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Urinary cystatin C as a biomarker for diabetic nephropathy and its immunohistochemical localization in kidney in Zucker diabetic fatty (ZDF) rats
Experimental and Toxicologic Pathology 65 (2013) 615–622
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Urinary cystatin C as a biomarker for diabetic nephropathy and its immunohistochemical localization in kidney in Zucker diabetic fatty (ZDF) rats Yuko Togashi, Yohei Miyamoto ∗ Toxicology and Pharmacokinetics Laboratories, Pharmaceutical Research Laboratories, Toray Industries, Inc., 6-10-1, Tebiro, Kamakura, Kanagawa 248-8555, Japan
a r t i c l e
i n f o
Article history: Received 12 March 2012 Accepted 20 June 2012 Keywords: Renal biomarker Cystatin C Diabetic nephropathy Immunohistochemistry Zucker diabetic fatty (ZDF) rats
a b s t r a c t Cystatin C, a cysteine protease inhibitor, is a novel biomarker of renal damage. In the present study, we examined the usefulness of urinary cystatin C for the detection of diabetic nephropathy in Zucker diabetic fatty (ZDF) rats compared to other biomarkers (2-microglobulin, calbindin, clusterin, epidermal growth factor (EGF), alpha-glutathione S-transferase (GST-␣), mu-glutathione S-transferase (GST-), kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), osteopontin, tissue inhibitor of metalloprotease-1 (TIMP-1), and vascular endothelial growth factor (VEGF). Urinary levels of cystatin C were increased in ZDF rats where renal damage was not histopathologically observed, and then further increased with the progression of renal damage, demonstrating the usefulness of early detection and accurate assessment of diabetic nephropathy. Urinary 2-microglobulin, clusterin, GST-, KIM-1, and osteopontin had the potency to detect renal damage in ZDF rats as well as cystatin C. We also investigated immunohistochemical localization of cystatin C in the kidney according to progressive renal damage. Cystatin C expression was mainly observed in the proximal renal tubule in ZDF rats, and hardly changed with progression of nephropathy. When renal damage was remarkable, cystatin C expression was also observed in the tubular lumen of the cortex and medulla, which was considered to be characteristic of renal damage in diabetic nephropathy. In conclusion, urinary cystatin C, 2-microglobulin, clusterin, GST-, KIM-1, and osteopontin could be useful biomarkers of diabetic nephropathy in ZDF rats. Immunohistochemical cystatin C expression in the proximal renal tubule was hardly changed by the progression of diabetic nephropathy, but it was newly observed in the tubular lumen when renal damage was remarkable in ZDF rats. Crown Copyright © 2012 Published by Elsevier GmbH. All rights reserved.
1. Introduction Serum creatinine and urea nitrogen (UN), which reflect impaired kidney function, are routinely used as markers of kidney disease and injury. However, these markers are insensitive since functional changes in the kidney are not observed until damage has become widespread (Hewitt et al., 2004; Ferguson et al., 2008). Therefore, new biomarkers for sensitive and more accurate indication are needed (Marrer and Dieterle, 2010; Fuchs and Hewitt, 2011). The Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) endorsed 7 urinary nephrotoxic biomarkers: total protein, albumin, kidney injury molecule-1 (KIM1), clusterin, 2-microglobulin, cystatin C, and trefoil factor 3 as biomarkers of acute kidney injury (AKI) in non-clinical settings (FDA, 2008; EMEA, 2008). The International Life Sciences Institute Health and Environmental Science (ILSI-HESI) also reported 4 urinary nephrotoxic biomarkers: alpha-glutathione S-transferase
∗ Corresponding author. Tel.: +81 467 32 9665; fax: +81 467 32 9768. E-mail address: Youhei [email protected] (Y. Miyamoto).
(GST-␣), mu-glutathione S-transferase (GST-), renal papillary antigen-1 (RPA-1), and clusterin (ILSI-HESI, 2008). In addition, several other biomarkers such as neutrophil gelatinase-associated lipocalin (NGAL) and tissue inhibitor of metalloprotease-1 (TIMP1) were reported to be able to detect nephrotoxicity, leading to the detection of AKI (Hoffmann et al., 2010). Cystatin C is a non-glycosylated 13 kDa cysteine protease inhibitor. It is produced at a constant rate by all karyocytes, directly and freely filtrated from the blood into the glomerulus, and is completely reabsorbed and catabolized in the proximal renal tubules (Abrahamson et al., 1990; Grubb, 1992; Tenstad et al., 1996). Because of these features, urinary cystatin C is an ideal estimator of the glomerular filtration rate (GFR), and its clinical use has been increasing recently (Coll et al., 2000; Madero and Sarnak, 2009). Tubular damage induces impairment of cystatin C reabsorption to the proximal renal tubule. In addition, glomerular alteration causes high molecular weight protein leakage, which leads to competition with tubular uptake of cystatin C. Therefore, urinary cystatin C, one of the qualified renal biomarkers for AKI, has been considered to have the potency to detect both glomerular and proximal renal injury (Conti et al., 2006; Dieterle et al., 2010; Parikh et al.,
0940-2993/$ – see front matter. Crown Copyright © 2012 Published by Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.etp.2012.06.005
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2010). We also showed it could detect CDDP-induced AKI in rats earlier than conventional markers (Togashi et al., in press). Diabetic nephropathy is a common microvascular complication of diabetic mellitus. It is characterized by the progressive impairment of glomerular function, leading to end-stage renal failure. Microalbuminuria has been considered to indicate renal damage at initial stages; however, morphological changes may occur earlier (Brocco et al., 1997; Fu et al., 2012). Recently, renal biomarkers such as cystatin C, NGAL, NAG, and KIM-1 in urine were reported to be useful for detection of chronic disease kidney (CKD) including diabetic nephropathy in clinical settings (Fassett et al., 2011; Jeon et al., 2011; Tramonti and Kanwar, 2011), but little is known in animal models. Zucker diabetic fatty (ZDF) rats, which have defective leptin receptors, exhibit metabolic abnormalities associated with type 2 diabetes and develop diabetic complications such as nephropathy, cataracts, and neuropathy (Shibata et al., 2000; Toblli et al., 2009). In this study, to clarify the usefulness of urinary cystatin C for the detection of diabetic nephropathy, we measured urinary C levels and compared them to other biomarkers (2-microglobulin, calbindin, clusterin, epidermal growth factor (EGF), GST-␣, GST, KIM-1, NGAL, osteopontin, TIMP-1, and vascular endothelial growth factor (VEGF)) in ZDF rats. Furthermore, we investigated the localization of cystatin C in the kidney according to the progression of renal damage.
2.3. Biomarker analysis Cystatin C, 2-microglobulin, calbindin, clusterin, EGF, GST-␣, GST-, KIM-1, NGAL, osteopontin, TIMP-1, and VEGF were measured by immunoassay using the rat kidney Multi-Analyte Profile kit (Rules Based Medicine, Austin, TX, USA), as described previously (Heuer et al., 2004). 2.4. Histopathology and immunohistochemistry After fixation, the kidney and pancreas were rinsed in tap water, dehydrated in a graded alcohol series, embedded in paraffin, and cut into 3 m thick sections. Paraffin sections were processed for hematoxylin–eosin (HE), Masson trichrome, and periodic acidSchiff staining (PAS), and histopathological changes were observed under a light microscope. In immunohistochemistry, an anti-rat cystatin C rabbit monoclonal antibody (Abcam Ltd., Cambridge, UK) and the Vectastain ABC kit (Vector Laboratories, Inc., Burlingame, CA, USA) were used. 2.5. Statistical analysis The differences between lean and ZDF rats at each age were analyzed with the Student’s t-test or Aspin–Welch’s t-test. The level of significance was set at 1% and 5%.
2. Materials and methods 3. Results 2.1. Animals 3.1. Fundamental metabolic parameters in diabetic model rats Male Zucker diabetic fatty rats (ZDF/CrlCrlj-Leptfa/fa , Charles River Laboratories Japan, Inc., Yokohama, Japan) and male nondiabetic lean rats (ZDF/CrlCrlj-Lept?/+ , Charles River Laboratories, Yokohama, Japan), which were described as ZDF and lean rats, were housed at 2 or 3 animals per cage under controlled temperature, lighting, and relative humidity conditions (19–25 ◦ C; 12 h on/off light/dark cycle; 40–60%, respectively). They were given a standard diet (Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water ad libitum, but were fasted during the urine collection period. Urine was collected and cooled for 24 h using individual metabolic cages with no food but water. Urine volumes were recorded, and the collected urine was centrifuged at 1870 × g for 15 min at 4 ◦ C and then stored at −80 ◦ C until analysis. After final body weighing, blood was collected from the abdominal aorta under pentobarbital anesthesia. The plasma was separated by centrifugation for 15 min at 4 ◦ C and stored at −80 ◦ C until analysis. After the collection of urine and blood, the rats were euthanized by bleeding under pentobarbital anesthesia, and then the kidneys were removed and fixed in 10% neutral buffered formalin. All experiments were conducted according to the Guidelines for Animal Experiments, Research & Development Division, Toray Industries, Inc. 2.2. Blood chemistry Creatinine, glucose, phospholipids, total cholesterol, triglycerides, UN in plasma, and albumin in urine were determined using a Hitachi 7070 automatic analyzer (Hitachi Instruments Service Co., Ltd, Tokyo, Japan), and autosera kits were used to determine creatinine (urease – UV method), glucose (hexokinase – UV method), phospholipids (phospholipase – HSDA method), total cholesterol (cholesterol oxidase method), triglycerides (free glycerol elimination method), UN (urease – GLDH method), and albumin (BCGs method). Plasma insulin was determined using a rat insulin ELISA kit (Shibayagi Co., Gunma, Japan).
Body weight and fundamental metabolic parameters were measured in lean and ZDF rats at 12, 20, and 25 weeks. Body weight in ZDF rats was significantly higher than lean rats at 12 weeks, but there was no apparent difference between both groups at 20 and 25 weeks. Plasma levels of glucose, phospholipids, total cholesterol, and triglycerides in ZDF rats were significantly higher than those in lean rats at 12 weeks, and increased thereafter. In contrast, plasma levels of insulin in ZDF rats were almost the same as lean rats at 12 weeks, and slightly decreased at 20 and 25 weeks (Table 1). Consistent with metabolic abnormalities, focal fibrosis of pancreatic islets was histopathologically pronounced in ZDF rats at 20 and 25 weeks, whereas no remarkable change was observed in lean rats (Fig. 1). 3.2. Renal functional and morphological changes in ZDF rats Renal functional parameters and histopathological changes were examined in lean and ZDF rats at 12, 20, and 25 weeks. Urine output for 24 hr and kidney weight were significantly higher in ZDF rats than those in lean rats at 12 weeks, and reached a plateau at 20 weeks. Excretion of urine albumin increased age-dependently (12 weeks, 2.9-fold; 20 weeks, 7.1-fold; and 25 weeks, 22-fold). On the other hand, plasma creatinine in ZDF rats did not increase relative to lean rats at all ages. Plasma UN was slightly increased in ZDF rats relative to lean ZDF rats, but the increase ratios were low and unchanged from 12 to 25 weeks (1.5–1.7-fold) (Table 2). In histopathology of the kidney, increased mesangial matrix and glomerular exudate were observed in ZDF rats at 20 and 25 weeks. In addition, PAS-positive hyaline droplets in the glomerulus were observed in ZDF rats from 12 weeks. Renal tubular alternations such as tubular dilatation, hyaline cast, basophilic changes with regeneration, and renal tubular vacuolation were noted in ZDF rats at 20 and 25 weeks. Pelvic dilatation was macroscopically apparent in most lean and ZDF rats, but it worsened in ZDF rats with age (Table 3, Figs. 2 and 3).
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Table 1 Fundamental metabolic parameters in lean and ZDF rats. 12 weeks Lean Body weight (g) Glucose (mg/dL) Insulin (ng/mL) Phospholipid (mg/dL) Total cholesterol (mg/dL) Triglyceride (mg/dL)
282 206 3.7 116 64 27
± ± ± ± ± ±
20 weeks ZDF
19 19 1.2 6 5 13
340 519 3.9 215 98 295
Lean ± ± ± ± ± ±
9** 50** 1.0 19** 8** 78**
376 222 5.8 137 90 56
25 weeks ZDF
± ± ± ± ± ±
15 27 0.5 8 5 7
384 653 3.1 305 186 269
Lean ± ± ± ± ± ±
16 48** 1.0** 26** 16** 41**
410 233 5.2 155 99 71
± ± ± ± ± ±
ZDF 9 12 1.7 9 7 25
404 657 2.5 361 225 333
± ± ± ± ± ±
30 80** 0.4* 40** 31** 79**
Each data is the mean ± S.D. of 5 animals. * Significant differences between lean and ZDF rats at each point: P < 0.05. ** Significant differences between lean and ZDF rats at each point: P < 0.01.
Fig. 1. Histopathological changes in the pancreas of lean (A–C) and ZDF (D–F) rats. No marked changes are seen in lean rats at 12, 20, and 25 weeks. Islet fibrosis is seen in ZDF rats at 20 weeks, which worsened at 25 weeks. (A and D) 12 weeks; (B and E) 20 weeks; and (C and F) 25 weeks. Masson trichrome staining. Each bar shows 100 m.
3.3. Cystatin C and other renal biomarkers in ZDF rats Urinary cystatin C levels in lean and ZDF rats were measured at 12, 20, and 25 weeks and compared to other renal biomarkers. Urinary cystatin C levels were significantly higher (2.3-fold) in ZDF rats than those in lean rats at 12 weeks, and further increased at 20 and 25 weeks (20 weeks; 4.0-fold, 25 weeks; 9.5-fold). From 12 weeks, urinary 2-microglobulin, clusterin, GST-, KIM-1,
and osteopontin levels increased age-dependently, by 3.2–27.1fold, 2.4–24.8-fold, 2.2–12.7-fold, 1.7–5.6-fold, and 2.7–9.3-fold, respectively. Calbindin and TIMP-1 in ZDF rats were significantly increased at 12, 20, and 25 weeks, but the increase ratios were unchanged. EGF was significantly decreased at 20 and 25 weeks. On the other hand, GST-␣, NGAL, and VEGF in ZDF rats were unchanged at all ages, though significant differences were sporadically seen (Table 4, Fig. 4).
Table 2 Renal function parameters in lean and ZDF rats. 12 weeks Lean Kidney (g/100 g body weight) Urine (mL/24 h) Plasma creatinine (mg/dL) Plasma UN (mg/dL) Urine albumin (mg/24 h)
0.70 9.3 0.32 15.6 4.2
20 weeks ZDF
± ± ± ± ±
0.04 1.4 0.02 1.1 1.2
0.96 15.1 0.26 23.7 12.2
Lean ± ± ± ± ±
**
0.02 2.2** 0.03** 2.3** 3.0**
Each data is the mean ± S.D. of 5 animals. * Significant differences between lean and ZDF rats at each point: P < 0.05. ** Significant differences between lean and ZDF rats at each point: P < 0.01.
0.76 6.8 0.38 16.2 4.0
25 weeks ZDF
± ± ± ± ±
0.04 2.2 0.04 0.7** 0.7
1.25 41.6 0.29 24.2 28.4
Lean ± ± ± ± ±
**
0.07 12.5** 0.02** 1.1** 7.6**
0.80 6.0 0.34 13.7 4.0
ZDF ± ± ± ± ±
0.02 2.1 0.03 1.7 0.7
1.26 31.4 0.27 23.9 90.1
± ± ± ± ±
0.11** 11.7** 0.02** 3.3** 29.1**
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Table 3 Histopathological changes of kidney in lean and ZDF rats. 12 weeks
(No. of animals) Histopathological findings
20 weeks
25 weeks
Lean (5)
ZDF (5)
Lean (5)
ZDF (5)
Lean (5)
ZDF (5)
Grade*
Glomerular exudate
0 1 2 3
0 0 0 0
0 0 0 0
0 0 0 0
2 3 0 0
0 0 0 0
0 5 0 0
Glomerular hyaline droplet
0 1 2 3
0 0 0 0
2 3 0 0
0 0 0 0
2 3 0 0
0 0 0 0
0 5 0 0
Increased mesangial matrix
0 1 2 3
0 0 0 0
0 0 0 0
0 0 0 0
3 2 0 0
0 0 0 0
0 5 0 0
Basophilic renal tubule
0 1 2 3
0 0 0 0
0 0 0 0
0 0 0 0
1 2 0 0
0 0 0 0
0 2 2 0
Hyaline cast of renal tubule
0 1 2 3
0 0 0 0
0 0 0 0
0 0 0 0
0 4 1 0
0 0 0 0
0 2 3 0
Tubular dilatation
0 1 2 3
0 0 0 0
0 0 0 0
0 0 0 0
2 0 0 0
0 0 0 0
0 5 0 0
Vacuolation of renal tubule
0 1 2 3
0 0 0 0
0 0 0 0
0 0 0 0
1 3 0 0
0 0 0 0
1 3 0 0
Pelvic dilatation
0 1 2 3
4 0 0 0
0 2 1 1
0 3 0 0
0 0 3 2
2 1 1 0
1 0 2 3
*
0, very slight; 1, slight; 2, moderate; and 3, marked.
Fig. 2. Histopathological changes in the kidney of lean (A–C) and ZDF (D–F) rats. No marked changes are seen in lean rats at all ages. Glomerular exudate and renal tubular alterations such as hyaline cast and basophilic changes with regeneration are apparent in ZDF rats at 20 and 25 weeks. In addition, renal tubular vacuolation is seen from 12 weeks. (A and D) 12 weeks; (B and E) 20 weeks; and (C and F) 25 weeks. HE staining. Each bar shows 200 m.
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Fig. 3. Histopathological changes in the kidney of lean (A–C, G) and ZDF (D–F, H) rats. No marked changes are seen in lean rats at all ages. Increased mesangial matrix is seen in ZDF rats at 20 weeks, which worsened at 25 weeks. PAS-positive hyaline droplet in the glomerulus (arrows) is seen only in ZDF rats from 12 weeks. (A and D) 12 weeks; (B and E) 20 weeks; and (C, F–H) 25 weeks. PAS (A–G) and HE (H) staining. Each bar shows 50 m.
Table 4 Urinary levels of renal biomarkers in lean and ZDF rats. 12 weeks
20 weeks
Lean Cystatin C (ng/24 h) 2-microglobulin (g/24 h) Calbindin (ng/24 h) Clusterin (g/24 h) EGF (ng/24 h) GST-␣ (ng/24 h) GST- (ng/24 h) KIM-1 (ng/24 h) NGAL (ng/24 h) Osteopontin (ng/24 h) TIMP-1 (ng/24 h) VEGF (pg/24 h)
2809 65.3 1690 5.1 81,846 426 157 3.40 674 71 22.2 6610
ZDF ± ± ± ± ± ± ± ± ± ± ± ±
519 17.3 472 1.7 24,402 108 35 0.91 280 31 9.8 2265
6486 212.1 7307 13.6 52,756 450 344 5.77 1154 189 68.0 9681
25 weeks
Lean ± ± ± ± ± ± ± ± ± ± ± ±
712** 81.8* 811** 5.1* 7502 217 52** 1.65* 516 74* 32.2* 3129
Each data is the mean ± S.D. of 5 animals. * Significant differences between lean and ZDF rats at each point: P < 0.05. ** Significant differences between lean and ZDF rats at each point: P < 0.01.
2620 106.6 2413 6.3 124,740 224 105 2.76 335 153 19.8 6004
ZDF ± ± ± ± ± ± ± ± ± ± ± ±
347 19.8 889 2.8 9827 105 74 1.23 136 41 8.9 2528
10,368 603.8 7505 30.2 34,290 668 737 9.5 535 470 71.0 13,515
Lean ± ± ± ± ± ± ± ± ± ± ± ±
1952** 77.1** 1639** 7.9** 5346** 197** 177** 2.8** 751 148** 27.4* 5417*
2404 54.8 1628 3.9 176,116 262 112 2.8 358 87 17.6 5738
ZDF ± ± ± ± ± ± ± ± ± ± ± ±
826 9.9 233 1.2 52,846 196 44 1.2 273 17 8.5 2373
22,916 1485.7 8142 97.0 35,055 834 1421 15.4 1552 811 86.7 11,701
± ± ± ± ± ± ± ± ± ± ± ±
7384** 702.1* 2652** 41.6** 6349** 443* 722* 5.4** 411** 304** 25.1** 4999
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Fig. 4. Urinary levels of cystatin C and other renal biomarkers in lean and ZDF rats. From 12 weeks, urinary cystatin C, 2-microglobulin, clusterin, GST-, KIM-1, and osteopontin increased with age in ZDF rats. Calbindin and TIMP-1 in ZDF rats increased at all ages, but the increase ratios were unchanged. EGF decreased from 20 weeks in ZDF rats. Each column represents the mean ± S.D. of 5 animals. Significant differences between the corresponding lean and ZDF rats at each point: *P < 0.05 and **P < 0.01. Lean rats (open column) and ZDF rats (closed column).
3.4. Immunohistochemical localization of cystatin C in the kidney of ZDF rats In both lean and ZDF rats, cystatin C was mainly localized in the proximal renal tubule of the cortex, but hardly observed in the medulla. In ZDF rats, cystatin C was also observed in the renal tubular lumen of the cortex and medulla from 20 weeks, but this finding was not observed in lean rats at all ages (Fig. 5).
4. Discussion In this study, we measured urinary cystatin C levels in ZDF rats, a type 2 diabetic nephropathy model, to evaluate its usefulness in detection of renal damage compared to urinary biomarkers (2-microglobulin, calbindin, clusterin, EGF, GST-␣, GST-, KIM1, NGAL, osteopontin, TIMP-1, and VEGF). In this study, ZDF rats showed hyperglycemia and hyperlipidemia from 12 weeks and slight decreases in insulin levels and fibrosis of pancreatic islets from 20 weeks, which indicated the onset of diabetic mellitus. As for renal function, urine volume and albumin excretion were increased from 12 weeks, and glomerular changes such as increased mesangial matrix and renal tubular changes were histopathologically observed from 20 weeks. In addition, renal tubular vacuolation, which is indicative of persistent diabetes and resembles human Armanni-Ebstein lesions (Zhou et al., 2011), and glomerular hyaline droplets were seen in ZDF rats. Therefore, ZDF rats were considered to establish incipient diabetic nephropathy, which is consistent with previous reports (Toblli et al., 2009; Zoja et al., 2011).
Urinary cystatin C was significantly higher in ZDF rats than that of lean rats at 12 weeks, prior to histopathological changes in the kidney, and further increased at 20 and 25 weeks. This result indicated that urinary cystatin C would be useful for early detection and accurate assessment of diabetes nephropathy. However, albuminuria, the most sensitive marker for diabetic nephropathy in clinical settings (Herrera et al., 2010; Martin, 2011), developed in ZDF rats from 12 weeks, indicating that the potential of urinary cystatin C and albumin for early detection of diabetic nephropathy is similar in the rat model. Urinary cystatin C was considered to have the potential to detect both glomerular and renal tubular damage in rats and humans. In this study, increased urinary cystatin C reflected damage to both the glomerulus and proximal renal tubule, since they were histopathologically observed in ZDF rats. Serum cystatin C is recognized as a superior marker of GFR and is able to detect mild nephropathy (CKD stages 1 and 2) in humans (Ozer, 2010; Inker and Okparavero, 2011). On the other hand, the usefulness of urinary cystatin C for the prediction of CKD has hardly been known, though recent studies suggested that it was a possible marker for renal dysfunction in type 2 diabetic patients (Jeon et al., 2011). The present study demonstrated for the first time that urinary cystatin C could be used as an indicator of diabetic nephropathy in ZDF rats. Urinary levels of 2-microglobulin, clusterin, GST-, KIM-1, and osteopontin were also higher in ZDF rats than those in lean rats at 12 weeks, and became even higher with progressive renal damage, indicating that they could be used as candidate biomarkers of diabetic nephropathy similar to cystatin C. KIM-1, an extracellular protein anchored in the proximal tubule membrane, is highly
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Fig. 5. Immunohistochemical localization of cystatin C in the kidney of lean (A–C, G–I) and ZDF (D–F, J–L) rats. Cystatin C is seen in the proximal renal tubules in the cortex (A–C), but hardly in the medulla (G–I) in lean rats. Cystatin C is seen in the proximal renal tubules in the cortex (D–F) in ZDF rats at all ages, and was also noted in the lumens of the renal tubules in the cortex (F) and medulla (K and L) at 20 and 25 weeks. Arrows represent cystatin C in the tubular lumen. Each bar represents 1000 m. (A–F) Cortex and (G–L) medulla.
induced and secreted in urine after renal damage, and is widely considered to be a sensitive biomarker to detect AKI (Waring and Moonie, 2011). In addition, recent studies reported that urinary excretion of KIM-1 could be used as a reliable marker for the progression of diabetic nephropathy in humans (Nielsen et al., 2011), which is consistent with the results of our present study in rats. NGAL, a ubiquitous lipocalin iron-carrying protein, has been identified as an early biomarker of AKI, and has great promise as a biomarker of CDK including diabetic nephropathy (Fassett et al., 2011; Tramonti and Kanwar, 2011). However, urinary levels were not increased in ZDF rats contrary to our expectations. Furthermore, our data could provide new insights into the potential role of 2-microglobulin, clusterin, GST-, and osteopontin as biomarkers of diabetic nephropathy, since little is known about their urinary
levels in CKD. TIMP-1, a tissue remodeling protein (Ishii et al., 2007; Wasilewska and Zoch-Zwierz, 2008), and calbindin, an intracellular calcium-binding protein (Kumar et al., 1994; Lambers et al., 2006), were increased in urine when renal damage was not evident, but these levels were not changed at any age in ZDF rats. The reason for this is unclear, but interesting findings could be provided if urinary levels are measured before 12 weeks. We also investigated the immunohistochemical localization of cystatin C in lean and ZDF rats. In lean rats, cystatin C was predominantly localized in the renal proximal tubules of the cortex, but were hardly observed in the medulla, which is consistent with previous studies using rats and humans (Jacobsson et al., 1995; Baran et al., 2006). Cystatin C is reabsorbed to proximal tubular epithelial cells, thus, the immunoreactive cystatin C detected in the epithelial
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cells was most likely caused by reabsorption from primary urine. Cystatin C expression was also observed in renal tubular epithelial cells in the cortex in ZDF rats, and was not apparently changed with progression of renal damage. Therefore, it is not considered that immunohistochemical expression of cystatin C in proximal renal tubules may be more sensitive for diabetic nephropathy than its urinary level. Consistent with this study, immunohistochemical expression of cystatin C in proximal renal tubules was not changed by CDDP-induced AKI in rats in our previous study (Togashi et al., in press), though there was a contrary report showing it was altered by some glomerular and/or tubular nephrotoxicants in rats (Dieterle et al., 2010). In ZDF rats, cystatin C was observed in not only the proximal renal tubule but also the tubular lumen of the cortex and medulla from 20 weeks. Cystatin C immunoreactivity in the renal tubular lumen was considered to be caused by cystatin C remaining in primary urine that had not been reabsorbed into the proximal tubule due to its dysfunction. These findings are considered to be characteristic of renal tubules in ZDF rats. In conclusion, urinary cystatin C, 2-microglobulin, clusterin, GST-, KIM-1, and osteopontin were increased in ZDF rats before renal damage was observed, and increased further with the progression of diabetic nephropathy, demonstrating the usefulness for early detection and accurate assessment of diabetic nephropathy. Furthermore, immunohistochemical cystatin C expression was predominantly localized in the proximal renal tubules, but it was also observed in the tubular lumen in the cortex and medulla when renal damage occurred, which is considered to be characteristic of the renal tubular damage of diabetic nephropathy. References Abrahamson M, Olafsson I, Palsdottir A, Ulvsbäck M, Lundwall A, Jensson O, et al. Structure and expression of the human cystatin C gene. Biochemical Journal 1990;268:287–94. Baran D, Tenstad O, Aukland K. Localization of tubular uptake segment of filtered cystatin C and aprotinin in the rat kidney. Acta physiologica (Oxford) 2006;186:209–21. Brocco E, Fioretto P, Mauer M, Saller A, Carraro A, Frigato F, et al. Renal structure and function in non-insulin dependent diabetic patients with microalbuminuria. Kidney International Supplement 1997;63(December):S40–4. Coll E, Botey A, Alvarez L, Poch E, Quintó L, Saurina A. Serum cystatin C as a new marker for noninvasive estimation of glomerular filtration rate and as a marker for early renal impairment. American Journal of Kidney Diseases 2000;36:29–34. Conti M, Moutereau S, Zater M, Lallali K, Durrbach A, Manivet P, et al. Urinary cystatin C as a specific marker of tubular dysfunction. Clinical Chemistry and Laboratory Medicine 2006;44:288–91. Dieterle F, Perentes E, Cordier A, Roth DR, Verdes P, Grenet O, et al. Urinary clusterin, cystatin C, beta2-microglobulin and total protein as markers to detect druginduced kidney injury. Nature Biotechnology 2010;28:463–9. European Medicines Agency (EMEA). Final report on the pilot joint EMEA/FDA VXDs experience on qualification of nephrotoxicity biomarkers; 2008, http://www.emea.europa.eu/docs/en GB/document library/Report/2010/01/ WC500069676.pdf. Fassett RG, Venuthurupalli SK, Gobe GC, Coombes JS, Cooper MA, Hoy WE. Biomarkers in chronic kidney disease: a review. Kidney International 2011;80:806–21. Ferguson MA, Vaidya VS, Bonventre JV. Biomarkers of nephrotoxic acute kidney injury. Toxicology 2008;245:182–93. Food and Drug Administration (FDA). Final report on the pilot joint EMEA/FDA VXDs experience on qualification of nephrotoxicity biomarkers; 2008, http://www.emea.europa.eu/docs/en GB/document library/Report/2010/01/ WC500069676.pdf. Fu WJ, Li BL, Wang SB, Chen ML, Deng RT, Ye CQ, et al. Changes of the tubular markers in type 2 diabetes mellitus with glomerular hyperfiltration. Diabetes Research and Clinical Practice 2012;95:105–9. Fuchs TC, Hewitt P. Preclinical perspective of urinary biomarkers for the detection of nephrotoxicity: what we know and what we need to know. Biomarkers in Medicine 2011;5:763–79.
Grubb A. Diagnostic value of analysis of cystatin C and protein HC in biological fluids. Clinical Nephrology 1992;38(suppl. 1):S20–7. Herrera R, Almaguer M, Chipi J, Martínez O, Bacallao J, Rodríguez N, et al. Albuminuria as a marker of kidney and cardio-cerebral vascular damage. Isle of Youth Study (ISYS), Cuba. MEDICC Reviews 2010;12:20–6. Heuer JG, Sharma GR, Gerlitz B, Zhang T, Bailey DL, Ding C, et al. Evaluation of protein C and other biomarkers as predictors of mortality in a rat cecal ligation and puncture model of sepsis. Critical Care Medicine 2004;32:1570–8. Hewitt SM, Dear J, Star RA. Discovery of protein biomarkers for renal diseases. Journal of the American Society of Nephrology 2004;15:1677–89. Hoffmann D, Fuchs TC, Henzler T, Matheis KA, Herget T, Dekant W, et al. Evaluation of a urinary kidney biomarker panel in rat models of acute and subchronic nephrotoxicity. Toxicology 2010;277:49–58. Inker LA, Okparavero A. Cystatin C as a marker of glomerular filtration rate: prospects and limitations. Current Opinion in Nephrology and Hypertension 2011;20:631–9. International Life Sciences Institute Health and Environmental Sciences Institute (ILSI-HESI). Project committee on the development and application of biomarkers of nephrotoxicity; 2008, http://www.hesiglobal.org/i4a/ pages/index.cfm?pageID=3464. Ishii A, Sakai Y, Nakamura A. Molecular pathological evaluation of clusterin in a rat model of unilateral ureteral obstruction as a possible biomarker of nephrotoxicity. Toxicologic Pathology 2007;35:376–82. Jacobsson B, Lignelid H, Bergerheim US. Transthyretin and cystatin C are catabolized in proximal tubular epithelial cells and the proteins are not useful as markers for renal cell carcinomas. Histopathology 1995;26:559–64. Jeon YK, Kim MR, Huh JE, Mok JY, Song SH, Kim SS, et al. as an early biomarker of nephropathy in patients with type 2 diabetes. Journal of Korean Medical Science 2011;26:258–63. Kumar R, Schaefer J, Grande JP, Roche PC. Immunolocalization of calcitriol receptor, 24-hydroxylase cytochrome P-450, and calbindin D28k in human kidney. American Journal of Physiology 1994;266:F477–85. Lambers TT, Bindels RJ, Hoenderop JG. Coordinated control of renal Ca2+ handling. Kidney International 2006;69:650–4. Madero M, Sarnak MJ. Association of cystatin C with adverse outcomes. Current Opinion in Nephrology and Hypertension 2009;18:258–63. Marrer E, Dieterle F. Impact of biomarker development on drug safety assessment. Toxicology and Applied Pharmacology 2010;243:167–79. Martin H. Laboratory measurement of urine albumin and urine total protein in screening for proteinuria in chronic kidney disease. Clinical Biochemistry Reviews 2011;32:97–102. Nielsen SE, Andersen S, Zdunek D, Hess G, Parving HH, Rossing P. Tubular markers do not predict the decline in glomerular filtration rate in type 1 diabetic patients with overt nephropathy. Kidney International 2011;79:1113–8. Ozer JS. A guidance for renal biomarker lead optimization and use in translational pharmacodynamics. Drug Discovery Today 2010;15:142–7. Parikh CR, Lu JC, Coca SG, Devarajan P. Tubular proteinuria in acute kidney injury: a critical evaluation of current status and future promise. Annals of Clinical Biochemistry 2010;47:301–12. Shibata T, Takeuchi S, Yokota S, Kakimoto K, Yonemori F, Wakitani K. Effects of peroxisome proliferator-activated receptor-alpha and -gamma agonist, JTT-501, on diabetic complications in Zucker diabetic fatty rats. British Journal of Pharmacology 2000;130:495–504. Tenstad O, Roald AB, Grubb A, Aukland K. Renal handling of radiolabelled human cystatin C in the rat. Scandinavian Journal of Clinical and Laboratory Investigation 1996;56:409–14. Toblli JE, Ferrini MG, Cao G, Vernet D, Angerosa M, Gonzalez-Cadavid NF. Antifibrotic effects of pioglitazone on the kidney in a rat model of type 2 diabetes mellitus. Nephrology, Dialysis, Transplantation 2009;24:2384–91. Togashi Y, Sakaguchi Y, Miyamoto M, Miyamoto Y. Urinary cystatin C as a biomarker for acute kidney injury and its immunohistochemical localization in kidney in the CDDP-treated rats. Experimental and Toxicologic Pathology, in press. Tramonti G, Kanwar YS. Tubular biomarkers to assess progression of diabetic nephropathy. Kidney International 2011;79:1042–4. Waring WS, Moonie A. Earlier recognition of nephrotoxicity using novel biomarkers of acute kidney injury. Clinical Toxicology (Philadelphia) 2011;49:720–8. Wasilewska AM, Zoch-Zwierz WM. Urinary levels of matrix metalloproteinases and their tissue inhibitors in nephrotic children. Pediatric Nephrology 2008;23:1795–802. Zhou C, Gilbert JD, Byard RW. How useful is basal renal tubular epithelial cell vacuolization as a marker for significant hyperglycemia at autopsy? Journal of Forensic Sciences 2011;56:1531–3. Zoja C, Cattaneo S, Fiordaliso F, Lionetti V, Zambelli V, Salio M, et al. Distinct cardiac and renal effects of ETA receptor antagonist and ACE inhibitor in experimental type 2 diabetes. American Journal of Physiology Renal Physiology 2011;301:F1114–23.
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Experimental and Toxicologic Pathology journal homepage: www.elsevier.de/etp
Urinary cystatin C as a biomarker for diabetic nephropathy and its immunohistochemical localization in kidney in Zucker diabetic fatty (ZDF) rats Yuko Togashi, Yohei Miyamoto ∗ Toxicology and Pharmacokinetics Laboratories, Pharmaceutical Research Laboratories, Toray Industries, Inc., 6-10-1, Tebiro, Kamakura, Kanagawa 248-8555, Japan
a r t i c l e
i n f o
Article history: Received 12 March 2012 Accepted 20 June 2012 Keywords: Renal biomarker Cystatin C Diabetic nephropathy Immunohistochemistry Zucker diabetic fatty (ZDF) rats
a b s t r a c t Cystatin C, a cysteine protease inhibitor, is a novel biomarker of renal damage. In the present study, we examined the usefulness of urinary cystatin C for the detection of diabetic nephropathy in Zucker diabetic fatty (ZDF) rats compared to other biomarkers (2-microglobulin, calbindin, clusterin, epidermal growth factor (EGF), alpha-glutathione S-transferase (GST-␣), mu-glutathione S-transferase (GST-), kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), osteopontin, tissue inhibitor of metalloprotease-1 (TIMP-1), and vascular endothelial growth factor (VEGF). Urinary levels of cystatin C were increased in ZDF rats where renal damage was not histopathologically observed, and then further increased with the progression of renal damage, demonstrating the usefulness of early detection and accurate assessment of diabetic nephropathy. Urinary 2-microglobulin, clusterin, GST-, KIM-1, and osteopontin had the potency to detect renal damage in ZDF rats as well as cystatin C. We also investigated immunohistochemical localization of cystatin C in the kidney according to progressive renal damage. Cystatin C expression was mainly observed in the proximal renal tubule in ZDF rats, and hardly changed with progression of nephropathy. When renal damage was remarkable, cystatin C expression was also observed in the tubular lumen of the cortex and medulla, which was considered to be characteristic of renal damage in diabetic nephropathy. In conclusion, urinary cystatin C, 2-microglobulin, clusterin, GST-, KIM-1, and osteopontin could be useful biomarkers of diabetic nephropathy in ZDF rats. Immunohistochemical cystatin C expression in the proximal renal tubule was hardly changed by the progression of diabetic nephropathy, but it was newly observed in the tubular lumen when renal damage was remarkable in ZDF rats. Crown Copyright © 2012 Published by Elsevier GmbH. All rights reserved.
1. Introduction Serum creatinine and urea nitrogen (UN), which reflect impaired kidney function, are routinely used as markers of kidney disease and injury. However, these markers are insensitive since functional changes in the kidney are not observed until damage has become widespread (Hewitt et al., 2004; Ferguson et al., 2008). Therefore, new biomarkers for sensitive and more accurate indication are needed (Marrer and Dieterle, 2010; Fuchs and Hewitt, 2011). The Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) endorsed 7 urinary nephrotoxic biomarkers: total protein, albumin, kidney injury molecule-1 (KIM1), clusterin, 2-microglobulin, cystatin C, and trefoil factor 3 as biomarkers of acute kidney injury (AKI) in non-clinical settings (FDA, 2008; EMEA, 2008). The International Life Sciences Institute Health and Environmental Science (ILSI-HESI) also reported 4 urinary nephrotoxic biomarkers: alpha-glutathione S-transferase
∗ Corresponding author. Tel.: +81 467 32 9665; fax: +81 467 32 9768. E-mail address: Youhei [email protected] (Y. Miyamoto).
(GST-␣), mu-glutathione S-transferase (GST-), renal papillary antigen-1 (RPA-1), and clusterin (ILSI-HESI, 2008). In addition, several other biomarkers such as neutrophil gelatinase-associated lipocalin (NGAL) and tissue inhibitor of metalloprotease-1 (TIMP1) were reported to be able to detect nephrotoxicity, leading to the detection of AKI (Hoffmann et al., 2010). Cystatin C is a non-glycosylated 13 kDa cysteine protease inhibitor. It is produced at a constant rate by all karyocytes, directly and freely filtrated from the blood into the glomerulus, and is completely reabsorbed and catabolized in the proximal renal tubules (Abrahamson et al., 1990; Grubb, 1992; Tenstad et al., 1996). Because of these features, urinary cystatin C is an ideal estimator of the glomerular filtration rate (GFR), and its clinical use has been increasing recently (Coll et al., 2000; Madero and Sarnak, 2009). Tubular damage induces impairment of cystatin C reabsorption to the proximal renal tubule. In addition, glomerular alteration causes high molecular weight protein leakage, which leads to competition with tubular uptake of cystatin C. Therefore, urinary cystatin C, one of the qualified renal biomarkers for AKI, has been considered to have the potency to detect both glomerular and proximal renal injury (Conti et al., 2006; Dieterle et al., 2010; Parikh et al.,
0940-2993/$ – see front matter. Crown Copyright © 2012 Published by Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.etp.2012.06.005
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2010). We also showed it could detect CDDP-induced AKI in rats earlier than conventional markers (Togashi et al., in press). Diabetic nephropathy is a common microvascular complication of diabetic mellitus. It is characterized by the progressive impairment of glomerular function, leading to end-stage renal failure. Microalbuminuria has been considered to indicate renal damage at initial stages; however, morphological changes may occur earlier (Brocco et al., 1997; Fu et al., 2012). Recently, renal biomarkers such as cystatin C, NGAL, NAG, and KIM-1 in urine were reported to be useful for detection of chronic disease kidney (CKD) including diabetic nephropathy in clinical settings (Fassett et al., 2011; Jeon et al., 2011; Tramonti and Kanwar, 2011), but little is known in animal models. Zucker diabetic fatty (ZDF) rats, which have defective leptin receptors, exhibit metabolic abnormalities associated with type 2 diabetes and develop diabetic complications such as nephropathy, cataracts, and neuropathy (Shibata et al., 2000; Toblli et al., 2009). In this study, to clarify the usefulness of urinary cystatin C for the detection of diabetic nephropathy, we measured urinary C levels and compared them to other biomarkers (2-microglobulin, calbindin, clusterin, epidermal growth factor (EGF), GST-␣, GST, KIM-1, NGAL, osteopontin, TIMP-1, and vascular endothelial growth factor (VEGF)) in ZDF rats. Furthermore, we investigated the localization of cystatin C in the kidney according to the progression of renal damage.
2.3. Biomarker analysis Cystatin C, 2-microglobulin, calbindin, clusterin, EGF, GST-␣, GST-, KIM-1, NGAL, osteopontin, TIMP-1, and VEGF were measured by immunoassay using the rat kidney Multi-Analyte Profile kit (Rules Based Medicine, Austin, TX, USA), as described previously (Heuer et al., 2004). 2.4. Histopathology and immunohistochemistry After fixation, the kidney and pancreas were rinsed in tap water, dehydrated in a graded alcohol series, embedded in paraffin, and cut into 3 m thick sections. Paraffin sections were processed for hematoxylin–eosin (HE), Masson trichrome, and periodic acidSchiff staining (PAS), and histopathological changes were observed under a light microscope. In immunohistochemistry, an anti-rat cystatin C rabbit monoclonal antibody (Abcam Ltd., Cambridge, UK) and the Vectastain ABC kit (Vector Laboratories, Inc., Burlingame, CA, USA) were used. 2.5. Statistical analysis The differences between lean and ZDF rats at each age were analyzed with the Student’s t-test or Aspin–Welch’s t-test. The level of significance was set at 1% and 5%.
2. Materials and methods 3. Results 2.1. Animals 3.1. Fundamental metabolic parameters in diabetic model rats Male Zucker diabetic fatty rats (ZDF/CrlCrlj-Leptfa/fa , Charles River Laboratories Japan, Inc., Yokohama, Japan) and male nondiabetic lean rats (ZDF/CrlCrlj-Lept?/+ , Charles River Laboratories, Yokohama, Japan), which were described as ZDF and lean rats, were housed at 2 or 3 animals per cage under controlled temperature, lighting, and relative humidity conditions (19–25 ◦ C; 12 h on/off light/dark cycle; 40–60%, respectively). They were given a standard diet (Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water ad libitum, but were fasted during the urine collection period. Urine was collected and cooled for 24 h using individual metabolic cages with no food but water. Urine volumes were recorded, and the collected urine was centrifuged at 1870 × g for 15 min at 4 ◦ C and then stored at −80 ◦ C until analysis. After final body weighing, blood was collected from the abdominal aorta under pentobarbital anesthesia. The plasma was separated by centrifugation for 15 min at 4 ◦ C and stored at −80 ◦ C until analysis. After the collection of urine and blood, the rats were euthanized by bleeding under pentobarbital anesthesia, and then the kidneys were removed and fixed in 10% neutral buffered formalin. All experiments were conducted according to the Guidelines for Animal Experiments, Research & Development Division, Toray Industries, Inc. 2.2. Blood chemistry Creatinine, glucose, phospholipids, total cholesterol, triglycerides, UN in plasma, and albumin in urine were determined using a Hitachi 7070 automatic analyzer (Hitachi Instruments Service Co., Ltd, Tokyo, Japan), and autosera kits were used to determine creatinine (urease – UV method), glucose (hexokinase – UV method), phospholipids (phospholipase – HSDA method), total cholesterol (cholesterol oxidase method), triglycerides (free glycerol elimination method), UN (urease – GLDH method), and albumin (BCGs method). Plasma insulin was determined using a rat insulin ELISA kit (Shibayagi Co., Gunma, Japan).
Body weight and fundamental metabolic parameters were measured in lean and ZDF rats at 12, 20, and 25 weeks. Body weight in ZDF rats was significantly higher than lean rats at 12 weeks, but there was no apparent difference between both groups at 20 and 25 weeks. Plasma levels of glucose, phospholipids, total cholesterol, and triglycerides in ZDF rats were significantly higher than those in lean rats at 12 weeks, and increased thereafter. In contrast, plasma levels of insulin in ZDF rats were almost the same as lean rats at 12 weeks, and slightly decreased at 20 and 25 weeks (Table 1). Consistent with metabolic abnormalities, focal fibrosis of pancreatic islets was histopathologically pronounced in ZDF rats at 20 and 25 weeks, whereas no remarkable change was observed in lean rats (Fig. 1). 3.2. Renal functional and morphological changes in ZDF rats Renal functional parameters and histopathological changes were examined in lean and ZDF rats at 12, 20, and 25 weeks. Urine output for 24 hr and kidney weight were significantly higher in ZDF rats than those in lean rats at 12 weeks, and reached a plateau at 20 weeks. Excretion of urine albumin increased age-dependently (12 weeks, 2.9-fold; 20 weeks, 7.1-fold; and 25 weeks, 22-fold). On the other hand, plasma creatinine in ZDF rats did not increase relative to lean rats at all ages. Plasma UN was slightly increased in ZDF rats relative to lean ZDF rats, but the increase ratios were low and unchanged from 12 to 25 weeks (1.5–1.7-fold) (Table 2). In histopathology of the kidney, increased mesangial matrix and glomerular exudate were observed in ZDF rats at 20 and 25 weeks. In addition, PAS-positive hyaline droplets in the glomerulus were observed in ZDF rats from 12 weeks. Renal tubular alternations such as tubular dilatation, hyaline cast, basophilic changes with regeneration, and renal tubular vacuolation were noted in ZDF rats at 20 and 25 weeks. Pelvic dilatation was macroscopically apparent in most lean and ZDF rats, but it worsened in ZDF rats with age (Table 3, Figs. 2 and 3).
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Table 1 Fundamental metabolic parameters in lean and ZDF rats. 12 weeks Lean Body weight (g) Glucose (mg/dL) Insulin (ng/mL) Phospholipid (mg/dL) Total cholesterol (mg/dL) Triglyceride (mg/dL)
282 206 3.7 116 64 27
± ± ± ± ± ±
20 weeks ZDF
19 19 1.2 6 5 13
340 519 3.9 215 98 295
Lean ± ± ± ± ± ±
9** 50** 1.0 19** 8** 78**
376 222 5.8 137 90 56
25 weeks ZDF
± ± ± ± ± ±
15 27 0.5 8 5 7
384 653 3.1 305 186 269
Lean ± ± ± ± ± ±
16 48** 1.0** 26** 16** 41**
410 233 5.2 155 99 71
± ± ± ± ± ±
ZDF 9 12 1.7 9 7 25
404 657 2.5 361 225 333
± ± ± ± ± ±
30 80** 0.4* 40** 31** 79**
Each data is the mean ± S.D. of 5 animals. * Significant differences between lean and ZDF rats at each point: P < 0.05. ** Significant differences between lean and ZDF rats at each point: P < 0.01.
Fig. 1. Histopathological changes in the pancreas of lean (A–C) and ZDF (D–F) rats. No marked changes are seen in lean rats at 12, 20, and 25 weeks. Islet fibrosis is seen in ZDF rats at 20 weeks, which worsened at 25 weeks. (A and D) 12 weeks; (B and E) 20 weeks; and (C and F) 25 weeks. Masson trichrome staining. Each bar shows 100 m.
3.3. Cystatin C and other renal biomarkers in ZDF rats Urinary cystatin C levels in lean and ZDF rats were measured at 12, 20, and 25 weeks and compared to other renal biomarkers. Urinary cystatin C levels were significantly higher (2.3-fold) in ZDF rats than those in lean rats at 12 weeks, and further increased at 20 and 25 weeks (20 weeks; 4.0-fold, 25 weeks; 9.5-fold). From 12 weeks, urinary 2-microglobulin, clusterin, GST-, KIM-1,
and osteopontin levels increased age-dependently, by 3.2–27.1fold, 2.4–24.8-fold, 2.2–12.7-fold, 1.7–5.6-fold, and 2.7–9.3-fold, respectively. Calbindin and TIMP-1 in ZDF rats were significantly increased at 12, 20, and 25 weeks, but the increase ratios were unchanged. EGF was significantly decreased at 20 and 25 weeks. On the other hand, GST-␣, NGAL, and VEGF in ZDF rats were unchanged at all ages, though significant differences were sporadically seen (Table 4, Fig. 4).
Table 2 Renal function parameters in lean and ZDF rats. 12 weeks Lean Kidney (g/100 g body weight) Urine (mL/24 h) Plasma creatinine (mg/dL) Plasma UN (mg/dL) Urine albumin (mg/24 h)
0.70 9.3 0.32 15.6 4.2
20 weeks ZDF
± ± ± ± ±
0.04 1.4 0.02 1.1 1.2
0.96 15.1 0.26 23.7 12.2
Lean ± ± ± ± ±
**
0.02 2.2** 0.03** 2.3** 3.0**
Each data is the mean ± S.D. of 5 animals. * Significant differences between lean and ZDF rats at each point: P < 0.05. ** Significant differences between lean and ZDF rats at each point: P < 0.01.
0.76 6.8 0.38 16.2 4.0
25 weeks ZDF
± ± ± ± ±
0.04 2.2 0.04 0.7** 0.7
1.25 41.6 0.29 24.2 28.4
Lean ± ± ± ± ±
**
0.07 12.5** 0.02** 1.1** 7.6**
0.80 6.0 0.34 13.7 4.0
ZDF ± ± ± ± ±
0.02 2.1 0.03 1.7 0.7
1.26 31.4 0.27 23.9 90.1
± ± ± ± ±
0.11** 11.7** 0.02** 3.3** 29.1**
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Table 3 Histopathological changes of kidney in lean and ZDF rats. 12 weeks
(No. of animals) Histopathological findings
20 weeks
25 weeks
Lean (5)
ZDF (5)
Lean (5)
ZDF (5)
Lean (5)
ZDF (5)
Grade*
Glomerular exudate
0 1 2 3
0 0 0 0
0 0 0 0
0 0 0 0
2 3 0 0
0 0 0 0
0 5 0 0
Glomerular hyaline droplet
0 1 2 3
0 0 0 0
2 3 0 0
0 0 0 0
2 3 0 0
0 0 0 0
0 5 0 0
Increased mesangial matrix
0 1 2 3
0 0 0 0
0 0 0 0
0 0 0 0
3 2 0 0
0 0 0 0
0 5 0 0
Basophilic renal tubule
0 1 2 3
0 0 0 0
0 0 0 0
0 0 0 0
1 2 0 0
0 0 0 0
0 2 2 0
Hyaline cast of renal tubule
0 1 2 3
0 0 0 0
0 0 0 0
0 0 0 0
0 4 1 0
0 0 0 0
0 2 3 0
Tubular dilatation
0 1 2 3
0 0 0 0
0 0 0 0
0 0 0 0
2 0 0 0
0 0 0 0
0 5 0 0
Vacuolation of renal tubule
0 1 2 3
0 0 0 0
0 0 0 0
0 0 0 0
1 3 0 0
0 0 0 0
1 3 0 0
Pelvic dilatation
0 1 2 3
4 0 0 0
0 2 1 1
0 3 0 0
0 0 3 2
2 1 1 0
1 0 2 3
*
0, very slight; 1, slight; 2, moderate; and 3, marked.
Fig. 2. Histopathological changes in the kidney of lean (A–C) and ZDF (D–F) rats. No marked changes are seen in lean rats at all ages. Glomerular exudate and renal tubular alterations such as hyaline cast and basophilic changes with regeneration are apparent in ZDF rats at 20 and 25 weeks. In addition, renal tubular vacuolation is seen from 12 weeks. (A and D) 12 weeks; (B and E) 20 weeks; and (C and F) 25 weeks. HE staining. Each bar shows 200 m.
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Fig. 3. Histopathological changes in the kidney of lean (A–C, G) and ZDF (D–F, H) rats. No marked changes are seen in lean rats at all ages. Increased mesangial matrix is seen in ZDF rats at 20 weeks, which worsened at 25 weeks. PAS-positive hyaline droplet in the glomerulus (arrows) is seen only in ZDF rats from 12 weeks. (A and D) 12 weeks; (B and E) 20 weeks; and (C, F–H) 25 weeks. PAS (A–G) and HE (H) staining. Each bar shows 50 m.
Table 4 Urinary levels of renal biomarkers in lean and ZDF rats. 12 weeks
20 weeks
Lean Cystatin C (ng/24 h) 2-microglobulin (g/24 h) Calbindin (ng/24 h) Clusterin (g/24 h) EGF (ng/24 h) GST-␣ (ng/24 h) GST- (ng/24 h) KIM-1 (ng/24 h) NGAL (ng/24 h) Osteopontin (ng/24 h) TIMP-1 (ng/24 h) VEGF (pg/24 h)
2809 65.3 1690 5.1 81,846 426 157 3.40 674 71 22.2 6610
ZDF ± ± ± ± ± ± ± ± ± ± ± ±
519 17.3 472 1.7 24,402 108 35 0.91 280 31 9.8 2265
6486 212.1 7307 13.6 52,756 450 344 5.77 1154 189 68.0 9681
25 weeks
Lean ± ± ± ± ± ± ± ± ± ± ± ±
712** 81.8* 811** 5.1* 7502 217 52** 1.65* 516 74* 32.2* 3129
Each data is the mean ± S.D. of 5 animals. * Significant differences between lean and ZDF rats at each point: P < 0.05. ** Significant differences between lean and ZDF rats at each point: P < 0.01.
2620 106.6 2413 6.3 124,740 224 105 2.76 335 153 19.8 6004
ZDF ± ± ± ± ± ± ± ± ± ± ± ±
347 19.8 889 2.8 9827 105 74 1.23 136 41 8.9 2528
10,368 603.8 7505 30.2 34,290 668 737 9.5 535 470 71.0 13,515
Lean ± ± ± ± ± ± ± ± ± ± ± ±
1952** 77.1** 1639** 7.9** 5346** 197** 177** 2.8** 751 148** 27.4* 5417*
2404 54.8 1628 3.9 176,116 262 112 2.8 358 87 17.6 5738
ZDF ± ± ± ± ± ± ± ± ± ± ± ±
826 9.9 233 1.2 52,846 196 44 1.2 273 17 8.5 2373
22,916 1485.7 8142 97.0 35,055 834 1421 15.4 1552 811 86.7 11,701
± ± ± ± ± ± ± ± ± ± ± ±
7384** 702.1* 2652** 41.6** 6349** 443* 722* 5.4** 411** 304** 25.1** 4999
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Fig. 4. Urinary levels of cystatin C and other renal biomarkers in lean and ZDF rats. From 12 weeks, urinary cystatin C, 2-microglobulin, clusterin, GST-, KIM-1, and osteopontin increased with age in ZDF rats. Calbindin and TIMP-1 in ZDF rats increased at all ages, but the increase ratios were unchanged. EGF decreased from 20 weeks in ZDF rats. Each column represents the mean ± S.D. of 5 animals. Significant differences between the corresponding lean and ZDF rats at each point: *P < 0.05 and **P < 0.01. Lean rats (open column) and ZDF rats (closed column).
3.4. Immunohistochemical localization of cystatin C in the kidney of ZDF rats In both lean and ZDF rats, cystatin C was mainly localized in the proximal renal tubule of the cortex, but hardly observed in the medulla. In ZDF rats, cystatin C was also observed in the renal tubular lumen of the cortex and medulla from 20 weeks, but this finding was not observed in lean rats at all ages (Fig. 5).
4. Discussion In this study, we measured urinary cystatin C levels in ZDF rats, a type 2 diabetic nephropathy model, to evaluate its usefulness in detection of renal damage compared to urinary biomarkers (2-microglobulin, calbindin, clusterin, EGF, GST-␣, GST-, KIM1, NGAL, osteopontin, TIMP-1, and VEGF). In this study, ZDF rats showed hyperglycemia and hyperlipidemia from 12 weeks and slight decreases in insulin levels and fibrosis of pancreatic islets from 20 weeks, which indicated the onset of diabetic mellitus. As for renal function, urine volume and albumin excretion were increased from 12 weeks, and glomerular changes such as increased mesangial matrix and renal tubular changes were histopathologically observed from 20 weeks. In addition, renal tubular vacuolation, which is indicative of persistent diabetes and resembles human Armanni-Ebstein lesions (Zhou et al., 2011), and glomerular hyaline droplets were seen in ZDF rats. Therefore, ZDF rats were considered to establish incipient diabetic nephropathy, which is consistent with previous reports (Toblli et al., 2009; Zoja et al., 2011).
Urinary cystatin C was significantly higher in ZDF rats than that of lean rats at 12 weeks, prior to histopathological changes in the kidney, and further increased at 20 and 25 weeks. This result indicated that urinary cystatin C would be useful for early detection and accurate assessment of diabetes nephropathy. However, albuminuria, the most sensitive marker for diabetic nephropathy in clinical settings (Herrera et al., 2010; Martin, 2011), developed in ZDF rats from 12 weeks, indicating that the potential of urinary cystatin C and albumin for early detection of diabetic nephropathy is similar in the rat model. Urinary cystatin C was considered to have the potential to detect both glomerular and renal tubular damage in rats and humans. In this study, increased urinary cystatin C reflected damage to both the glomerulus and proximal renal tubule, since they were histopathologically observed in ZDF rats. Serum cystatin C is recognized as a superior marker of GFR and is able to detect mild nephropathy (CKD stages 1 and 2) in humans (Ozer, 2010; Inker and Okparavero, 2011). On the other hand, the usefulness of urinary cystatin C for the prediction of CKD has hardly been known, though recent studies suggested that it was a possible marker for renal dysfunction in type 2 diabetic patients (Jeon et al., 2011). The present study demonstrated for the first time that urinary cystatin C could be used as an indicator of diabetic nephropathy in ZDF rats. Urinary levels of 2-microglobulin, clusterin, GST-, KIM-1, and osteopontin were also higher in ZDF rats than those in lean rats at 12 weeks, and became even higher with progressive renal damage, indicating that they could be used as candidate biomarkers of diabetic nephropathy similar to cystatin C. KIM-1, an extracellular protein anchored in the proximal tubule membrane, is highly
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Fig. 5. Immunohistochemical localization of cystatin C in the kidney of lean (A–C, G–I) and ZDF (D–F, J–L) rats. Cystatin C is seen in the proximal renal tubules in the cortex (A–C), but hardly in the medulla (G–I) in lean rats. Cystatin C is seen in the proximal renal tubules in the cortex (D–F) in ZDF rats at all ages, and was also noted in the lumens of the renal tubules in the cortex (F) and medulla (K and L) at 20 and 25 weeks. Arrows represent cystatin C in the tubular lumen. Each bar represents 1000 m. (A–F) Cortex and (G–L) medulla.
induced and secreted in urine after renal damage, and is widely considered to be a sensitive biomarker to detect AKI (Waring and Moonie, 2011). In addition, recent studies reported that urinary excretion of KIM-1 could be used as a reliable marker for the progression of diabetic nephropathy in humans (Nielsen et al., 2011), which is consistent with the results of our present study in rats. NGAL, a ubiquitous lipocalin iron-carrying protein, has been identified as an early biomarker of AKI, and has great promise as a biomarker of CDK including diabetic nephropathy (Fassett et al., 2011; Tramonti and Kanwar, 2011). However, urinary levels were not increased in ZDF rats contrary to our expectations. Furthermore, our data could provide new insights into the potential role of 2-microglobulin, clusterin, GST-, and osteopontin as biomarkers of diabetic nephropathy, since little is known about their urinary
levels in CKD. TIMP-1, a tissue remodeling protein (Ishii et al., 2007; Wasilewska and Zoch-Zwierz, 2008), and calbindin, an intracellular calcium-binding protein (Kumar et al., 1994; Lambers et al., 2006), were increased in urine when renal damage was not evident, but these levels were not changed at any age in ZDF rats. The reason for this is unclear, but interesting findings could be provided if urinary levels are measured before 12 weeks. We also investigated the immunohistochemical localization of cystatin C in lean and ZDF rats. In lean rats, cystatin C was predominantly localized in the renal proximal tubules of the cortex, but were hardly observed in the medulla, which is consistent with previous studies using rats and humans (Jacobsson et al., 1995; Baran et al., 2006). Cystatin C is reabsorbed to proximal tubular epithelial cells, thus, the immunoreactive cystatin C detected in the epithelial
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cells was most likely caused by reabsorption from primary urine. Cystatin C expression was also observed in renal tubular epithelial cells in the cortex in ZDF rats, and was not apparently changed with progression of renal damage. Therefore, it is not considered that immunohistochemical expression of cystatin C in proximal renal tubules may be more sensitive for diabetic nephropathy than its urinary level. Consistent with this study, immunohistochemical expression of cystatin C in proximal renal tubules was not changed by CDDP-induced AKI in rats in our previous study (Togashi et al., in press), though there was a contrary report showing it was altered by some glomerular and/or tubular nephrotoxicants in rats (Dieterle et al., 2010). In ZDF rats, cystatin C was observed in not only the proximal renal tubule but also the tubular lumen of the cortex and medulla from 20 weeks. Cystatin C immunoreactivity in the renal tubular lumen was considered to be caused by cystatin C remaining in primary urine that had not been reabsorbed into the proximal tubule due to its dysfunction. These findings are considered to be characteristic of renal tubules in ZDF rats. In conclusion, urinary cystatin C, 2-microglobulin, clusterin, GST-, KIM-1, and osteopontin were increased in ZDF rats before renal damage was observed, and increased further with the progression of diabetic nephropathy, demonstrating the usefulness for early detection and accurate assessment of diabetic nephropathy. Furthermore, immunohistochemical cystatin C expression was predominantly localized in the proximal renal tubules, but it was also observed in the tubular lumen in the cortex and medulla when renal damage occurred, which is considered to be characteristic of the renal tubular damage of diabetic nephropathy. References Abrahamson M, Olafsson I, Palsdottir A, Ulvsbäck M, Lundwall A, Jensson O, et al. Structure and expression of the human cystatin C gene. Biochemical Journal 1990;268:287–94. Baran D, Tenstad O, Aukland K. Localization of tubular uptake segment of filtered cystatin C and aprotinin in the rat kidney. Acta physiologica (Oxford) 2006;186:209–21. Brocco E, Fioretto P, Mauer M, Saller A, Carraro A, Frigato F, et al. Renal structure and function in non-insulin dependent diabetic patients with microalbuminuria. Kidney International Supplement 1997;63(December):S40–4. Coll E, Botey A, Alvarez L, Poch E, Quintó L, Saurina A. Serum cystatin C as a new marker for noninvasive estimation of glomerular filtration rate and as a marker for early renal impairment. American Journal of Kidney Diseases 2000;36:29–34. Conti M, Moutereau S, Zater M, Lallali K, Durrbach A, Manivet P, et al. Urinary cystatin C as a specific marker of tubular dysfunction. Clinical Chemistry and Laboratory Medicine 2006;44:288–91. Dieterle F, Perentes E, Cordier A, Roth DR, Verdes P, Grenet O, et al. Urinary clusterin, cystatin C, beta2-microglobulin and total protein as markers to detect druginduced kidney injury. Nature Biotechnology 2010;28:463–9. European Medicines Agency (EMEA). Final report on the pilot joint EMEA/FDA VXDs experience on qualification of nephrotoxicity biomarkers; 2008, http://www.emea.europa.eu/docs/en GB/document library/Report/2010/01/ WC500069676.pdf. Fassett RG, Venuthurupalli SK, Gobe GC, Coombes JS, Cooper MA, Hoy WE. Biomarkers in chronic kidney disease: a review. Kidney International 2011;80:806–21. Ferguson MA, Vaidya VS, Bonventre JV. Biomarkers of nephrotoxic acute kidney injury. Toxicology 2008;245:182–93. Food and Drug Administration (FDA). Final report on the pilot joint EMEA/FDA VXDs experience on qualification of nephrotoxicity biomarkers; 2008, http://www.emea.europa.eu/docs/en GB/document library/Report/2010/01/ WC500069676.pdf. Fu WJ, Li BL, Wang SB, Chen ML, Deng RT, Ye CQ, et al. Changes of the tubular markers in type 2 diabetes mellitus with glomerular hyperfiltration. Diabetes Research and Clinical Practice 2012;95:105–9. Fuchs TC, Hewitt P. Preclinical perspective of urinary biomarkers for the detection of nephrotoxicity: what we know and what we need to know. Biomarkers in Medicine 2011;5:763–79.
Grubb A. Diagnostic value of analysis of cystatin C and protein HC in biological fluids. Clinical Nephrology 1992;38(suppl. 1):S20–7. Herrera R, Almaguer M, Chipi J, Martínez O, Bacallao J, Rodríguez N, et al. Albuminuria as a marker of kidney and cardio-cerebral vascular damage. Isle of Youth Study (ISYS), Cuba. MEDICC Reviews 2010;12:20–6. Heuer JG, Sharma GR, Gerlitz B, Zhang T, Bailey DL, Ding C, et al. Evaluation of protein C and other biomarkers as predictors of mortality in a rat cecal ligation and puncture model of sepsis. Critical Care Medicine 2004;32:1570–8. Hewitt SM, Dear J, Star RA. Discovery of protein biomarkers for renal diseases. Journal of the American Society of Nephrology 2004;15:1677–89. Hoffmann D, Fuchs TC, Henzler T, Matheis KA, Herget T, Dekant W, et al. Evaluation of a urinary kidney biomarker panel in rat models of acute and subchronic nephrotoxicity. Toxicology 2010;277:49–58. Inker LA, Okparavero A. Cystatin C as a marker of glomerular filtration rate: prospects and limitations. Current Opinion in Nephrology and Hypertension 2011;20:631–9. International Life Sciences Institute Health and Environmental Sciences Institute (ILSI-HESI). Project committee on the development and application of biomarkers of nephrotoxicity; 2008, http://www.hesiglobal.org/i4a/ pages/index.cfm?pageID=3464. Ishii A, Sakai Y, Nakamura A. Molecular pathological evaluation of clusterin in a rat model of unilateral ureteral obstruction as a possible biomarker of nephrotoxicity. Toxicologic Pathology 2007;35:376–82. Jacobsson B, Lignelid H, Bergerheim US. Transthyretin and cystatin C are catabolized in proximal tubular epithelial cells and the proteins are not useful as markers for renal cell carcinomas. Histopathology 1995;26:559–64. Jeon YK, Kim MR, Huh JE, Mok JY, Song SH, Kim SS, et al. as an early biomarker of nephropathy in patients with type 2 diabetes. Journal of Korean Medical Science 2011;26:258–63. Kumar R, Schaefer J, Grande JP, Roche PC. Immunolocalization of calcitriol receptor, 24-hydroxylase cytochrome P-450, and calbindin D28k in human kidney. American Journal of Physiology 1994;266:F477–85. Lambers TT, Bindels RJ, Hoenderop JG. Coordinated control of renal Ca2+ handling. Kidney International 2006;69:650–4. Madero M, Sarnak MJ. Association of cystatin C with adverse outcomes. Current Opinion in Nephrology and Hypertension 2009;18:258–63. Marrer E, Dieterle F. Impact of biomarker development on drug safety assessment. Toxicology and Applied Pharmacology 2010;243:167–79. Martin H. Laboratory measurement of urine albumin and urine total protein in screening for proteinuria in chronic kidney disease. Clinical Biochemistry Reviews 2011;32:97–102. Nielsen SE, Andersen S, Zdunek D, Hess G, Parving HH, Rossing P. Tubular markers do not predict the decline in glomerular filtration rate in type 1 diabetic patients with overt nephropathy. Kidney International 2011;79:1113–8. Ozer JS. A guidance for renal biomarker lead optimization and use in translational pharmacodynamics. Drug Discovery Today 2010;15:142–7. Parikh CR, Lu JC, Coca SG, Devarajan P. Tubular proteinuria in acute kidney injury: a critical evaluation of current status and future promise. Annals of Clinical Biochemistry 2010;47:301–12. Shibata T, Takeuchi S, Yokota S, Kakimoto K, Yonemori F, Wakitani K. Effects of peroxisome proliferator-activated receptor-alpha and -gamma agonist, JTT-501, on diabetic complications in Zucker diabetic fatty rats. British Journal of Pharmacology 2000;130:495–504. Tenstad O, Roald AB, Grubb A, Aukland K. Renal handling of radiolabelled human cystatin C in the rat. Scandinavian Journal of Clinical and Laboratory Investigation 1996;56:409–14. Toblli JE, Ferrini MG, Cao G, Vernet D, Angerosa M, Gonzalez-Cadavid NF. Antifibrotic effects of pioglitazone on the kidney in a rat model of type 2 diabetes mellitus. Nephrology, Dialysis, Transplantation 2009;24:2384–91. Togashi Y, Sakaguchi Y, Miyamoto M, Miyamoto Y. Urinary cystatin C as a biomarker for acute kidney injury and its immunohistochemical localization in kidney in the CDDP-treated rats. Experimental and Toxicologic Pathology, in press. Tramonti G, Kanwar YS. Tubular biomarkers to assess progression of diabetic nephropathy. Kidney International 2011;79:1042–4. Waring WS, Moonie A. Earlier recognition of nephrotoxicity using novel biomarkers of acute kidney injury. Clinical Toxicology (Philadelphia) 2011;49:720–8. Wasilewska AM, Zoch-Zwierz WM. Urinary levels of matrix metalloproteinases and their tissue inhibitors in nephrotic children. Pediatric Nephrology 2008;23:1795–802. Zhou C, Gilbert JD, Byard RW. How useful is basal renal tubular epithelial cell vacuolization as a marker for significant hyperglycemia at autopsy? Journal of Forensic Sciences 2011;56:1531–3. Zoja C, Cattaneo S, Fiordaliso F, Lionetti V, Zambelli V, Salio M, et al. Distinct cardiac and renal effects of ETA receptor antagonist and ACE inhibitor in experimental type 2 diabetes. American Journal of Physiology Renal Physiology 2011;301:F1114–23.