Hypertrophy and Loss of Podocytes in Diabetic Nephropathy

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1 ORIGINAL ARTICLE Hypertrophy and Loss of Podocytes in Diabetic Nephropathy Masaaki Miyauchi, Masao Toyoda, Keiko Kobayashi, Makiko Abe, Takako Kobayashi, Mayuko Kato, Naoyuki Yamamoto, Moritsugu Kimura, Tomoya Umezono and Daisuke Suzuki Abstract Objective The loss of podocytes has been reported to have a role in the onset and progression of diabetic nephropathy (DN). Although structural changes such as podocyte hypertrophy are considered to be associated with podocyte loss, the relationship has not been thoroughly investigated using human DN renal tissues. Methods The subjects were 17 patients with DN diagnosed histopathologically by renal biopsy. Immunostaining was performed with antibodies for Wilm s tumor 1 (WT1) and synaptopodin (SPD), which are markers of podocytes, to determine the number of podocytes and assess podocyte hypertrophy. Results The number of podocytes was decreased in DN patients compared with the controls. An inverse correlation was observed between the number of podocytes and both the urinary protein excretion and the extent of mesangial expansion. Podocyte hypertrophy was also more marked in DN patients compared with controls. Conclusion Based on these results, podocyte loss and hypertrophy were suggested to be involved in the development and progression of human DN. Key words: podocyte loss, podocyte hypertrophy, diabetic nephropathy () () Introduction The number of patients requiring dialysis due to diabetic nephropathy (DN) is increasing year by year in Japan, and it has currently become the number one reason for starting dialysis, followed by chronic glomerulonephritis. The mechanisms leading to the onset and progression of DN should be urgently clarified in order to improve the quality of life of patients with renal failure and to reduce medical costs. The relationship between podocyte damage and renal disease has attracted attention in recent years and reports about involvement of podocyte loss in the progression of DN have been published (1-5). The association of podocyte hypertrophy with podocyte loss has also been reported (6, 7), however, this relationship has not been examined in detail using human DN renal tissues. Recently, some podocyte specific proteins have been reported. The Wilm s tumor-1 (WT1) gene encodes the WT1 protein, which is expressed throughout urogenital development and continues to be highly expressed in podocytes. Furthermore, WT1 may play an important role in the maintenance of podocyte function (8, 9). On the other hand, synaptopodin (SPD) is a proline-rich protein, intimately associated with the actin microfilament, and it is exclusively expressed in the foot process of podocytes (10). While SPD is expressed in the foot processes, WT1 is exclusively expressed in the nucleus of the podocyte. A recent study using an experimental diabetic rat model reported down regulation in both WT1 and SPD rats compared with normal condition rats, and these results may be related podocyte loss (11). In the present study, we aimed to clarify the relationship between podocyte loss and podocyte hypertrophy based on various clinical indexes and immunostaining of human DN Division of Nephrology and Metabolism, Department of Internal Medicine, Tokai University School of Medicine, Shimokasuya, Isehara Received for publication February 7, 2009; Accepted for publication May 27, 2009 Correspondence to Dr. Daisuke Suzuki, daisuke@is.icc.u-tokai.ac.jp 1615

2 renal tissues for WT1 and SPD, which are different distribution markers of podocytes. Table1. ClinicalCharacteristicsbetween DN and Control Subjects and Methods Patients The subjects were 17 patients in whom kidney biopsy was performed at Tokai University Hospital and DN was histologically diagnosed based on the results of light microscopy [with periodic-acid-schiff (PAS), hematoxylin-eosin (HE), and periodic acid methenamine silver (PAM) staining, fluorescence microscopy, and electron microscopy]. We also examined control samples obtained from 10 subjects using uninvolved portions of surgically removed kidneys afflicted with malignancies (Table 1). The gender, age, blood pressure, urinary protein excretion (U-Prot), creatinine clearance (Ccr), and angiotensinconverting enzyme inhibitors (ACE-Is) and/or angiotensin II receptor blocker (ARBs) administration were investigated. The extent of mesangial expansion was investigated as a pathological index of the progression of DN. In the present study, we excluded severe mesangial expansion in which Kimmelstiel-Wilson nodules were observed. Mesangial expansion was evaluated by randomly selecting at least 5 glomeruli in which the vascular pole could be identified, and calculating the PAS-positive area in each glomerulus by automated computer analysis with WinRoof software (Mitani Co., Fukui, Japan). Written informed consent was obtained from each patient for the use of their clinical data and the residual renal tissue after a pathological diagnosis had been made by renal biopsy. Immunohistochemistry WT1 and SPD double staining Paraffin-embedded tissues were cut into 4 μm sections. After deparaffinization and dehydration in an ethanol series, antigen retrieval was performed by heating in a microwave oven (750 W for 5 min 5 times in citrate buffer). After cooling, endogenous peroxidase was inhibited by incubation in MeOH-H2O2 (100 : 1) for 15 min. The specimens were rinsed in phosphate-buffered saline (PBS), blocked in the normal goat serum (15 : 1,000) for 10 min, followed by incubation with rabbit anti-wt1 antibody (1:50) (sc-192, Santa Cruz Biotechnology, CA, USA) overnight at 4. Then, they were rinsed in PBS, incubated in biotin-anti rabbit IgG (made in goat) for 1 hour, rinsed in PBS, and reacted with Vectastain ABC Reagent (Vector Laboratories, Burlingame, CA, USA). After rinsing with PBS, the specimens were stained with Ni-Co-diaminobentidine (DAB) (Nichirei Bioscience, Tokyo, Japan). Then, the specimens were incubated again in MeOH-H2O2 (100 : 1) for 15 min, rinsed in PBS, incubated in avidin solution, and then in biotin solution. After blocking the specimens with normal goat serum, incubation was done overnight at 4 with mouse anti-synaptopodin (SPD) antibody (1 : 100) (G1D4, Progen, Heidelberg, Germany). After rinsing with PBS, the specimens were reacted with the biotin-anti mouse IgG (made in goat) and Vectastain ABC reagent. After rinsing with PBS, DAB staining was performed for a predetermined duration to avoid any influence of changes in the duration of staining. At least 5 glomeruli, in which the vascular pole could be identified, were randomly selected from each patient. WT1- positive cells located in the SPD-positive area or proximal to the urinary space (WT1-positive/SPD-positive cells) were defined as podocytes (Fig. 1). The number of these cells in each glomerulus was counted. The SPD-positive area of each glomerulus was calculated by automated computer analysis with WinRoof software (Fig. 2). The ratio of the SPD-positive area to the number of WT1-positive/SPDpositive cells were calculated as indexes of podocyte hypertrophy. Statistical analysis Results are shown as the mean ± SD, and the Man- Whitney U-test was used for comparison of 2 groups. The Kruskal-Wallis test was used for comparison of 3 or more groups. Spearman s rank correlation analysis was used to assess the correlation between each group. In all analyses, p< 0.05 was considered statistically significant. Stat view 5.0 software (SAS Institute Inc., Cary, NC, USA) was used for analysis. Results Podocytes were identified by immunostaining for WT1 and SPD (Fig. 1). The number of WT1-positive/SPDpositive cells (podocyte count) was significantly decreased in DN patients compared with the controls (Figs. 3, 4). An inverse correlation was observed between the number of WT 1-positive/SPD-positive cells and U-Prot (Fig. 5A). There was no significant correlation between the number of WT1- positive/spd-positive cells and Ccr (Fig. 5B). An inverse correlation was observed between the number of WT1-1616

Figure1. ImmunohistochemistryofWT1andSPDdoublestaining.(A)WT1stainingshoweda nuclearpaternandspdstainingshowedalinearpatern(originalmagnification 100).

Theexpresionlevelofsynaptopodin proteinwasquantitatedbypercentageofimmmunohistochemicaly-positiveareainaglomerulus [(A)/(B)

finaly,spd-positiveareaperthenumberofwt1-positive/spdpositivecels(numberofpodocytes)wascalculatedasanindexofpodocytehypertrophy. Figure3.

3 Figure1. ImmunohistochemistryofWT1andSPDdoublestaining.(A)WT1stainingshoweda nuclearpaternandspdstainingshowedalinearpatern(originalmagnification 100).(B)WT1- positivecels,locatedinthespd-positiveareaorproximaltotheurinaryspace,weredefinedaspodocytes(arrow, 200). Figure2. ThecalculationmethodoftheSPD-positivearea.Theexpresionlevelofsynaptopodin proteinwasquantitatedbypercentageofimmmunohistochemicaly-positiveareainaglomerulus [(A)/(B) 100,where(A)istheimmmunohistochemicaly-positiveareaintheglomerulusand(B)is thetotalareaoftheglomerulus].finaly,spd-positiveareaperthenumberofwt1-positive/spdpositivecels(numberofpodocytes)wascalculatedasanindexofpodocytehypertrophy. Figure3. ImmunohistochemistryofWT1andSPDdoublestaininginDN(A)andcontrol(B). positive/spd-positive cells and the PAS-positive area (Fig. 5C). The ratio of the SPD-positive area to the number of WT1-positive/SPD-positive cells, an index for evaluating hypertrophy of podocytes, was significantly increased in DN 1617

4 patients (Fig. 6). Discussion Figure4. ThenumberofWT1-positive/SPD-positivecels (podocyte)wasdecreasedindncomparedwiththecontrol. In this study, we identified podocytes by double-staining for WT1 and SPD, and counted the number of such cells. Our results again showed evidence of podocyte loss in patients with DN. Because podocyte loss was correlated with indexes of clinical and pathological progression (i.e., proteinuria and mesangial expansion), it was suggested to also be correlated with the progression of DN. Various reports on the relationship between podocyte loss and progression of renal disease have been published in recent years (12-15). Kritz et al reported that podocyte loss occurs because of lack of proliferation of these terminally differentiated cells in the glomeruli, which leads to denudation of the glomerular basement membrane (GBM) and finally results in glomerulosclerosis after adhesion with Bowman s capsule (16-19). Progression of DN was reported to be related to podocyte loss by other authors (1-5), in agreement with the results of this study. Podocyte loss was reported to show a stronger relationship with the renal prognosis than GBM thickening or mesangial expansion, which are pathological features of DN (2). Based on such results, it may be important to prospectively investigate the severity of podocyte loss in relation to the renal prognosis of individual patients in the future. Investigation of the ratio of the SPD-positive area to the number of WT1-positive/SPD-positive cells in this study suggested that podocyte hypertrophy occurs in DN patients. Regarding the mechanism of podocyte hypertrophy, it has been suggested that residual podocytes on the GBM after partial loss may enlarge to cover the denuded areas caused by lack of proliferation of mature podocyte, leading to hypertrophy (1). In contrast, Wiggins et al studied aging rats and suggested that podocyte hypertrophy already occurs before proteinuria and podocyte loss (20). Further assessment of the above reports is necessary to determine whether podocyte loss and podocyte hypertrophy is the cause or result. The patients with DN in this study had overt nephropathy rather than microalbuminuria, and both podocyte loss and Figure5. (A)CorrelationbetweenthenumberofWT1-positive/SPD-positivecelsandu-prot. (B)CorrelationbetweenthenumberofWT1-positive/SPD-positivecelsandCcr.(C)Correlation betweenthenumberofwt1-positive/spd-positivecelsandpas-positivearea. 1618

5 Table2. Diferences between ACE-Is/ARBs Prescribed GroupandnotPrescribedGroup Figure6. TheratiooftheSPD-positiveareatothenumber ofwt1-positive/spd-positivecels(podocytehypertrophy)in DNcomparedwiththecontrol. podocyte hypertrophy were observed. It was therefore difficult to determine which event was the earlier event and this remains to be elucidated in the future. However, the results of this study are useful because detailed evaluation of podocyte loss and podocyte hypertrophy has not been performed in Japanese patients with DN. Future investigation of podocyte loss and podocyte hypertrophy in early DN (including normo-albuminuria) might contribute to the elucidation of the mechanism underlying the onset and progression of DN. It is important to clarify the detailed mechanism of podocyte hypertrophy for development of treatment. In addition to the report of Pagtalunan et al (1), some authors have reported that mechanical stretching due to increased internal glomerular pressure causes hypertrophy by influencing the cell cycle via the cyclin-dependent kinase inhibitors such as P21 (7, 21), whereas others have suggested that high glucose activates ERK1/2 and Akt/PKB via production of reactive oxygen species (ROS) and induces hypertrophy that is further potentiated by angiotensin II (22). However, all of these results were obtained in animal experiments. A recent clinical study revealed that ACE-Is and ARBs play an important role in remission or regression of diabetic nephropathy (23). However, from our results, both clinical indexes and pathological indexes were not significantly different between ACE-Is and/or ARBs prescribed group and the non prescribed group (Table 2). Although podocyte hypertrophy was suggested to be targeted by antioxidant therapy, ACE-Is and ARBs, further research into the detailed mechanisms of podocyte loss and podocyte hypertrophy is needed. Conclusion Double staining for WT1 and SPD suggested the occurrences of podocyte loss and podocyte hypertrophy in patients with DN. It was also suggested that these changes are possibly related to the onset and progression of DN. It is necessary to confirm these results in a large series of patients with early DN. References 1. Pagtalunan ME, Miller PL, Jumping-Eagle S, et al. Podocyte loss and progressive glomerular injury in type II diabetes. J Clin Invest 99: , Meyer TW, Bennett PH, Nelson RG. Podocyte number predicts long-term urinary albumin excretion in Pima Indians with type II diabetes and microalbuminuria. Diabetologia 42: , Steffes MW, Schmidt D, McCrery R, Basgen JM; International Diabetic Nephropathy Study Group. Glomerular cell number in normal subjects and in type I diabetic patients. Kidney Int 59: , White KE, Bilous RW, Marshall SM, et al; The European Study for the Prevention of Renal Disease in Type I diabetes (ESPRIT). Podocyte number in normotensive type I diabetic patients with albuminuria. Diabetes 51: , Dalla Vestra M, Masiero A, Roiter AM, Saller A, Crepaldi G, Fioretto P. Is podocyte injury relevant in diabetic nephropathy? Studies in patients with type 2 diabetes. Diabetes 52: , Mundel P, Shankland SJ. Podocyte biology and response to injury. J Am Soc Nephrol 13: , Hoshi S, Shu Y, Yoshida F, et al. Podocyte injury promotes progressive nephropathy in zucker diabetic fatty rats. Lab Invest 82: 25-35, Natoli TA, Liu J, Eremina V, et al. A mutant form of the Wilms tumor suppressor gene WT1 observed in Denys-Drash syndrome interferes with glomerular capillary development. J Am Soc Nephrol 13: , Srichai MB, Konieczkowski M, Padiyar A, et al. A WT1 coregulator controls podocyte phenotype by shuttling between adhesion structures and nucleus. J Biol Chem 279: , Mundel P, Heid HW, Mundel TM, Kruger M, Reiser J, Kriz W. Synaptopodin: an actin-associated protein in telencephalic dendrites and renal podocytes. J Cell Biol 139: , Menini S, Iacobini C, Oddi G, et al. Increased glomerular cell (podocyte) apoptosis in rats with streptozotocin-induced diabetes mellitus: role in the development of diabetic glomerular disease. Diabetologia 50: , Kriz W, Kretzler M, Nagata M, et al. A frequent pathway to 1619

6 glomerulosclerosis: Deterioration of tuft architecture-podocyte damage-segmental sclerosis. Kidney Blood Press Res 19: , Kim YH, Goyal M, Kurnit D, et al. Podocyte depletion and glomerulosclerosis have a direct relationship in the PAN-treated rat. Kidney Int 60: , Shirato I, Hosser H, Kimura K, et al. The development of focal segmental glomerulosclerosis in masugi nephritis is based on progressive podocyte damage. Virchow Arch 429: , Lemley KV, Lafayette RA, Safari M, et al. Podocytepenia and disease activity in IgA nephropathy. Kidney Int 61: , Kritz W, Gretz N, Lemley KV. Progression of glomerular disease: is the podocyte the culprit? Kidney Int 54: , Kritz W. Podocyte is the major culprit accounting for the progression of chronic renal disease. Microsc Res Tech 57: , Kritz W. Progressive renal failure inability of podocytes to replicate and the consequences for development of gomerulosclerosis. Nephrol Dial Transplant 11: , Kihara I, Yaoita E, Kawasaki K, Yamamoto T. Limitations of podocyte adaptation for glomerular injury in puromycin aminonucleoside nephrosis. Pathol Int 45: , Wiggins JE, Goyal M, Sanden SK, et al. Podocyte hypertrophy, Adaptation, and Decompensation associated with glomerular enlargement and glomerulosclerosis in the aging rat: Prevention by calorie restriction. J Am Soc Nephrol 16: , Pettermann AT, Pippin J, Durvasula R. Mechanical stretch induces podocyte hypertrophy in vitro. Kidney Int 67: , Kim NH, Rincon-Choles H, Bhandari B, Choudhury GG, Abboud HE, Gorin Y. Redox dependence of glomerular epithelial cell hypertrophy in response to glucose. Am J Physiol Renal Physiol 290: , Makino H, Nakayama Y, Wada J. Remission and regression of diabetic nephropathy. Hypertension Res 26: , The Japanese Society of Internal Medicine

Expression of human nephrin mrna in diabetic nephropathy

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