Tyrosine kinase inhibitor, genistein, reduces renal inflammation and injury in streptozotocin-induced diabetic mice

Vascular Pharmacology 55 (2011) 149–156

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Vascular Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / v p h

Tyrosine kinase inhibitor, genistein, reduces renal inflammation and injury in streptozotocin-induced diabetic mice Ahmed A. Elmarakby a, b, c, f,⁎, Ahmed S. Ibrahim c, d, g, Jessica Faulkner a, Mahmood S. Mozaffari a, Gregory I. Liou c, d, Rafik Abdelsayed e a

Department of Oral Biology, Georgia Health Sciences University, Augusta, GA. 30912, United States Pharmacology & Toxicology, Georgia Health Sciences University, Augusta, GA. 30912, United States Vascular Biology Center, Georgia Health Sciences University, Augusta, GA. 30912, United States d Ophthalmology, Georgia Health Sciences University, Augusta, GA. 30912, United States e Oral Health and Diagnostic Sciences, Georgia Health Sciences University, Augusta, GA. 30912, United States f Department of Pharmacology & Toxicology, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt g Department of Biochemistry, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt b c

a r t i c l e

i n f o

Article history: Received 14 February 2011 Received in revised form 7 July 2011 Accepted 8 July 2011 Keywords: Diabetes P-tyrosine Nephrinuria Albuminuria gp91phox ICAM-1

a b s t r a c t Tyrosine kinase inhibition is known to reduce diabetes-induced end-organ damage but the mechanisms remain elusive. We hypothesized that inhibition of tyrosine kinase reduces renal inflammation and injury in streptozotocin-induced diabetes. Male C57BL/6 mice were given daily injections of streptozotocin (45 mg/kg/day, i.p. for 5 days); control animals received the vehicle (citrate buffer). Thereafter, streptozotocin-treated mice were treated with genistein (10 mg/kg, i.p three times a week for 10 weeks, n = 8–10/group) or the vehicle (5% DMSO). The streptozotocin-treated mice displayed significant elevation in blood glucose level and decrease in plasma insulin level compared to their vehicle-treated controls. Treatment with genistein reduced blood glucose level (~15%; p b 0.05) without a significant effect on plasma insulin level; however, blood glucose remained significantly higher than the control group. The development of diabetes was associated with significant increases in total protein, albumin, nephrin and collagen excretions compared to their controls. In addition, the diabetic mice displayed increased urinary MCP-1 excretion in association with increased renal ICAM-1 expression and apoptotic cells. Furthermore, renal gp91 expression levels and urinary Thio-Barbituric Acid Reactive Substances (TBARs) excretion, indices of oxidative stress, were also elevated in diabetic mice. These changes were associated with increased renal phospho-tyrosine expression and renal phospho-ERK/ERK ratio. Importantly, treatment with genistein reduced all these parameters towards control values. Collectively, the results suggest that the reno-protective effect of genistein likely relates to reduced renal inflammation, oxidative stress and apoptosis in diabetic mice. © 2011 Elsevier Inc. All rights reserved.

1. Introduction Diabetes mellitus is a global health problem and its worldwide prevalence is expected to increase to 366 million by the year 2030 (Wild et al., 2004). Persistent hyperglycemia leads to several complications including retinopathy, nephropathy, and neuropathy despite existing therapeutic measures. Diabetic nephropathy is a devastating complication, affecting one out of three patients with type 1 diabetes (Falk et al., 1983). Signs and symptoms of diabetic nephropathy include nephrotic syndrome with excessive filtration of proteins into the urine and progressive impairment in kidney function which could further progress to end-stage renal disease. Therefore,

⁎ Corresponding author at: Medical College of Georgia, Augusta, GA. 30912, United States. Tel.: + 1 706 721 2748; fax: + 1 706 721 6252. E-mail address: [email protected] (A.A. Elmarakby). 1537-1891/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.vph.2011.07.007

new therapeutic strategies that prevent diabetic renal complications and progression to end-stage renal failure are required. The etiology of diabetic renal injury is complex and multi-factorial including hyperglycemia, hypertension, dyslipidemia, production of inflammatory cytokines and oxidative stress (Sasser et al., 2007). Hyperglycemia is known to increase oxidative stress and inflammation in diabetic animal models and NAD(P)H oxidase has emerged as a major source of superoxide production (Sasser et al., 2007). Over the past decade, inflammation has received considerable attention in relation to the pathogenesis of diabetic renal injury (Kaul et al., 2010). Clinical studies indicate that infiltration of mononuclear cells is a prominent feature in the glomeruli of patients with diabetic nephropathy (Furuta et al., 1993). The infiltrated macrophages release reactive oxygen species (ROS), transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein (MCP-1) that serve to initiate subsequent immune response leading to fibrosis, matrix

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deposition and progressive renal injury (Chow et al., 2004; Wada et al., 2000; Tesch, 2010). Thus, Pharmacological regulation of inflammatory reaction is a rational approach to modulate early pathological changes to prevent loss of renal function. Tyrosine kinases are implicated in renal inflammation as they are important components of signaling pathways for cytokine production. Many cytokines, including TNF-α, interleukin 6 and 10 (IL-6 and IL-10), utilize tyrosine kinases in their signaling pathways, making them critical not only for cytokine production but also for their function (Page et al., 2009). Downstream signaling of tyrosine kinases involves activation of extracellular signal-regulated kinase (ERK) which is a critical player in early extracellular matrix accumulation and glomerular mesangial cells expansion in diabetic kidney (Stevens et al., 2007; Ohashi et al., 2010). Given the crucial role of inflammation in the development of diabetic renal injury and the fact that tyrosine kinase plays a pivotal role in initiating the activation of various inflammatory cells (Correll et al., 2004), we hypothesized that tyrosine kinase inhibition attenuates diabetes-induced renal injury. Genistein, an isoflavonoid, is a naturally occurring tyrosine kinase inhibitor that has attracted considerable attention because of its anti-inflammatory properties (Polkowski and Mazurek, 2000). Genistein reduces pro-inflammatory factor-induced vascular endothelial barrier dysfunction and inhibits leukocyte-endothelium interaction, the major events in the pathogenesis of microvascular lesions (Si and Liu, 2007). Furthermore, genistein protects the mouse kidney from cisplatin-induced injury, improves functional changes in aortic vascular reactivity in diabetic rats and inhibits retinal vascular leakage observed in diabetic rats (Sung et al., 2008; Baluchnejadmojarad and Roghan, 2008; Nakajima et al., 2001). However, the potential effects of genistein in diabetesinduced renal complications have not been addressed. Thus, we evaluated the efficacy of genistein in alleviating diabetes-induced renal injury in the current study in the context of its potential effects on several inflammatory markers. 2. Materials and methods All procedures with animals were performed in accordance with the Public Health Service Guide for the Care and Use of Laboratory Animals (Department of Health, Education, and Welfare publication, NIH 80–23) and Georgia Health Sciences University guidelines. Eightweek-old male C57BL/6 mice were given daily injection of streptozotocin (45 mg/kg; i.p.) for 5 days after a 4 hour fast; control animals received the vehicle (citrate buffer; 0.01 mol/L, pH: 4.5). Diabetes was confirmed by measurement of fasting blood glucose levels of N250 mg/dl three days after streptozotocin injection. Thereafter, diabetic mice were randomly subdivided to receive genistein (10 mg/kg, i.p., Sung et al., 2008; Ruetten and Thiemermann, 1997) three times a week or injections of the vehicle, 5% DMSO (n = 810/group). Ten weeks after initiation of genistein treatment, mice were individually placed in metabolic cages for collection of 24-hour urine samples for determination of urinary electrolytes, total protein, albumin, Thio-Barbituric Acid Reactive Substances (TBARs), collagen, nephrin and MCP-1 excretions. Mice were then anesthetized with sodium pentobarbital (45–50 mg/kg i.p.) and blood samples obtained for glucose determination. Plasma was separated and used to assess insulin levels using ELISA kit (Millipore Corporation, Billerica, MA). This was followed by removal of kidneys for histopathological examination and Western blot analysis. 3. Renal histopathology Four mice were used from each group. Kidney was embedded and frozen in Optimal Cutting Temperature (Tissue-Tek, Hatfield, PA) and then sliced into 5-μm sections for the terminal dUTP nick end-labeling (TUNEL) assay. The other kidney was fixed in 4% paraformaldehyde in

PBS and embedded in paraffin. Five micrometer-thick sections were obtained, deparaffinized, and stained with Periodic acid-Schiff (PAS) and Masson trichrome. Images were obtained using light-microscopy at 40× and 10× magnification power, respectively. Pancreas was also isolated from each group and fixed in 10% formalin, paraffin embedded, and cut into 4–5 μm sections. The sections were stained with Hematoxylin and Eosin stain (H & E to detect morphological changes in pancreatic islet using light-microscopy at 20× magnification power. 4. Terminal dUTP nick end-labeling analysis (TUNEL) TUNEL analysis was performed using the ApopTag Fluorescein InSitu Apoptosis Detection Kit (Millipore Temecula, CA) following the manufacturer's directions. Briefly, OCT-frozen kidney sections (5 μm) from each group were fixed using paraformaldehyde and ethanol: acetic acid solution (2:1). Then, the samples were incubated with Terminal Deoxynucleotidyl Transferase followed by incubation with anti-digoxigenin conjugate. Propidium iodide 1 μg/ml was added as a nuclear counter stain. Upon completion of the TUNEL assay, coverslips were applied using vectashield mounting medium for fluorescence (Vector Laboratories, Burlingame, CA). Each section was systematically scanned for positive green fluorescent cells in kidney sections indicating apoptosis. Images were obtained using confocal microscopy (LSM 510, Carl Zeiss, Oberkochen, Germany) with 10X magnification power. 5. Human mesangial cells culture Human mesangial cells (Lonza CC-2559) were cultured in MsBM media (Lonza CC-3146) supplemented with 5% fetal bovine serum and antibiotics till reached to passage six. Once reached 70–80% confluency, cells were placed in a serum-free MsBM media supplemented only with antibiotics for 12–18 hours. Cells were then treated with normal (5 mM glucose) or high (30 mM glucose) glucose condition with either vehicle (DMSO) or 100 μM genistein treatment for three days. Additional cells were also treated with 30 mM mannitol with either vehicle or 100 μM genistein in DMSO to examine the effect of osmolarity and media was changed after 48 hours. On the fourth day of treatment, cells were harvested and homogenized in RIPA buffer supplemented with protease and phosphatase inhibitors for Western blotting. 6. Homogenization of the renal cortex for protein expression using western blotting Renal lysates were subjected to Western blot analysis as previously described (Elmarakby et al., 2010). Briefly, kidney samples were homogenized in ice cold modified RIPA buffer supplemented with inhibitors for proteases and phosphatases. Protein concentrations were determined by standard Bradford assay (Bio-Rad, Hercules, CA) using bovine serum albumin as the standard. Kidney protein samples (50 μg) were separated by SDS-PAGE then transferred onto nitrocellulose membrane and incubated with primary antibodies. Antibodies for ICAM-1 (R & D, Minneapolis, MN), gp91phox (Santa Cruz Biotechnology, Santa Cruz, CA), β-actin (Sigma), phosphotyrosine, phospho-ERK and ERK MAPK (Cell Signaling Technology, Beverly, MA) were detected with a horseradish peroxidase-conjugated antibody and ECL chemiluminescence (Amersham BioSciences, Buckinghamshire, UK). Intensity of immunoreactivity was measured by densitometry and β-Actin was used to verify equal protein loading. 7. Assays Urinary sodium and potassium levels were measured using Easy Electrolytes (Medica Corporations, Bedford, MA) and used to calculate

A.A. Elmarakby et al. / Vascular Pharmacology 55 (2011) 149–156 Table 1 Effects of genistein treatment on body weight, urine volume, food and water intake, and urinary sodium and potassium excretion in steptozotocin-induced diabetic mice. Mice were placed in metabolic cages for 24 h after 10 weeks of induction of diabetes and genistein treatment (n = 7–8/group, * P b 0.05 vs. control mice). Group

Control

Diabetic

Diabetic/genistein

Body weight (g) Urine volume(ml/day) Water intake (ml/day) Food intake (g/day) Na⁎ excretion (mmol/day) K⁎ excretion (mmol/day)

26.8 ± 1.7 1 ± 0.2 4.3 ± 0.7 3.3 ± 0.6 0.12 ± 0.03 0.17 ± 0.05

24.8 ± 1 24.6 ± 3.6⁎ 29.4 ± 3.6⁎ 7.7 ± 0.7⁎ 1.4 ± 0.2⁎ 1.2 ± 0.2⁎

25.3 ± 0.8 23.2 ± 2.2⁎ 27.9 ± 2.3⁎ 7.8 ± 0.3⁎ 1.1 ± 0.1⁎ 0.95 ± 0.1⁎

excretion rates. Urinary total protein and albumin excretions were determined using commercially available kits [(Biorad, Hercules, CA) and (Exocell, Philadelphia, PA), respectively]. Urinary TBARs excretion was assessed as a marker of oxidative stress (Cayman Chemical, Ann Arbor, MI). Urinary excretions of collagen, nephrin and MCP-1 were measured using ELISA kits (Exocell, Philadelphia, PA and BD Bioscience, San Jose, CA). 8. Data analyses Statistical analyses were performed using Prism software (GraphPad, San Diego, CA, USA). Data are reported as means ± SEM. All data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test for comparison of groups; p b 0.05 was considered significant. 9. Results 9.1. Effect of genistein on body weight, water intake, food intake, urine volume, and electrolyte excretion in diabetic mice The streptozotocin-treated mice displayed a slight non-significant decrease in mean body weight 10 weeks after induction of diabetes

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compared to their control counterparts (Table 1); genistein did not affect body weight. Hyperglycemia was accompanied with increased food intake, water intake, urine volume, sodium and potassium excretions compared to control mice (Table 1). These changes were attenuated, albeit non-significantly in genistein-treated diabetic mice (Table 1). Blood pressure was not affected by either diabetes or genistein treatment (data not shown). 9.2. Effect of genistein on pancreatic islet, blood glucose and plasma insulin in diabetic mice Microscopic evaluation of pancreas revealed that the number of islet cells is reduced and the remaining islets are smaller and distorted in appearance in streptozotocin-induced diabetic mice compared to controls (Fig. 1A). These morphological changes were associated with a significant increase in blood glucose level and decrease in plasma insulin levels in streptozotocin-induced diabetic mice compared to control (Fig. 1B, P b 0.05). Although genistein treatment did not seem to have an overt effect on the number and appearance of islet cell or on plasma insulin levels in streptozotocin-induced diabetic mice, genistein treatment significantly reduced blood glucose levels of diabetic mice compared to the untreated diabetic group (P b 0.05); however, blood glucose level remained significantly higher than the control group (Fig. 1B, P b 0.05). 9.3. Renal injury The development of diabetes in mice was associated with increase (~3.5 fold) in daily total protein and albumin excretions. Genistein treatment of diabetic mice resulted in a significant decrease (40–45%) in proteinuria and albuminuria compared to their untreated counterparts (Fig. 2A–B). Next we examined whether the effect of genistein on albumin excretion is associated with the protection of slit diaphragm from hyperglycemia-induced injury. Thus, we measured urinary excretion of nephrin, a key component of the slit diaphragm in the glomerular filtration barrier (Saleh et al., 2010). As

Fig. 1. A. Representative images of H&E-stained pancreas from control, untreated diabetic and diabetic mice treated with the tyrosine kinase inhibitor genistein (n = 3–4/group). Also shown are blood glucose and plasma insulin levels in control and diabetic mice with or without genistein treatment (panel B). Blood glucose significantly increased and plasma insulin decreased in diabetic mice compared to control group (*P b 0.05 vs. control mice). Although genistein treatment reduced blood glucose levels, it remained significantly higher than the control group; however, genistein did not significantly affect plasma insulin levels of diabetic mice (#P b 0.05 vs. diabetic mice, n = 8–10/group).

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Fig. 2. Indices of renal injury in streptozotocin-induced diabetic mice. Total protein and albumin excretions increased significantly in diabetic than control mice (*P b 0.05 vs. control mice). Inhibition of tyrosine kinase with genistein treatment resulted in significant reduction in these parameters in diabetic mice (#P b 0.05 vs. diabetic mice; n = 8–10/group).

shown in Fig. 3A, while control mice excreted negligible amount of nephrin, a dramatic increase in its excretion rate was observed in diabetic mice (P b 0.05). Importantly, nephrin excretion was reduced in genistein-treated diabetic mice although the changes were not significant (Fig. 3A, P b 0.1). This observation is consistent with lower albumin excretion in genistein-treated mice as nephrin plays a critical role in the regulation of albumin loss in urine (Saleh et al., 2010). Similarly, while diabetes increased collagen excretion, genistein treatment significantly reduced it (Fig. 3B). The increase in collagen excretion in diabetic mice was consistent with increased collagen deposition in kidney sections stained with Masson trichrome (blue staining) from diabetic mice and this increase was markedly reduced with genistein treatment (Fig. 4A). The kidney sections were also assessed by PAS and TUNEL staining to examine the presence of glomerular deposits and apoptotic cells, respectively. In control sections, PAS highlighted the capillary tufts as thin wispy lines, which demarcated the vascular walls and were separated by lumina containing small aggregates of red blood cells (Fig. 4B). In diabetic sections, there were moderate-to-intense patchy, irregular glomerular PAS-positive deposits which spaced out many of the vascular lumina; however, occasionally, remaining thin-walled capillaries were seen. Genistein treatment attenuated PAS-positive deposits that developed in diabetic mice compared to their vehicle-treated mice, manifested by mild patchy PAS-positive glomerular deposits between occasional capillaries (Fig. 4B). TUNEL staining revealed large numbers of apoptotic cells in proximal tubules, distal tubules, and interstitial cells in kidneys from streptoztocin-injected mice, an effect that was mitigated by genistein treatment (Fig. 4C).

9.4. Inflammatory markers and oxidative stress Urinary MCP-1 excretion and renal ICAM-1 protein expression were measured as markers for inflammation in diabetic mice after 10 weeks of genistein treatment. As shown in Fig. 5 A and B, urinary MCP-1 excretion and renal tissue ICAM-1 were significantly higher in diabetic mice compared to non-diabetic controls and genistein treatment significantly reduced these changes (P b 0.05). In light of genistein's anti-inflammatory effect, interest in its mechanisms of action was expanded to include oxidative stress which contributes importantly to the pathogenesis of diabetic nephropathy. Thus, urinary TBARs levels were assessed as an index of oxidative stress in diabetic mice. The vehicle-treated diabetic mice displayed increased urinary TBARs compared with control which was significantly reduced by genistein treatment (Fig. 6A, P b 0.05). Furthermore, to investigate the molecular mechanism responsible for the regulation of oxidative stress by genistein, renal protein expression levels of NADPH oxidase subunit (gp91phox) were determined. As shown in Fig. 6B, diabetes significantly up-regulated (~ 2-fold) the expression of gp91phox compared with control mice and genistein treatment effectively reduced the expression of gp91phox in diabetic mice (P b 0.05). To verify whether the inhibitory effects of genistein on diabetesinduced renal inflammation could be, at least in part, mediated by protein tyrosine kinase inhibition, we examined the effects of genistein on renal tyrosine phosphorylation. Fig. 7A shows that 10 weeks of diabetes increased renal tyrosine phosphorylation which was effectively blocked with genistein treatment. In parallel

Fig. 3. Urinary collagen and nephrin excretions in streptozotocin-induced diabetic rats with or without genistein treatment. Urinary collagen and nephrin excretion significantly increased in diabetic than control mice (*P b 0.05 vs. control mice). Genistein treatment significantly decreased collagen excretion (#P b 0.05 vs. diabetic mice) and reduced nephrin excretion (P b 0.01) in diabetic mice; nonetheless, collagen and nephrin excretions of genistein-treated diabetic mice remained higher than those of control mice (*P b 0.05 vs. control mice, n = 8–10/group).

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tion (5 mM) (P b 0.05). Genistein treatment prevented high glucoseinduced tyrosine kinase and ERK phosphorylation and decreased ICAM-1 expression. To examine the effect of osmolality on tyrosine and ERK phosphorylation and ICAM-1 expression, human mesangial cells were also cultured in 30 mM mannitol for three days with or without genistein treatment. Neither mannitol alone or with genistein had a significant effect on tyrosine and ERK phosphorylation or ICAM1 expression in human mesangial cells (Data not shown). 10. Discussion

Fig. 4. Representative images of Periodic acid-Schiff (PAS; panel A), Masson trichrome staining (panel B), and TUNNEL staining (panel C) for detection of kidney glomerular deposits, collagen deposition, and apoptotic cells, respectively, in kidney sections from control and diabetic mice with or without genistein treatment (data are representative of n = 4/group).

experiments, the effect of genistein on ERK1/2 phosphorylation was also examined because the ERK1/2 pathway is most closely associated with activation of tyrosine kinase receptors (Garrington and Johnson, 1999). As shown in Fig. 7B, genistein also significantly inhibited the increase in renal ERK phosphorylation in diabetic mice (P b 0.05). 9.5. In-vitro effects of genistein on high glucose-induced tyrosine kinase and ICAM-1 activations in human mesangial cells Fig. 8 showed that incubation of human mesangial cells with high glucose condition (30 mM) for three days significantly increased tyrosine phosphorylation and phospho-ERK/ERK ratio as well as increased ICAM-1 expression compared with normal glucose condi-

The present study shows that the tyrosine kinase inhibitor, genistein, ameliorates streptozotocin-induced renal abnormalities in mice. The reno-protective effect of genistein is associated with amelioration of inflammation and oxidative stress. These observations are of relevance and significance given the fact that many patients with diabetes mellitus develop progressive renal disease despite existing therapeutic regimens {e.g., inhibition of the renin-angiotensin system and good glycemic control (Mogensen et al., 1988)}. Genistein, a tyrosine kinase inhibitor, has a high therapeutic index with very low toxicity (Polkowski and Mazurek, 2000). Its efficacy has been demonstrated in several animal models of diseases. In relation to diabetes, genistein exerts beneficial effects in experimental animals. Chronic treatment with genistein improved abnormal vascular reactivity and inhibited retinal vascular leakage in diabetic rats (Baluchnejadmojarad and Roghan, 2008; Nakajima et al., 2001). Albuminuria is considered the earliest clinical indicator of diabetic nephropathy which heralds progressive renal disease. Inhibition of tyrosine kinase has also been shown to decrease albuminuria and restore decreased kidney nephrin in diabetic db/db mice (Sung et al., 2006). Consistent with these observations, we now show that genistein counteracts diabetes-induced renal injury in streptozotocin-induced diabetic mice as indicated by its ability to decrease proteinuria, albuminuria, collagen levels, nephrinuria and renal cell death. Previous studies suggest that genistein plays an important role in regulating glucose homeostasis in type 1 and type 2 diabetic animal models (Fu et al., 2010; Choi et al., 2008; Yang et al., 2010; Ae Park et al., 2006). Consistent with these reports, we observed significant reduction in blood glucose levels without improving insulinopenia or the morphology of pancreatic islets in genistein-treated diabetic mice. Thus, it is unlikely that the reduction in blood glucose levels underlies the reno-protective effects of genistein in diabetic mice because blood glucose remained very high in these animals indicating that other mechanisms could contribute to the reno-protective effects of genistein.

Fig. 5. Urinary MCP-1 excretion (A) and renal ICAM-1 expression relative to actin (B) in control, diabetic, and genistein-treated diabetic mice. Urinary MCP-1 excretion and renal ICAM-1 expression level were significantly elevated in diabetic compared with control mice (*P b 0.05 vs. control mice). Genistein treatment significantly decreased urinary MCP-1 excretion and renal ICAM-1 expression in diabetic mice (#P b 0.05 vs. diabetic mice, n = 8–10/group).

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Fig. 6. Urinary TBARs excretion (A) and renal gp91phox expression relative to β-actin (B) in steptozotocin-induced diabetic mice. Urinary TBARs excretion and renal gp91phox expression were significantly elevated in diabetic than control mice (*P b 0.05 vs. control mice) but were reduced with genistein treatment (#P b 0.05 vs. diabetic mice). However, urinary TBARs excretion remained significantly higher in diabetic mice treated with genistein compared with control (*P b 0.05 vs. control mice, n = 8–10/group).

Increasing evidence indicates a role for various inflammatory molecules, including chemokines, adhesion molecules, and proinflammatory cytokines, in diabetic complications. Also, activation of tyrosine kinase is recognized as a potential culprit contributing to the stimulation of inflammatory cells such as mast cells (Wong et al., 1997), eosinophils (Kato et al., 1995), macrophages (Dong et al., 1993) and neutrophils (Berkow and Dodson, 1991). In healthy cells, phospho-tyrosine kinase activity is tightly regulated and a balance between tyrosine kinases and phosphatases is essential for normal cell function. On the other hand, increased tyrosine phosphorylation is implicated in many pathological conditions such as inflammation and diabetic vascular complications (Page et al., 2009; Vlahovic and Crawford, 2003). Genistein has been shown to reverse the sciatic nerve pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 overproduction in streptozotocin-induced diabetic mice (Valsecchi et al., 2010). Ibrahim et al. recently showed that local intravitreal injection of genistein also suppressed diabetes-induced retinal inflammation

via inhibition of tyrosine kinase inflammatory signal activation, inhibition of ERK phosphorylation, and suppression of the release of TNF-α in steptozotocin-induced diabetic rats (Ibrahim et al., 2010). Consistent with these observations, genistein inhibited renal tyrosine phosphorylation and ERK phosphorylation together with attenuation of the renal inflammatory process, including pro-inflammatory cytokines ICAM-1 and urinary MCP-1 levels and in association with reduction of renal hypertrophy and oxidative stress in the diabetic mice. The ability of genistein to inhibit tyrosine and ERK phosphorylation and ICAM-1 expression was further confirmed in the current study using cultured human mesangial cell under high glucose condition in vitro. It is well known that TNF-α could increase MCP-1 levels via the activation of nuclear factor kappa B (NFκB) signaling and tyrosine kinase is important for TNF-α effects. Kakizaki et al. have reported that MCP-1 mRNA expression, induced by IL-1 beta and TNF-α, was suppressed by the tyrosine kinase inhibitor genistein in glomerular endothelial cells (Kakizaki et al., 1995). Thus it is plausible

Fig. 7. Renal phospho-tyrosine relative to β-actin (A) and phospho-ERK relative to ERK (B) in steptozotocin-induced diabetic mice. Renal phospho-tyrosine expression level and phospho-ERK/ERK ratio significantly increased in diabetic compared with control mice (*P b 0.05 vs. control mice). Genistein treatment not only decreased but also normalized renal phospho-ERK/ERK ratio and phospho-tyrosine expression in diabetic mice (#P b 0.05 vs. diabetic mice, n = 6/group).

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Fig. 8. Phospho-tyrosine expression relative to β-actin (A), phospho-ERK relative to ERK (B), and ICAM-1 expression relative to β-actin (C) in glomerular mesangial cell incubated in normal (5 mM) and high (30 mM) glucose condition for three days with or without genistein treatment (100 μM). High glucose condition significantly increased phospho-tyrosine, phospho-ERK/ERK ratio and ICAM-1 compared with normal glucose condition (*P b 0.05 vs. normal glucose condition) and genistein treatment decreased these changes (#P b 0.05 vs. high glucose condition, n = 4). NG indicates normal glucose, HG indicates high glucose and G indicates genistein.

that inhibition of tyrosine kinase by genistein decreases MCP-1 excretion via inhibition of TNF-α-induced NFκB activation. Collectively, these data suggest that genistein-induced inhibition of tyrosine kinase provides anti-inflammatory properties which could be a potential mechanism for alleviating diabetic-induced renal injury. In addition to inhibiting tyrosine kinase activity, geneistein's antiinflammatory effects could be attributed to its antioxidant properties (Ma et al., 2010). Hyperglycemia is well-known to generate reactive oxygen species which in turn increases lipid peroxidation and membrane damage (Sasser et al., 2007; Lee, 2006). Hepatic TBARs levels increased and antioxidant enzymes levels decreased in streptozotocin-induced diabetic rats and these changes were reversed with genistein treatment (Lee, 2006). Genistein treatment also improved antioxidant enzyme activities and decreased reactive oxygen species production and lipoperoxide levels in the brain and liver of streptozotocin-induced diabetic mice (Valsecchi et al., 2010). NAD(P)H oxidase activity is enhanced in diabetic vascular smooth muscle cells with subsequent increase in superoxide production. Inhibition of tyrosine kinase, with genistein, attenuated NAD(P)H oxidase activation and superoxide production in diabetic vascular smooth muscle cells suggesting that tyrosine kinase is upstream of NAD(P)H oxidase activation in diabetic vascular smooth muscle cells (Jeong et al., 2005). Consistent with these data, genistein treatment reduced the elevation in urinary TBARs excretion and renal NAD(P)H oxidase subunit gp91phox expression in diabetic mice highlighting the potential antioxidant ability of genistein as a reno-protective mechanism in diabetes. Emerging evidence indicates that hyperglycemia-induced NAD(P) H oxidase-dependent superoxide production (Li and Wang, 2010) might subsequently decrease the activity of tyrosine phosphatases which normally antagonize tyrosine kinase activity (Herrlich and Bohmer, 2000) resulting in accumulation of phosphorylated tyrosine residues. Thus, genistein's ability to reduce tyrosine phosphorylation could be also explained in part by blockade of tyrosine phosphatase

inactivation via its antioxidant activity. Furthermore, alterations in the activity of transcription factors involved in inflammation have been reported as an important component of NAD(P)H oxidase-dependent redox signaling (Kim et al., 2010). Based on our observation that genistein treatment lowered levels of renal ICAM-1 and MCP-1 in diabetic mice, we suggest that the ability of genistein to attenuate renal inflammation and injury could be explained at least in part by its antioxidant effects. Previous studies demonstrated that MAPK activation is necessary for secretion of pro-inflammatory cytokines (Nakajima et al., 2004; Yeh et al., 2001). In our study, genistein had an appreciable inhibitory effect on hyperglycemia-induced ERK phosphorylation in the kidney of diabetic mice as well as in cultured glomerular mesangial cell incubated under high glucose condition demonstrating the involvement of tyrosine kinase as a critical upstream signaling event in hyperglycemia-mediated ERK activation. Since ERK pathway is most closely associated with activation of tyrosine kinase receptors (Frey and Singletary, 2003; Garrington and Johnson, 1999), blockade of hyperglycemia-induced tyrosine phosphorylation by genistein is consistent with its inhibition of ERK activation. Aside from its anti-inflammatory properties, genistein has been shown to inhibit synthesis of hyperglycemia-induced extracellular matrix components, collagen IV and fibronectin, and secretion of TGF-β in cultured rodent renal mesangial cells (Yuan et al., 2009). Genistein also inhibited butylhydroperoxide-induced neuronal cell death in human cortical cell lines and its neuroprotective effect is mainly mediated via its regulation of anti-apoptotic protein Bcl-2 (Sonee et al., 2004). Oxidative stress has been shown to increase apoptosis and inhibit cell proliferation in cultured human umbilical vein endothelial cells incubated with high glucose or hydrogen peroxide and genistein treatment reduced diabetes-induced apoptosis and endothelial injury via up-regulation of antiapoptotic proteins and modulation of cell survival signaling (Xu et al., 2009). We observed that genistein reduced collagen deposition and excretion

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which reflects renal overproduction of extracellular matrix proteins. Further, genistein decreased renal apoptotic cells in diabetic mice suggesting that inhibition of tyrosine kinase could also provide renal protection during diabetes via modulation of apoptosis and renal extracellular matrix components. In summary, the present study demonstrates the ability of genistein to reduce renal injury and apoptosis in a rat model of type 1 diabetes. The study highlights that the reno-protective effects of genistein could be attributed to its antioxidant and anti-inflammatory properties as well as its ability to inhibit tyrosine kinase. However, the study did not directly determine whether the inhibition of tyrosine kinase mediates genisteins' antioxidant and anti-inflammatory effects. Nonetheless, a plausible scenario is diabetes-associated oxidative stress which would activate tyrosine kinase thereby triggering the activation of pro-inflammatory cytokines and exacerbating renal injury. 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