Characteristics of Podocyte Injury in Malignant Hypertensive Nephropathy of Rats (MSHRSP/Kpo Strain)

Biomedical Research (Tokyo) 36 (5) 313-321, 2015

Characteristics of podocyte injury in malignant hypertensive nephropathy of rats (MSHRSP/Kpo strain)

1, 3 2 3 Takashi KATO , Nobuyuki MIZUGUCHI , and Akihiko ITO 1 Department of Pharmacology, Faculty of Medicine, Kinki University, Osaka, Japan; 2 Life Science Research Institute, Kinki University, Osaka, Japan; and 3 Department of Pathology, Faculty of Medicine, Kinki University, Osaka, Japan (Received 2 July 2015; and accepted 6 August 2015)

ABSTRACT Proteinuria is not only a hallmark of renal complication in malignant hypertension, but is also a major deteriorating factor for the progression to end-stage renal disease. Podocyte injury plays a crucial role in the renal damage associated with hypertensive nephropathy, but the underlying mechanism remains unclear. Malignant stroke-prone spontaneously hypertensive rats (MSHRSP/ Kpo) represent an original and useful model of human malignant hypertension. In this study, we disclosed the glomerular injuries in the MSHRSP/Kpo. MSHRSP/Kpo exhibited elevated blood pressure at 6 weeks along with renal dysfunction and proteinuria. Histological analysis of the MSHRSP/Kpo glomeruli revealed a severe atrophy, but no change was found in the podocyte number. The expression levels of podocyte-specific proteins, nephrin, podocin, and synaptopodin were decreased in the MSHRSP/Kpo glomeruli, though another podocyte-specific protein, CD2AP, in the MSHRSP/Kpo glomeruli exhibited a similar extent of staining as in normotensive WKY/ Kpo rats. Furthermore, desmin was not markedly detected in the WKY/Kpo glomeruli, but was strongly positive in MSHRSP/Kpo. By electron microscopy, well-formed foot processes (FP) were replaced by effacement in MSHRSP/Kpo. An original malignant hypertension strain MSHRSP/ Kpo exhibits podocyte injuries associated with the decrease of some podocyte-specific proteins and the upregulation of desmin, along with FP effacement and proteinuria.

Malignant hypertension is critical for various kinds rosis, and tubular cell atrophy (26). A number of of vascular event and subsequent organ damage, factors including a defective renal autoregulatory re- such as stroke, cardiac hypertrophy, nephrosis, and sponse to hypertension, renal susceptibility genes, atherosclerosis. Among these, hypertensive nephro- and environmental factors such as salt, smoking, sclerosis from long-standing uncontrolled hyperten- and lead exposure have been suggested to contribute sion is a major cause of chronic kidney disease and to the development of hypertensive nephrosclerosis accounts for significant mortality (3). The histologi- (17). In particular, proteinuria is not only a hallmark cal lesions of hypertensive nephrosclerosis are well of renal complication in hypertension, but is also a recognized and characterized by hyalinization and major deteriorating factor for the progression to end- sclerosis of interlobular and afferent arterioles, to- stage renal diseases (23, 24). gether with tubulointerstitial fibrosis, glomeruloscle- Glomerular epithelial cells, podocytes, which are located on the outside of the glomerulus and cover the capillary wall, play an important role in the glo- Address correspondence to: Dr. Takashi Kato Department of Pharmacology, Faculty of Medicine, Kinki merular filtration of the kidney. The podocytes do University, 377-2 Ohno-higashi, Osakasayama, Osaka not proliferate after birth under normal conditions 589-8511, Japan and are terminally differentiated to maintain an in- Tel: +81-72-366-0221, Fax: +81-72-367-1820 tricate and polarized cellular organization consisting E-mail: [email protected] of a cell body, major processes, and foot process 314 T. Kato et al.

(FP). Dysfunction of the podocyte, such as FP ef- sections were used. For synaptopodin, desmin, facement, is associated with the development of pro- CD2AP, and nestin staining, 10% formalin-fixed teinuria and nephrotic syndrome (25). Furthermore, sections were used, as reported previously (11, 12). podocyte injury likely plays a crucial role in the re- Antigen retrieval was performed by using 1 mM nal damage associated with proteinuria and hyper- EDTA at pH 8.0 in microwave for 20 min for these tensive nephrosclerosis (2). Despite the progress staining except PECAM-1 staining. Immunostaining made by many studies in understanding renal dam- assays were performed as described previously (13). age induced by malignant hypertension, the underly- WT-1-positive and total cells in the glomerulus were ing mechanism of podocyte injury remains unclear. counted as described previously (11). Stroke-prone spontaneously hypertensive rats (SHRSP/Kpo) represent a useful model of human Electron microscopy. Deeply anesthetized animals essential hypertension (12, 19). In addition to the tu- were transcardially perfused with PBS followed by bular lesions, we previously reported the thickened ice-cold 1% glutaraldehyde (GA)/1% paraformalde- capillary walls in the SHRSP/Kpo glomeruli (12). hyde (PFA) in PBS. For transmission electron mi- However, we could not find the distinct injuries in croscopy (TEM) analysis, the extracted kidneys the SHRSP/Kpo podocytes. We found that some were fixed in the same solution at 4°C overnight. rats of the SHRSP/Kpo exhibited blood pressure of Tissue specimens were post-fixed in osmium tetrox-

230 mmHg at 10 weeks, continued to inbreed them ide (OsO4) for 2 h at 4°C, dehydrated through an and recently established malignant SHRSP/Kpo ethanol gradient, and embedded in Epon. Ultrathin (MSHRSP/Kpo). In the present study, we therefore sections (90 nm) were cut with an ultra-microtome, disclosed the glomerular injuries, especially in podo- stained with 4% uranyl acetate and 1% lead citrate cytes of MSHRSP/Kpo strain. (Pb), and then examined by TEM (HT7700; Hitachi, Tokyo, Japan). For scanning electron microscopy (SEM) analysis, the specimens were fixed in 2.5% MATERIALS AND METHODS GA overnight at 4°C. After washing with 0.1 M phos-

Animal experiment. We maintained MSHRSP/Kpo phate buffer, the specimens were post-fixed in OsO4 and WKY/Kpo (control) rats and measured the for 1 h at 4°C. The specimens were subsequently blood pressure as described previously (12). All ani- dehydrated in a graded series of ethanol and t-butyl mal experiments were carried out according to the alcohol, and then freeze-dried. The dried specimens Guideline for Experimental Animal Care issued by were finally mounted on stubs, coated with gold pal- the Prime Minister’s Office of Japan and approved by ladium, and examined by SEM (SU3500, Hitachi). the Committee on Animal Experimentation of Kinki University School of Medicine. SDS-PAGE analysis Statistical analysis. Student’s t-test was used to de- of urine was performed as described previously (10, termine statistical significance. 11). The concentrations of urinary albumin, blood urea nitrogen (BUN) and serum creatinine were ex- RESULTS amined as described previously (10, 12). Characterization of malignant SHRSP/Kpo Histological analysis. Right kidneys removed at We first examined the blood pressure of MSHRSP/ each autopsy were fixed in either cold 10%-buffered Kpo compared with that of normotensive WKY/Kpo formalin or 70% ethanol for 24 h, and were submit- rats. SHR/Kpo and SHRSP/Kpo strains had almost ted to a routine process for paraffin embedding. The the same level as the WKY/Kpo at 6 weeks of age renal sections were stained with periodic acid Schiff (12). However, MSHRSP/Kpo exhibited an increase (PAS). To detect podocyte proteins, anti-nephrin IgG of blood pressure at 6 weeks (Fig. 1A). MSHRSP/ (sc-19000; Santa Cruz Biotechnology (SCB), Dallas, Kpo further reached to 280 mmHg at 12 weeks TX, USA), anti-podocin IgG (sc-22298, SCB), anti- (Fig. 1A). We next investigated the urinary and se- WT-1 IgG (sc-7385, SCB), anti-synaptopodin IgG rum biochemical parameters in the MSHRSP/Kpo. (65194; Progene, Heidelberg, Germany), anti-CD2AP MSHRSP/Kpo exhibited proteinuria at 15 weeks by IgG (sc-9137, SCB), anti-nestin IgG (556309; BD SDS-PAGE analysis (Fig. 1B). Moreover, BUN and Bioscience, San Jose, CA, USA), anti-desmin IgG serum creatinine levels at 15-week-age MSHRSP/ (M0760; DAKO, Glostrup, Denmark), and anti- Kpo were 3.0- and 1.6-fold higher than in the age- PECAM-1 IgG (555025, BD Bioscience) were used. matched WKY/Kpo, respectively (Fig. 1C and 1D). For nephrin and podocin staining, 70% ethanol-fixed Podocyte in hypertensive rat model 315

Fig. 1 Blood pressure, urinary parameters, and ultrastructures of the glomeruli in MSHRSP/Kpo and WKY/Kpo. (A) Blood pressure of MSHRSP/Kpo and WKY/Kpo at 6 and 12 weeks of age. (B) SDS-PAGE shows albuminuria in MSHRSP/Kpo at 15 weeks. M: protein marker, Arrow head: major urinary protein. (C) BUN and (D) serum creatinine in MSHRSP/Kpo at 6, 10, and 15 weeks of age are compared with those of WKY/Kpo. Values are expressed as the mean ± SD. n = 5 per group, * P < 0.05, ** P < 0.01, *** P < 0.001.

Histological analysis of glomerulus Expression of podocyte-specific proteins in MSHRSP/ To evaluate glomerular lesions in MSHRSP/Kpo, we Kpo glomerulus performed histological examinations after PAS stain- Since we could not find any changes in the number ing. As shown in Fig. 2A, the WKY/Kpo glomeruli of podocytes, we focused on podocyte morphology. were normal, but we found a severe glomerular atro- Podocytes form FPs, highly dynamic cellular exten- phy in the MSHRSP/Kpo (Fig. 2B). The number of sions that are connected by a slit diaphragm (SD). glomerular cells was slightly decreased in MSHRSP/ Nephrin is a transmembrane protein of the immuno- Kpo [WKY/Kpo: 61.05 ± 3.23, (20W, n = 5) vs globulin family of cell-adhesion molecules, which is MSHRSP/Kpo: 55.56 ± 5.80, (19W, n = 3)]. Since localized to the SD and constitutes the filtration bar- Wilm’s Tumor-1 (WT-1) was shown to be localized rier of the kidney (16). In the WKY/Kpo glomeruli, in the nucleus of podocytes (11), we counted the nephrin was strongly detected along the extra-capil- WT-1-positive cells in the glomerulus (Fig. 2C and lary areas (Fig. 3A), but was weakly detected in 2D). However, the podocytes did not show signifi- MSHRSP/Kpo at 19 weeks (Fig. 3B). We next ex- cant differences between WKY/Kpo and MSHRSP/ amined the expression of podocin, an SD-associated Kpo [WKY/Kpo: 11.27 ± 1.52, (20W, n = 5) vs molecule (16). Podocin in the 20-week-old WKY/ MSHRSP/Kpo: 10.89 ± 0.53, (19W, n = 3)]. As the Kpo glomeruli was strongly detected (Fig. 3C), but SHRSP/Kpo glomeruli at 20 weeks (12), PECAM-1 in 19-week-old MSHRSP/Kpo glomeruli, it was staining was stronger in the MSHRSP/Kpo glomeru- faint (Fig. 3D). By determining the nephrin and li than that in the WKY/Kpo (Fig. 2E and 2F), and podocin levels in glomeruli, we found significant indicated the thickening of glomerular endothelial differences in these two slit-associated proteins be- cell layer in MSHRSP/Kpo kidneys. tween the two strains (Fig. 3E). We then examined the change in synaptopodin, a 316 T. Kato et al.

Fig. 2 Histopathology of the glomeruli in the WKY/Kpo (A, C, E) and MSHRSP/Kpo (B, D, F) glomeruli. (A, B) Periodic acid Schiff (PAS) staining. (C, D) WT-1 immunostaining. (E, F) PECAM-1 immunofluorescence. key player in FP regulation in podocytes (31). Con- Expression of intermediate filaments in MSHRSP/ comitantly with attenuations of nephrin and podocin, Kpo podocyte the synaptopodin level decreased in the 19-week-old In addition to the decrease of podocyte-specific pro- MSHRSP/Kpo glomeruli (Fig. 4B) compared with teins, injured podocytes are reported to exhibit the that in 20-week-old WKY/Kpo (Fig. 4A). We next upregulation of mesenchymal proteins (17, 32). Nes- investigated the glomerular CD2AP expression. tin is a cytoskeleton-associated intermediate filament CD2AP is a multifunctional adaptor protein local- protein that is reported to be stably expressed in the ized to the cytoplasm and membrane ruffles in glo- podocytes of mature glomeruli; it also plays an im- merular podocytes, and plays a role in cytoskeletal portant role in maintaining the normal morphology remodeling, cell survival, and endocytosis (16). Of and function of podocytes (11, 15). Like 20-week- interest, CD2AP in the 19-week-old MSHRSP/Kpo old WKY/Kpo, 19-week-old MSHRSP/Kpo also glomeruli exhibited a strong staining pattern as in strongly expressed the nestin in podocytes (Fig. 5A the 20-week-old WKY/Kpo glomeruli (Fig. 4C and and 5B). We next examined desmin expression in 4D). Histological quantification analysis revealed the MSHRSP/Kpo glomeruli. Up-regulated desmin significant differences in synaptopodin, but the same has been to be suggested as a podocyte injury marker levels of CD2AP were found between the two (17). Although desmin signals were not sufficiently strains (Fig. 4E). detected in the glomeruli of 20-week-old WKY/Kpo, Podocyte in hypertensive rat model 317

Fig. 3 Immunofluorescence assay of slit-associated proteins in the glomeruli of WKY/Kpo (A, C) and MSHRSP/Kpo (B, D). (E) Quantification of the expression levels of nephrin and podocin proteins. Values are expressed as themean ± SD. * P < 0.05 (WKY/Kpo, n = 5; MSHRSP/Kpo, n = 3).

Fig. 4 Immunofluorescence assay of synaptopodin and CD2AP proteins in the glomeruli of WKY/Kpo (A, C) and MSHRSP/Kpo (B, D). (E) Quantification of the expression levels of podocyte proteins. Values are expressed as the mean ± SD. * P < 0.05 (WKY/Kpo, n = 5; MSHRSP/Kpo, n = 3). multiple glomeruli were positive for desmin along the cal quantification analysis revealed that these pro- capillary tufts in 19-week-old MSHRSP/Kpo (Fig. 5C teins displayed significant differences at 15 weeks. and 5D). Actually, statistical evaluation confirmed Of note, the desmin levels were increased in the that the expression of nestin was unchanged between MSHRSP/Kpo glomeruli as early as 10 weeks. 20-week-old WKY/Kpo and 19-week-old MSHRSP/ Thus, we could nominate desmin as a desirable Kpo glomeruli, but the desmin level was remarkably marker for podocyte-injury detection. enhanced in the MSHRSP/Kpo glomeruli (Fig. 5E). We furthermore investigated the expression levels Ultrastructural analysis of podocytes in MSHRSP/ of nephrin, podocin, synaptopodin, and desmin at Kpo glomerulus earlier age as 10 and 15 weeks to elucidate the time We finally performed ultrastructural analysis by course of podocyte injuries (Table 1). The histologi- transmission electron microscopy on 15-week-old 318 T. Kato et al.

Fig. 5 Immunofluorescence assay of intermediate filament (IF) proteins in the glomeruli of WKY/KpoA ( , C) and MSHRSP/ Kpo (B, D). (E) Quantification of the expression levels of IF proteins in the glomerulus. Values are expressed as the mean ± SD. ** P < 0.01 (WKY/Kpo, n = 5; MSHRSP/Kpo, n = 3).

Table 1 Expression levels of podocyte-specific proteins in the glomerulus Weeks of age Strain (n = 5) Nephrin Podocin Synaptopodin Desmin 10 WKY/Kpo 3.42 ± 0.43 3.65 ± 0.20 3.84 ± 0.16 1.01 ± 0.14 MSHRSP/Kpo 3.27 ± 0.32 3.22 ± 0.63 3.38 ± 0.29 1.30 ± 0.27* 15 WKY/Kpo 3.54 ± 0.31 3.66 ± 0.38 3.80 ± 0.19 0.56 ± 0.13 MSHRSP/Kpo 2.41 ± 0.29* 2.18 ± 0.60* 2.52 ± 0.39** 2.09 ± 0.16** Values are expressed as mean ± SD, *P < 0.05, **P < 0.01 compared with the age-matched WKY/Kpo

WKY/Kpo and 6-, 15-, and 19-week-old MSHRSP/ ized by molecular alterations of the SD or by reor- Kpo strains. Normotensive WKY/Kpo rats displayed ganization of the FP structure with the fusion of a normal, intact arrangement of interdigitating FPs slits. In our study, MSHRSP/Kpo exhibited hyper- and preserved filtration slit (Fig. 6A). Ultrastructural tensive nephropathy and proteinuria, which were in examination of MSHRSP/Kpo podocytes revealed accordance with podocyte injuries. Of note, the morphology indistinguishable from the WKY/Kpo podocyte number was not significantly different, but podocytes at 6 weeks (Fig. 6B). However, MSHRSP/ the expression levels of nephrin, podocin, and syn- Kpo podocytes showed partial FP effacement at 15 aptopodin were attenuated in the MSHRSP/Kpo weeks (Fig. 6C), and FP effacement was complete at glomeruli. Some reports have revealed that human 19 weeks (Fig. 6D). Under scanning electron mi- and experimental hypertensive nephrosis gave rise croscopy, well-formed FPs were built in the WKY/ to a decrease of WT-1-positive podocytes in the Kpo glomeruli (Fig. 6E), but FP effacement was glomerulus. The expression levels of nephrin, podo- found in the MSHRSP/Kpo (Fig. 6F). cin, and synaptopodin mRNA were down-regulated in patients with hypertensive nephrosclerosis together with podocyte loss (28). Moreover, salt-loaded DISCUSSION Dahl rats developed severe hypertension and pro- Podocytes are injured in many human and experi- teinuria, and the loss of podocytes and nephrin oc- mental glomerular diseases, including minimal- curred (21). Meanwhile, other reports have described change disease, focal segmental glomerulosclerosis, the reduction of podocyte-specific proteins without collapsing glomerulopathy, diabetic nephropthay, podocyte attenuation. Glomeruli of two-kidney one- membranous glomerulopathy, crescentic glomerulo- clip (2K1C) hypertensive rats showed reduced levels nephritis, and lupus nephritis (1). Independent of the of proteins that are important for the SD, nephrin underlying disease, the early events are character- and podocin, but the synaptopodin level was un- Podocyte in hypertensive rat model 319

Fig. 6 Severe podocyte FP effacement in the MSHRSP/Kpo glomeruli by transmission microscopy (TEM) and scanning electron microscopy (SEM). (A) TEM image of the WKY/Kpo glomerulus at 20 weeks. (B–D) TEM images of the MSHRSP/ Kpo glomeruli in MSHRSP/Kpo at 6 (B), 15 (C), and 19 weeks (D). (E, F) SEM images of glomeruli in WKY/Kpo at 20 weeks (E) and MSHRSP/Kpo at 19 weeks (F). Scale bars: 10 μm (A–D), 50 μm (E, F). changed (5). In women with preeclampsia, a preg- pression could protect against apoptosis during PAN nancy-specific disorder characterized by hypertension nephrosis and this process is mediated by maintain- and proteinuria, the nephrin level was down-regulat- ing the regular arrangement of the actin cytoskeleton ed, but that of podocin was unchanged (6). Since (29). Of interest, CD2AP also inhibits the apoptosis the MSHRSP/Kpo strain started to die by spontane- of podocytes in albumin overload-induced endoplas- ous hemorrhage or ischemic stroke at 15 weeks mic reticulum (ER) stress (7). We found that the (manuscript in preparation), we further plan to in- podocyte number was not significantly decreased, vestigate the podocyte injuries in prone-suppressed and thus think that nestin and CD2AP might protect MSHRSP/Kpo kidneys. podocytes against apoptosis in the MSHRSP/Kpo It is reported that nestin expression of podocytes glomeruli. was significantly reduced in nephropathic patients It is important to elucidate the mechanisms of with proteinuria (9). By contrast, in the puromycin podocyte injuries in MSHRSP/Kpo. As glomeruli aminonucleoside (PAN)-induced rat nephrosis mod- become irreversibly scarred and a decline in the el, nestin expression was up-regulated in the ab- functional nephron mass ensues, the remaining sence of podocyte apoptosis, even though the FP nephrons are subjected to an elevated pressure gra- was significantly effaced (29). Increased nestin ex- dient across the capillary wall, resulting in an exces- 320 T. Kato et al. sive mechanical stress. Independent of the inciting CONFLICTS OF INTEREST events, glomerular capillary hypertension ensures further damage to the podocytes and represents a fi- The authors have no conflicts of interest to report. nal common pathway to glomerulosclerosis and end- Although the study was supported by external stage renal failure (14). Thus, current therapy for grants, there was no direct benefit (commercial/non- hypertensive patients is aimed at prohibiting the commercial) to the sponsors. vascular events caused by hypertension rather than decreasing blood pressure (22). It is important to as- REFERENCES sess the correlation of podocyte injuries with glomer- 1. Barisoni L and Mundel P (2003) Podocyte biology and the ular endothelial cells in hypertensive nephropathy. emerging understanding of podocyte diseases. Am J Nephrol In addition, the renin-angiotensin system (RAS) is 23, 353–360. closely correlated with the pathogenesis of hyperten- 2. D’Agati VD (2008) The spectrum of focal segmental glomer- sion and its sequence. Mechanical stretch has an im- ulosclerosis: new insights. Curr Opin Nephrol Hypertens 17, 271–281. portant role on the increase of angiotensin II (ANG 3. Dasgupta I, Porter C, Innes A and Burden R (2007) “Benign” II) production in conditionally immortalized podo- hypertensive nephrosclerosis. 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