Renal Protective Effect of Xiao-Chai-Hu-Tang on Diabetic Nephropathy of Type 1-Diabetic Mice

Xiao-Chai-Hu-Tang (XCHT), a traditional Chinese medicine formula consisting of seven medicinal plants, is used in the treatment of various diseases. We show here that XCHT could protect type-1 diabetic mice against diabetic nephropathy, using streptozotocin (STZ)-induced diabetic mice and high-glucose (HG)-exposed rat mesangial cell (RMC) as models. Following 4 weeks of oral administration with XCHT, renal functions and renal hypertrophy significantly improved in the STZ-diabetic mice, while serum glucose was only moderately reduced compared to vehicle treatment. Treatment with XCHT in the STZ-diabetic mice and HG-exposed RMC resulted in a decrease in expression levels of TGF-β1, fibronectin, and collagen IV, with concomitant increase in BMP-7 expression. Data from DPPH assay, DHE stain, and CM-H2DCFDA analysis indicated that XCHT could scavenge free radicals and inhibit high-glucose-induced ROS in RMCs. Taken together, these results suggest that treatment with XCHT can improve renal functions in STZ-diabetic mice, an effect that is potentially mediated through decreasing oxidative stress and production of TGF-β1, fibronectin, and collagen IV in the kidney during development of diabetic nephropathy. XCHT, therefore merits further investigation for application to improve renal functions in diabetic disorders.


Introduction
Increasing prevalence of nephropathy and/or end-stage renal disease (ESRD) in diabetic patients is becoming a serious social and health problem worldwide [1,2]. Hyperglycemia is considered as the main factor to induce diabetic nephropathy (DN), and clinical strategies for management of DN include glycemic control and blood pressure regulation [3,4]. However, current therapeutic effect for DN remains unsatisfactory, thus resulting a year-by-year increase in the number of diabetic patients with nephropathy [5]. Pathological changes of DN are characterized by structural abnormalities including renal-cell hypertrophy, increase in thickness of glomerular basement membranes, and progressive accumulation of extracellular matrix components [6].
Due to hyperglycemia, diabetes exhibits high oxidative stress which depletes the activity of antioxidative defense system and in turn promotes de novo free radicals generation; thus, strategies to reduce oxidative stress in diabetes mellitus may exert favorable effects on the progression of diabetic glomerulosclerosis [7,8]. Hyperglycemia and oxidative stress during DN induce abnormal production and stimulation of TGF-β1-resident renal cells [4,6,8,9]. TGF-β1 causes augmented deposition of extracellular matrix proteins, such as collagen IV and fibronectin, in the glomeruli, thus inducing mesangial expansion and glomerular basement membrane thickening [1]. The renal protective protein, bone morphogenetic protein-7 (BMP-7), can reduce glomerular and tubulointerstitial fibrosis and prevent the pathogenesis in diabetic nephropathy [10,11]. Expression or treatment 2 Evidence-Based Complementary and Alternative Medicine with recombinant BMP-7 has been shown to improve renal damage from hyperglycemia-induced oxidative stress in the kidney of diabetic animals [10,12]. Xiao-Chai-Hu-Tang (Japanese name, Shosaiko-to) is a herbal drug formula widely used in traditional Chinese medicine and Japanese kampo medicine. XCHT consists of a mixture of seven different medicinal plants (Table 1) and has several experimentally proven pharmacological activities, including prevention of experimental hepatotoxicity [13], immunomodulatory effect [14][15][16], antineoplastic activity [17] and promotion of liver regeneration [18]. XCHT has also been documented to decrease fibrogenic protein expression, including TGF-β1 and collagen, to inhibit hepatic fibrosis [19][20][21]. However, it remains unclear whether XCHT has any renal protective activity during diabetic nephropathy and could prevent streptozotocin (STZ)-induced type-1 diabetic mice from renal fibrosis. We, therefore, investigated the treatment effect of XCHT on STZ-induced type-1 diabetic mice and high-glucose (HG)-exposed rat mesangial cell (RMC) models. Expression levels of TGF-β1, fibronectin, collagen IV, and BMP-7 as well as antioxidative effects of XCHT treatment were examined in order to explore the renal protective effect and mechanism of XCHT in diabetic nephropathy.  (Table 1). To prepare the aqueous extract of XCHT, 108 g of the dried material was extracted with 1 liter of boiling water for 2 h. After the supernatant of the first extraction was removed, an additional 1 liter of distilled water was added and the sample was further subjected to another 2 h of extraction. The same procedure was repeated for 3 times. The decoction obtained from the three extractions was combined, filtered, concentrated, and lyophilized. The yield of the dried extract was about 38%, which was collected and stored at −20 • C until use.

Animal Model for Diabetic
Nephropathy. BALB/c mice, 8-week old, 18-20 g in weight, were obtained from the Animal Center of National Cheng Kung University Medical College. STZ-diabetic mice were induced by intraperitoneal (i.p.) injecting STZ (200 mg/kg) (Sigma-Aldrich Inc., Saint Louis, MO, USA) into fasting mice as described previously [22]. Mice were considered to be diabetic if they had elevated plasma glucose concentrations of 350 mg/dL. All experiments were carried out at 9 weeks after STZ injection. STZ-diabetic mice were considered to have diabetic nephropathy (DN) when they had elevated levels of urea nitrogen (BUN) and creatinine. All animal procedures were performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Chang Jung Christian University approved the animal care protocol (IACUC approval no. CJCU-99-002) for the experiments performed in this study. STZ-diabetic mice were treated with 200 mg/kg of XCHT twice a day for four weeks. The vehicle (distilled water) used to disperse XCHT was given at the same volume. The STZ-diabetic mice were divided into 3 groups (n = 6), respectively: (1) Vehicle-treated normal mice; (2) Vehicle-treated STZ-diabetic mice and (3) XCHT-treated STZ-diabetic mice. At the end of the 4-week treatment, mice were euthanized by cervical dislocation after pentobarbital (40 mg/kg, i.p.) anesthesia, and blood was collected from their abdominal aorta for renal function analysis. Kidneys were dissected and rinsed with cold isotonic saline and then weighed, frozen in liquid nitrogen, and then kept for storage at −80 • C for further analysis. An index of renal hypertrophy was estimated by comparing the weight of the left kidney to the body weight.

Blood Glucose and Renal Functions
Determination. Blood samples were collected before the treatments and one hour after the last treatment for estimating the levels of plasma glucose, BUN, and creatinine. Blood samples from the mice were centrifuged at 3,000 g for 10 min. Samples were incubated with reagents from glucose, BUN, or creatinine kits (AppliedBio assay kits; Hercules, CA, USA), and the levels of blood glucose, BUN, and serum creatinine were then assessed by an autoanalyzer (Quik-Lab, Ames, Miles Inc., Elkhart, Indiana, USA), with samples run in duplicate.

Renal Histological Analysis.
For morphometric analysis, the kidney was removed and embedded in paraffin to prepare 4-μm tissue slices. The tissue slices were stained with periodic acid-Schiff (PAS). The mesangial expansion index (MEI) was scored in four levels from 0 to 3, with the index scores defined as follows [23]: 0, normal glomeruli; 1, matrix expansion occurred in up to 50% of a glomerulus; 2, matrix expansion occurred in 50 to 75% of a glomerulus; 3, matrix expansion occurred in 75 to 100% of a glomerulus. Scores were assigned for at least 30 glomeruli from kidney slices from each mouse, and the means were calculated. Each slide was scored by a pathologist who was unaware of the experimental details.

Immunohistochemical Staining
Kidney sections were dewaxed and hydrated through graded ethanol solutions and then rinsed with PBS. After treatment for 5 min in 3% H 2 O 2 and 30 min in blocking agent, slides were incubated with primary antibodies of TGF-β1, fibronectin, collagen IV, or BMP-7 (Santa Cruz, USA) diluted to 1 : 50 in PBS for 16 h at 4 • C. Then, slides were washed three times with PBS and incubated for 30 min with biotinconjugated secondary antibodies (Vector, UK) for 1 h. The slides were washed again with PBS and the signal was amplified with Vectastain ABC kit (Vector, UK). Finally, the samples were developed using diaminobenzidine (DAB). Hematoxylin was applied as the counter stain.

Western Blot Analysis for Kidney and RMCs
. TGF-β1, fibronectin, collagen IV, and BMP-7 expression levels in mouse kidney or RMCs were determined by Western blotting analysis. RIPA buffer was used to extract total protein. For Western analysis, proteins were separated by SDS-PAGE, transferred, and immobilized on a nitrocellulose membrane. The membrane was blocked with 5% nonfat dry milk in phosphate-buffered saline containing 0.1% Tween 20 (PBS-T) for 2 h at room temperature. The membrane was then washed in PBS-T and hybridized with primary antibodies diluted to proper concentration in PBS-T for 16 h. Specific antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) for TGF-β1, fibronectin, collagen IV, BMP-7 (1 : 200 dilution), and β-actin (1 : 1000 dilution) were used. Incubation with secondary antibodies and detection of the antigen-antibody complex were performed using the ECL kit (Amersham Biosciences, UK).

DPPH Radical Scavenging
Assay. The antioxidant activity of XCHT and ascorbic acid (vitamin C) was measured in terms of 1,1-diphenyl-2-picrylhydrazyl (DPPH) (Sigma-Aldrich Inc., Saint Louis, MO, USA) free radical scavenging ability. Vitamin C, an antioxidant, was used as a positive control. The highest tested concentration of Vitamin C was considered as 100% of scavenging activity. A solution of XCHT at different concentrations was placed in a cuvette and 1 mL of DPPH radical in methanol solution (23.7 μg/mL) was added, followed by incubation for 30 min. The decrease in absorbance at 517 nm was determined with a spectrophotometer. All determinations were performed in triplicates. The percent inhibition of DPPH radical by the samples was calculated according to the following formula: where A (c) is the absorbance of the control (without sample), and A (s) is the absorbance of the sample at t = 30 min.  3.6. Statistical Analysis. Results were expressed as mean ± SD. Statistical analysis was carried out by using ANOVA analysis and Dunnett post hoc analysis. Statistical significance was achieved if P < 0.05.
In the absence of intervention (vehicle only), these three blood chemistry parameters in the vehicle-treated STZdiabetic mice increased significantly when comparing with levels prior to treatment initiation. In contrast, STZ-diabetic mice that received XCHT had modest reduction in serum glucose but a significant decrease in serum levels of BUN and creatinine compared to that in vehicle control treatment after the 4-week oral administration (P < 0.05) (Figures 1(a), 1(b) and 1(c)). At the end of the in vivo experiment, vehicle-treated STZ-diabetic had enlarged kidneys, with the ratio of kidney weight to body weight being significantly higher than that of normal mice (Figure 2(a)). XCHT treatment, however, reduced the degree of renal hypertrophy in the STZ-diabetic mice (P < 0.05). When performing histological analysis in the vehicle-treated normal mice, the PAS stain shows nearly normal glomerular structure with only mild mesangial expansion and no significant tubular damage (Figure 2(b) A). In contrast, there is an increase in mesangial matrix, mesangial cellularity, and capillary basement membrane thickening in thediabetic mice that received vehicle treatment (Figure 2(b) B). In particular, remarkable changes were observed in the tubulointerstitial areas, including tubules dilation and lined by flattened epithelium. The glomerular hypertrophy is also prominent in the vehicle-treated STZ-diabetic mice (Figure 2(b) B). These histological changes, however, improved in the diabetic mice administered with XCHT, which showed reduction in mesangial expansion, basement membrane thickening, glomerular hypertrophy, and tubular damage (Figure 2(b) C). These observations are also reflected in the index of the glomerular matrix expansion assessed in each mice group (Figure 2(c)). Thus, XCHT treatment can effectively improve renal functions and prevent kidney injury in diabetic mice induced by STZ.

XCHT Treatment Reduces TGF-β1, Fibronectin, and Collagen IV Expression While Increasing BMP-7 Levels in STZ-Diabetic Mice
In order to explore the underlying mechanisms in XCHT's renal protective effects against diabetic nephropathy, changes in the mice kidney expression of TGF-β1, fibronectin, collagen IV, and BMP-7 were assessed using RT-PCR and Western blot analyses. Vehicle-treated STZ-diabetic mice showed higher expression of TGF-β1, fibronectin, and collagen IV than in vehicle-treated normal mice (Figures 3(a) and 3(b)). Furthermore, due to the diabetic induction, the expression of BMP-7 in the diabetic mice receiving control treatment was lower than that in the vehicle-treated normal mice. In contrast, after 4 weeks of XCHT treatment in STZ-diabetic mice, kidney expression of these markers reverted to levels similar to vehicle-treated normal mice, with augmented BMP-7 and reduced levels of TGF-β1, fibronectin, and collagen IV expression (Figures 3(a) and 3(b)). To further validate the above results, renal immunostaining for TGF-β1, fibronectin, collagen IV, and BMP-7 expression was carried out on kidneys of the test mice. As shown in Figure 4, the STZ-diabetic mice exhibited greater TGF-β1 immunostaining in the glomeruli (Figure 4(b)) whereas XCHT-treated mice showed reduced TGF-β1 protein (Figure 4(c)). The expression of fibronectin and collagen IV was observed to accumulate in the glomeruli and interstitial space of STZdiabetic mice (Figures 4(e) and 4(h)) relative to that of the normal mice (Figures 4(d) and 4(g)). Following the 4-week treatment, however, the extent of fibronectin and collagen IV expression in the glomeruli and interstitial space of XCHTtreated STZ-diabetic mice (Figures 4(f) and 4(i)) was clearly less than in the vehicle-treated STZ-diabetic mice (Figures 4(e) and 4(h)). Compared to the strong immunostaining of BMP-7 expression in normal mice (Figure 4(j)), STZdiabetic mice showed reduced immunostaining of BMP-7 (Figure 4(k)) and 4-week treatment with XCHT could reverse the pathophysiological effect by enhancing its expression (Figure 4(l)). From these results, it appears that XCHT mediates its renal protective effect on the pathophysiology of diabetes in part by reducing profibrotic protein expression and increasing BMP-7 level.

XCHT Inhibits Oxidative Stress Induced by High-Glucose Exposure in RMCs and Reduces Profibrotic Factors While
Increasing BMP-7 Expression. We have previously shown that exposing RMCs to high-glucose (HG) medium raises ROS production in these cells [12], and ROS induced by hyperglycemia can stimulate TGF-β1 and contribute to diabetes nephropathy [6]. To further determine whether oxidative stress is involved in XCHT's renal protective mechanism under diabetic condition, we evaluated the direct ROS scavenging effect of XCHT in HG-exposed RMCs. As shown in the DPPH radical scavenging assay ( Figure 5(a)), XCHT could scavenge the free radicals directly in a concentrationdependent manner similar to the antioxidant vitamin C. The anti-oxidative ability of XCHT was further visualized by DHE stain (Figure 5(b)) and measured by CM-H 2 DCFDA staining ( Figure 5(c)). In both instances, ROS was noted to increase in RMCs under high-glucose exposure compared with normal-glucose (NG) control ( Figures 5(b) A, 5(b) B and 5(c)), but its levels were markedly inhibited by XCHT, similar to effects observed with the antioxidant tiron ( Figures 5(b) C, 5(b) D, and 5(c)). XCHT also dosedependently reduced HG-stimulated ROS generation in the RMCs (Figure 5(c)).
Normal mice + Veh To assess whether the antioxidant effect from XCHT was related to its renal protective effects, we tested the mRNA and protein expression of TGF-β1, fibronectin, collagen IV, and BMP-7 in RMCs exposed to HG medium (Figures 6(a) and 6(b)). HG medium-treated RMCs showed lower levels of BMP-7 and increased TGF-β1, fibronectin, and collagen IV expression compared to NG medium-cultured RMCs. These effects, however, were reversed using the antioxidant tiron and XCHT treatments, whereby an enhancement in BMP-7 and a decrease TGF-β1, fibronectin, and collagen IV expressions were observed (Figures 6(a) and 6(b)).

Discussion
Although the main clinical strategies for diabetic nephropathy (DN) are glycemic control and blood pressure regulation [3,4], the therapeutic outcome remains unsatisfactory [5]. In the present study, we showed that XCHT could improve the renal functions and renal hypertrophy while decreasing serum glucose moderately in STZ-diabetic mice. Furthermore, we provide evidence that XCHT's renal protective mechanism in DN could potentially be mediated through (1) decreasing oxidative stress and production of TGF-β1, fibronectin, and collagen IV, and (2) increasing BMP-7 expression in the kidney of STZ-diabetic mice and HGexposed RMCs.
Recent search for new antifibrotic drugs has refocused on herbal medicine [27]. XCHT is widely prescribed to patients with liver cirrhosis for over a millennium in Asia, and the formula is established based on traditional clinical experience and practice [17,18,20]. TGF-β1 is involved in mediating the development of diabetic renal hypertrophy and is upregulated in diabetic kidney [28]. Hyperglycemiainduced upregulation of TGF-β1 stimulates mesangial cell proliferation and ECM induction (increased fibronectin and collagen production), which contributes to the major pathological changes observed in DN [28]. Thus, TGF-β1 has been considered as a therapeutic target in DN and other chronic kidney diseases [29]. XCHT has been documented to inhibit hepatic expression of TGF-β1 mRNA and protein in many experimental fibrogenic animal models [30,31]. The in vivo results from the present study also indicated that XCHT treatment could decrease TGF-β1, fibronectin, and collagen IV expression in the kidney of STZ-diabetic mice. In addition, in vitro, the expression of these profibrotic proteins was also reduced by XCHT treatment in highglucose-exposed RMCs. Thus, XCHT treatment appears to improve DN by reducing TGF-β1 and ECM production. Oxidative stress induced by hyperglycemia is implicated in the development of DN [6]. ROS generated from high-glucose condition can stimulate a number of growth factors and cytokines, including TGF-β1, and lead to excessive production and accumulation of ECM proteins [6,9,32]. In vitro studies have shown that mesangial cells are the major source of free radicals after exposure to high-glucose concentrations [4,33]. Previously, XCHT has also been reported to reduce the extracellular concentration of free radical by its O 2 − and NO scavenging activity in LPSstimulated cells [34]. Similarly, our results from the DPPH assay and intracellular ROS analyses have shown that not only XCHT could directly scavenge free radicals, but it could also decrease ROS generation in high glucose-exposed RMCs. The inhibition of ROS production by XCHT supposedly could prevent downstream stimulation of TGF-β1 activation and production, and ultimately avert the resulting aberrant ECM deposition. The in vitro results from the RMCs recapitulated our previous in vivo observations in the STZ-diabetic mice (Figures 3 and 4). Thus, it appears that the inhibitory effects of XCHT on high-glucose-induced ROS generation could potentially disturb stimulation of TGF-β1 and the resulting ECM proteins accumulation, thereby partly explaining the drug's renal protective effects in diabetic nephropathy. Thus, the observed effect of XCHT in decreasing the ECM proteins in vitro and in vivo is likely related to its antioxidative ability in impeding TGF-β1. BMP-7 helps to maintain normal renal structure and physiological function, and its expression is decreased in damaged kidney [35]. During the development of DN, hyperglycemia causes oxidative stress and leads to BMP-7 expression decrease in renal cells [10][11][12]. Exogenous and overexpression of BMP-7 has been shown to improve renal function and prevent glomerular sclerosis in diabetic rats [12,36]. The medicinal plant Angelica sinensis could also prevent STZ-diabetic rat from DN through increasing renal BMP-7 expression [37]. Our study showed that reduced renal BMP-7 expression in STZ-diabetic mice could be reversed by 4-week treatment with XCHT. Moreover, XCHT could increase BMP-7 expression in high-glucose-exposed RMCs. Our results therefore suggest that XCHT's protective effect against DN is at least in part mediated by increasing renal BMP-7 expression.
In conclusion, XCHT could prevent renal functions through augmenting BMP-7 expression and reducing ROS and ECM production. We suggest that XCHT could be applied for handling diabetic nephropathy and merits further study for its clinical application in diabetic nephropathy.