpyrocarbonate-treated water. In situ hybridization was performed according to the modified technique developed in our laboratory [11]. Briefly, freshly biopsied kidney tissue was embedded in compound OCT and stored at 70 C for future use, when specimens were cut to a thickness of 4 mm, and sections were fixed in 4% paraformaldehyde in phosphate-buffered saline and then deproteinized with HCl and digested with proteinase K (Sigma Chemical, St Louis, MO). The specimens were prehybridized in a hybridization buffer, drained, and then hybridized overnight with a DIG-labelled oligonucleotide probe in the hybridization buffer. After hybridization, the DIG-labelled probe was visualized by immunohistochemical staining using a mouse monoclonal anti-dig antibody (Boehringer Mannheim), horseradish peroxidase (HRP)- conjugated rabbit anti-mouse IgG antibody (Dako, Glostrup, Denmark), and HRP-conjugated swine anti-rabbit IgG antibody (Dako). Colour was developed with diaminobenzidine tetrahydrochloride in 0.05 M Tris HCl, ph 7.6, and

3 382 M. Toyoda et al. 0.03% H 2 O 2. Sections were briefly counterstained with haematoxylin, rinsed, dehydrated, cleared in xylene and mounted. To evaluate the specificity of the signals, two types of control experiments were carried out as described previously [11]. First, pre-treatment with RNase (Boehringer Mannheim Biochemica) was done after proteinase K digestion, following which sections were pre-hybridized and hybridized. Second, a competitive study was performed by adding a 100-fold excess amount of homologous or unrelated, unlabelled oligonucleotides to the hybridization buffer together with the antisense probe. Statistical analysis To quantify the expression of nephrin mrna, we counted the total number of nuclei and the subpopulation of nuclei surrounded by nephrin mrna-positive cytoplasm in at least 10 randomly selected cross-sections of non-sclerotic glomeruli. Quantification of nephrin mrna expression was done by three independent investigators who were blind to the histopathologic classification of each specimen. The results of the quantification were expressed as the percentage of mrna-positive cells relative to the total number of glomerular cells. All results are shown as mean ± SD. Differences between groups were analysed for statistical significance using the Mann Whitney U-test. The relationships between nephrin mrna expression and clinical parameters were examined using linear regression analysis. A P value of <0.05 was considered statistically significant. Results Detection of nephrin mrna by in situ hybridization Our in situ hybridization method demonstrated cells positive for nephrin mrna in renal tissues of DN and MCNS and in normal kidneys (normals). As shown in Figure 2, in situ hybridization followed by periodic acid-schiff staining clearly identified individual cells positive for nephrin mrna. High-magnification views indicated that these cells were exclusively glomerular epithelial cells (Figure 2a). As shown in Figure 2b e, the number of glomerular cells positive for nephrin mrna in DN2 was lower than in DN1, MCNS and normals. Fig. 2. In situ hybridization of nephrin mrna in glomeruli from normal human kidneys (normals), MCNS and diabetics. In situ hybridization followed by periodic acid-schiff staining revealed that nephrin mrna was localized in exclusively glomerular epithelial cells (arrows) [(a) normal, 200]. Cells positive for nephrin mrna in glomeruli of normals, MCNS and DN. The proportion of glomerular cells positive for nephrin mrna in DN2 was less than in normals and MCNS [(b) normal, 100; (c) MCNS, 100; (d) DN1, 100; (e) DN2, 100]. Furthermore, in a competitive experiment, the signals disappeared when a 100-fold excess of unlabelled homologous oligonucleotide was added to the standard hybridization mixture containing the labelled probe [(f) normal, 100].

4 Nephrin in diabetic nephropathy 383 The specificity of the nephrin mrna signal detected by in situ hybridization was confirmed by two control studies. The signal was diminished in tissues pre-treated with RNase prior to hybridization (data not shown). Furthermore, in a competitive experiment, the signals disappeared when a 100-fold excess of unlabelled homologous oligonucleotide was added to the standard hybridization mixture containing the labelled probe (Figure 2f). nephrin mrna expression and total protein (r 2 ¼ 0.460, P ¼ ), but there was no correlation with any other clinical parameter, including HbA1c (r 2 ¼ 0.079, P ¼ ), serum creatinine (r 2 ¼ 0.118, P ¼ ) and creatinine clearance (r 2 ¼ 0.030, P ¼ ). Discussion Comparison and correlation of nephrin mrna The results of staining for nephrin mrna are summarized in Figure 3. The percentage of cells positive for nephrin mrna in DN2 was significantly lower than in MCNS and normals. The percentage of cells positive for nephrin mrna in DN1 was not significantly different from that of MCNS and normals. Furthermore, there was an inverse correlation between the percentage of cells positive for nephrin mrna and the extent of proteinuria when all patients with DN were analysed (Figure 4). We also analysed the correlation between the percentage of glomerular cells positive for nephrin mrna and other clinical parameters. There was a significant correlation between Fig. 3. Percentage of cells positive for nephrin mrna in normals, MCNS, DN1 and DN2. *P <0.05 vs normals and MCNS. Fig. 4. Linear regression analysis of the percentages of cells positive for nephrin mrna and extent of proteinuria in all DN patients. The ultrafiltration of plasma components during the formation of primary urine in the glomerulus is one of the important functions of the human kidney. The glomerular filtration barrier is formed of three layers: the podocyte, the glomerular basement membrane (GBM) and the endothelium. The podocyte foot processes are connected just above the GBM by a special structure known as the slit diaphragm. This structure bridges the filtration pores between adjacent podocytic foot processes. Based on experimental animal models and some human data, it was for a long time believed that defects in the GBM were responsible for proteinuria. However, a number of studies investigated the direct role of the slit diaphragm in the pathogenesis of proteinuria. Their results suggest that the slit diaphragm is important for the maintenance of the glomerular filter [13]. Nephrin is a major protein in the podocyte slit diaphragm [2]. That it has a crucial role in maintaining the glomerular filter is suggested by the characteristic nephrin mutations in NPHS1 patients, and the remarkable down-regulation of nephrin mrna expression in experimental animal models [4]. However, the molecular pathogenesis of proteinuria in glomerular diseases is poorly known. DN is a major cause of glomerular injury and proteinuria. Increased proteinuria heralds a gradual deterioration of glomerular function [14]; therefore, it is important to elucidate the mechanisms of proteinuria in DN. In this study, we determined the expression of nephrin mrna in different severities of DN using non-radioactive in situ hybridization. Our results clearly demonstrate the presence in DN of renal cells positive for nephrin mrna. To distinguish individual cells in the glomeruli positive for nephrin mrna, we performed PAS staining after in situ hybridization, and then defined the topographical relationship of positively stained cells relative to the glomerular basement membrane and mesangial matrix. The signal for nephrin mrna was present among resident glomerular cells, in exclusively glomerular epithelial cells. This result is compatible with the findings of Kestila et al. [1] and Patrakka et al. [7]. In the experimental animal model of minimal change nephrosis, puromycin aminonucleoside nephrosis, a correlation has been shown between the low expression of nephrin mrna and proteinuria [3,4]. More recently, some studies that examined the expression of nephrin mrna in animal diabetes suggested that the low expression of nephrin mrna is linked to the development of proteinuria in DN [5,15]. Therefore,

5 384 M. Toyoda et al. we quantitated the relative number of glomerular cells positive for nephrin mrna. In glomeruli, no significant difference was seen between the expression levels in DN1, MCNS and normals; however, the proportion of cells positive for nephrin mrna in DN2 was significantly lower than in MCNS and normals. These results suggest that nephrin mrna expression correlates with renal tissue damage in DN. We found that the expression level of nephrin mrna was significantly different between DN and MCNS. This also suggests that the pathogenetic mechanisms underlying a particular disease may alter the expression of nephrin mrna. Furthermore, our results revealed a significant inverse correlation between the percentage of cells positive for nephrin mrna and the extent of proteinuria in DN. Doublier et al. [8] studied the expression of nephrin protein in biopsies from patients with primary acquired nephrotic syndrome. They reported that nephrin expression was significantly reduced in those patients, and there was an inverse correlation between nephrin expression and extent of proteinuria. Our results are in agreement with their findings: nephrin expression was decreased in advanced stages of DN and inversely correlated with the extent of proteinuria. A number of studies on the expression of nephrin mrna in animal diabetes report low nephrin mrna expression in the late stages of the disease [5,15]. Two mechanisms were proposed for the downregulation of nephrin mrna in DN. One is the characteristic structural alteration of glomerular epithelial cells, i.e. alteration of foot processes and filtration slits [16]. The other is loss of podocytes [17]. We counted the number of nephrin mrna-positive cells to evaluate nephrin mrna expression. In view of its exclusive expression, we should consider the possibility that these cell numbers themselves reflect the numbers of podocytes; in other words, it is also probable that the loss of podocytes is closely related to the extent of proteinuria or progressive glomerular injury, or both, in DN. From our results it is difficult to determine if reduced nephrin mrna expression in DN2 is due to the down-regulation of nephrin by the diabetic state or due to podocyte loss. The precise mechanisms of the down-regulation of nephrin mrna in advanced stages of DN still are not fully understood. Recent studies suggest that hyperglycaemia, hypertension, angiotensin II, reactive oxidants and protein kinase C [4,15,18] might be involved. More recently, Doublier et al. [19] reported that in DN nephrin protein had a reduced expression and altered distribution. They suggest that glycated albumin and angiotensin II contribute to the down-regulation mechanisms of nephrin. In the present study, nephrin mrna expression did not correlate with HbA1c or systolic and diastolic blood pressures. However, we recently reported down-regulation of PKC mrna expression in human late-stage DN [20]. The present study, however, could not determine whether downregulation of nephrin mrna expression is the cause of proteinuria or the result of renal damage. Therefore, further studies are necessary to identify the mechanisms of down-regulation of nephrin mrna. In conclusion, we used human renal tissue samples from patients with DN to demonstrate nephrin mrna expression by DIG-labelled in situ hybridization. mrna expression decreased with the progression of renal injury and inversely correlated with the extent of proteinuria. Our results suggest that in human DN a decrease in nephrin mrna expression may be closely linked to the development or progression of proteinuria, or to both. Conflict of interest statement. None declared. References 1. 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