Astragaloside IV synergizes with ferulic acid to inhibit renal tubulointerstitial fibrosis in rats with obstructive nephropathy

BACKGROUND AND PURPOSE The combination of Chinese herbs, Astragali Radix and Angelicae Sinensis Radix, could alleviate renal interstitial fibrosis. Astragaloside IV (AS-IV) and ferulic acid (FA) are the two major active constituents in this combination. In this study, we employed rats with unilateral ureteral obstruction to determine whether AS-IV and FA have the same renoprotective effects and investigated the mechanisms of this action. EXPERIMENTAL APPROACH Renal pathological changes were evaluated after treatment with AS-IV, FA or AS-IV + FA (AF) for 10 days. Meanwhile, the expression of transforming growth factor β1 (TGF-β1), fibronectin, α-smooth muscle actin (α-SMA), phosphorylation of c-Jun NH2-terminal kinase (p-JNK) and nitric oxide (NO) production in kidney were determined. The expressions of fibronectin, α-SMA, mitogen-activated protein kinases [JNK, extracellular signal-regulated kinases (ERK), P38] in TGF-β1-treated NRK-49F cells or interleukin-1-treated HK-2 cells after AS-IV, FA or AF were assessed. KEY RESULTS AF alleviated the infiltration of mononuclear cells, tubular atrophy and interstitial fibrosis; reduced the expression of fibronectin, α-SMA, TGF-β1 and p-JNK; and dramatically increased the production of NO in obstructed kidneys. Neither AS-IV nor FA alone improved renal damage, but both increased NO production. AF inhibited α-SMA and fibronectin expression in NRK-49F or HK-2 cells. Furthermore, AF significantly inhibited IL-1β-induced JNK phosphorylation, without affecting ERK or P38 phosphorylation. Neither AS-IV nor FA alone had any effect on the cells. CONCLUSIONS AND IMPLICATIONS AS-IV synergizes with FA to alleviate renal tubulointerstitial fibrosis; this was associated with inhibition of tubular epithelial–mesenchymal transdifferentiation (EMT) and fibroblast activation, as well as an increase in NO production in the kidney.


Introduction
Chronic kidney disease (CKD) is widely recognized as a major public health problem that progressively develops to endstage renal disease (ESRD) irrespective of the underlying cause. The persistent kidney injury induces renal inflammation, the overexpression of profibrotic factors and myofibroblast proliferation, followed by an exaggerated accumulation of extracellular matrix (ECM) components, destruction of the renal vasculature and ultimately renal fibrosis (Hewitson, 2009). Although the systematic approach used to treat this condition includes intensive reduction of blood pressure and dietary restriction, this is not effective enough to completely halt the progression of CKD (Ripley, 2009).
It is well accepted that tubulointerstitial fibrosis is a better indicator of the deterioration of renal function than glomerulosclerosis in renal diseases (Bohle et al., 1987). Inhibition of tubulointerstitial fibrosis has the potential to protect the kidney from the deleterious effects of CKD. In the progression of CKD, the activated interstitial fibroblast or myofibroblast is central to the development of tubulointerstitial fibrosis (Strutz and Zeisberg, 2006). A number of pro-inflammatory, profibrotic cytokines, such as transforming growth factor-b1 (TGF-b1) (Schnaper et al., 2009), interleukin-1 (Fan et al., 2001;Vesey et al., 2002) and connective tissue growth factor (Chen et al., 2009), as well as angiotensin II (Gaedeke et al., 2002) are released from the kidney in pathological conditions. These factors converge and establish a fibrogenic context that drives the resident cells, including fibroblasts (Qi et al., 2006), pericytes (Lin et al., 2008), tubular cells (Kalluri and Neilson, 2003) and endothelial cells (Zeisberg et al., 2008), to transform into myofibroblasts. The number of myofibroblasts is closely associated with the extent of tubulointerstitial fibrosis (Qi et al., 2006). Therefore, to intervene in the transition of these cellular phenotypes is pivotal in inhibiting tubulointerstitial fibrosis.
In China, many Chinese herbal medicines have been reported to slow down the progression of CKD (Li and Wang, 2005;Peng et al., 2005;Wojcikowski et al., 2006), including Astragali Radix [the root of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao] and Angelicae Sinensis Radix [the root of Angelia sinensis (Oliv.) Diels]. The combination of Astragali Radix and Angelicae Sinensis Radix was demonstrated to possess a renoprotective effect in animal models of CKD (Li et al., 1998;Wang et al., 2004;Zhang et al., 2006a;Meng et al., 2007;Song et al., 2009), and this was associated with a down-regulation of the expression of TGFb 1, inhibition of the epithelial-mesenchymal transdifferentiation (EMT), reduction of macrophage infiltration and myofibroblast proliferation, as well as an improvement in endothelial dysfunction.
There are about 150 components in the decoction of Astragali Radix and Angelicae Sinensis Radix (Xiao et al., 1999). To identify the chemicals responsible for its renoprotective effects, we gradually refined the mixture of the two herbs according to its ability to improve the renal pathological damage (Zhao et al., 2010). Using mass spectrometry analysis, we identified two major constituents, astragaloside IV (AS-IV) and ferulic acid (FA), from an extract of Astragali Radix and Angelicae Sinensis Radix, which preserve the antifibrotic effects in rats with unilateral ureteral obstruction (UUO). AS-IV was reported to inhibit the adhesion of glomerular podocyte dysfunction , alleviate tumor necrosis factor a (TNF-a)-induced endothelial cell apoptosis (Xu et al., 2006) and improve the barrier function of the endothelial cell . FA alleviates renal injury by decreasing urinary protein excretion and inhibiting the overexpression of TGF-b 1 (Fujita et al., 2008). Thus, AS-IV and FA may be the important active constituents for inhibiting tubulointerstitial fibrosis. In this study, we sought to identify the renal protective efficacy of AS-IV and FA in obstructive nephropathy and to assess the potential mechanisms of its renoprotective effects.

Methods
Drug preparation AS-IV (Lot No. 110781-200512) and FA (Lot No. 0773-9910) used in this study were of high purity (99%) as determined by high performance liquid chromatography (HPLC) analysis and were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The compounds used in the cellular experiments were dissolved in 1% DMSO (Sigma-Aldrich, St. Louis, MO, USA) in a concentration of 2 mg·mL -1 as a stock solution. The stock solution was diluted with incubation medium when used. The compounds were prepared as a suspension in the medium before administration to the animals. We have abbreviated the combination of AS-IV and FA to AF throughout the manuscript. The mixture of Astragali Radix and Angelicae Sinensis Radix (A&A) was prepared according to the protocol in our earlier study (Meng et al., 2007). The quality of A&A was controlled by HPLC-electrospray ionization and mass spectrometry .

Animal and experimental protocol
All experiments were performed with approval from the Animal Experimentation Ethics Committee of Peking University First Hospital. Male SPF Wistar rats (192 Ϯ 8 g) were obtained from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (Beijng). Animals were allowed free access to water and standard rat chow. Animals were housed at 25°C with 40% humidity with a 12/12 h light/dark cycle. Animals were randomly divided into eight groups as shown in Table 1. For the UUO groups, rats were anaesthetized with sodium pentobarbital (40 mg·kg -1 , i.p.), then the left ureter was exposed using a mid-abdominal incision and ligated with 3-0 silk suture. In sham-operated rats, the ureter was mobilized but not ligated. The test compounds were administered to the rats by gavage on the day of the operation. Because 0.45-0.85 mg AS-IV and 0.2-0.5 mg FA were determined·per gram dried weight of A&A, the low dose and high dose of AS-IV and FA used in this study are equivalent to the minimum and maximum contents determined in A&A.
Rats from each group were killed on day 10 after surgery. Serum was obtained from the abdominal aorta blood after centrifugation to detect serum creatinine and urea using a Beckman Coulter LX20 Automated Analyzer (Beckman Coulter Inc, Brea, CA, USA). The left kidneys were decapsulated, washed with ice-cold normal saline, then rapidly dissected. Coronal sections of 2-3 mm thick through the mid-portion of kidney were embedded in paraffin after being fixed in 10% neutral buffered formalin; the rest was snap froze in liquid nitrogen for further study.

Morphological and quantitative image analysis
Sections were stained with haematoxylin and eosin (HE) and Masson trichrome for morphological studies. Ten randomly selected separate non-overlapping microscopic fields of each kidney section were examined under the light microscope at a magnification (¥200) and averaged to yield the score of each kidney. Morphometric evaluation of the tubulointerstitial area was performed by two authors under the direction of an expert in experimental renal pathology. The renal pathology of animals was assessed using a severity index (Radford et al., 1997) that assigns points (0 to 3) for interstitial features of kidney. The extents of interstitial fibrosis, tubular atrophy (defined as luminal dilation and flattened tubular epithelial cells) and interstitial cell infiltration were semiquantitatively scored as absent (0), involving less than 25% of the field (1), involving 26% to 50% of the field (2) and involving greater than 50% of the field (3).

Immunohistochemistry
After formalin fixation and paraffin embedding, 4 mm sections were deparaffinized in xylene and rehydrated through graded ethanols. The endogenous peroxidase activity was suppressed by exposing slide-mounted tissue to 0.3% H2O2 after antigen retrieval with pepsin or boiling. Sections were then incubated with the following polyclonal antibodies overnight at 4°C in phosphate-buffered saline: TGF-b1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), fibronectin (Santa Cruz Biotechnology), a-SMA (Zymed Laboratories, San Francisco, CA, USA). After being washed, slides were incubated with biotinylated goat anti-rabbit IgG antibody and then peroxidase conjugated streptavidin. Sections were visualized with diaminobenzidine tetrahydrochloride (DAB) substrate and counterstained with haematoxylin.
The expression of TGF-b1, fibronectin or a-SMA in cortical interstitium was assessed by spectrophotometry. The stained sections were visualized on a computer display with an Olympus DP50 camera connected to a light Olympus BX51 microscope (Olympus Optical Co., Tokyo, Japan) at an original magnification of ¥200. The morphometric evaluation consisted of quantitative analyses of TGF-b1, fibronectin or a-SMA by using specific software (Image-Pro Plus 6.0, Media Cybernetics Inc. Bethesda, MD, USA). At least ten nonoverlapping fields at a magnification of ¥200 per section were analysed for each animal. The expression of TGF-b1, fibronectin or a-SMA was assessed by the ratio of the mean positively stained area to the mean whole area of the section.

Immunofluorescence
After formalin fixation and paraffin embedding, 4 mm sections were deparaffinized in xylene and rehydrated through graded ethanols. Sections were then incubated with a mouse monoclonal antibody of phosphorylation of c-Jun NH2terminal kinase (p-JNK, Santa Cruz Biotechnology) overnight at 4°C in PBS. After being washed, slides were incubated with Table 1 The groups of rats and their administrations in animal experiments

Determination of renal tissue nitric oxide (NO)
Kidney tissue (~100 mg) was pulverized using a chilled mortar and pestle and then resuspended in 3 mL modified radioimmunoprecipitation assay buffer containing 50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.25% Na deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethanesulphonyl fluoride, 1 mg·mL -1 aprotinin, 1 mg·mL -1 leupeptin, 1 mg·mL -1 pepstatin, 1 mM Na3VO4, and 1 mM NaF. After being incubated at 4°C for 1 h, the suspension was centrifuged (22 000¥ g, 4°C, 30 min), and the supernatant was aliquoted, frozen in liquid nitrogen and stored at -80°C until use. The measurement of NO in kidney homogenates was performed according to a method described previously (Meng et al., 2007). Briefly, 100 mL of the supernatant of kidney homogenates was incubated with nitrate reductase, which converted nitrate to nitrite, for 1 h at 37°C. Then, the samples were incubated for 10 min at room temperature after the addition of the Griess reagent. The optical density values of the samples were measured at 530 nm on a spectrophotometer (Beckman DU530, Beckman Coulter Inc, Brea, CA, USA). The concentration of total nitrite, expressed as mmol·mg -1 protein, represented NO production in the homogenates.

Western blot analysis
Proteins in cell lysates were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrophoretically transferred to nitrocellulose membrane. The nitrocellulose membrane was blocked with 5% skimmed milk in Tris-buffered saline-Tween-20 (TBS-T) for 1 h. The membrane was incubated with antibodies for a-SMA, fibronectin and mitogen-activated protein kinases (MAPKs) diluted in 2% (w/v) BSA dissolved in TBS-T at 4°C overnight. Blots were washed for 45 min in TBS-T before incubation with a horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG for 2 h at room temperature with rotation. Proteins were detected on X-ray film using chemiluminescence reagent plus (Perkin-Elmer Life Sciences, Waltham, MA, USA).  Table 2.

RNA extraction and quantitative reverse transcription-PCR analysis
Reaction specificity was confirmed by melting curve analysis. Gene expression profiles were normalized to GAPDH and calculated using the DDCt (2 -DDCt ) method for each sample.

Statistical analysis
Results are expressed as mean Ϯ SD. The results were assessed by one-way analysis of variance (ANOVA) for comparisons among groups. Differences between groups were determined by Tukey's multiple comparisons tests. Results were considered significant when P < 0.05. All analyses were performed with the SPSS14.0 statistical software package (SPSS Inc., Chicago, IL, USA).

AF improved renal function and renal interstitial injury
The serum creatinine and urea levels were higher in the UUO group than that in the sham control group (P < 0.05) and were all decreased after treatment with Enalapril, A&A, AF-H or AF-L (P < 0.05). There was no difference between the AS-IV or FA alone treated group and the UUO group. As shown in Figure 1 and Table 3, the semiquantitative histomorphometry analysis on HE-and Masson-stained sections revealed significant infiltration of mononuclear cells, damaged tubules and interstitial fibrosis in the obstructed kidneys, compared with that in sham controls (P < 0.05). The pathological changes were significantly alleviated by AF (high or low dose), with similar effects obtained with Enalapril or A&A (P < 0.05), but there was no significant change after treatment with either AS-IV or FA alone.

AF alleviated the expression of fibronectin, a-SMA, TGF-b 1 in obstructed kidneys
We examined the interstitial fibronectin accumulation in fibrotic kidney after AS-IV and FA treatment. As a major component of the ECM in the fibrotic kidney, fibronectin was prominently stained in the tubulointerstitium of obstructed kidneys compared with the sham-operated kidneys. The Table 2 The primers of a-SMA, fibronectin and GAPDH semiquantitative histomorphometric analysis showed that obstructive injury provoked a 7.5-fold induction of fibronectin (P < 0.05). Monotherapy with AS-IV or FA alone did not attenuate the accumulation of fibronectin, but AF treatment resulted in a significant reduction of fibronectin (P < 0.05, Figure 2). The expression of a-SMA is the molecular hallmark of myofibroblasts. As shown in Figure 2, a-SMA was expressed in the smooth muscle cells of renal arterioles but was rarely evident in the tubulointerstitium of sham-operated kidneys. Quantification of immunostaining in renal tissue indicated a striking increase in the a-SMA-positive area in the obstructed kidneys compared with that in the sham control (P < 0.05), and treatment with AF significantly reduced interstitial expression of a-SMA (P < 0.05).

Genes
TGF-b1 is an important pro-fibrotic growth factor in the progression of interstitial fibrosis. As shown in Figure 2, there was only occasional immunostaining of TGF-b1 in the tubular cells of the sham-operated kidneys but diffuse staining in the obstructive kidneys, especially the proximal tubular cells and infiltrating monocyte/macrophage. The expression of TGF-b1 in the AF group was low in the epithelial cells of the dilated tubuli. Semiquantitative analysis revealed that the expression of TGF-b1 was significantly increased in the UUO group compared with the sham group (P < 0.05) and was decreased after AF therapy compared with the UUO group (P < 0.05). Similar to fibronectin, AS-IV or FA treatment alone has no impact on the expression of a-SMA and TGF-b1.

AF increased renal tissue NO production
Astragali Radix and Angelicae Sinensis Radix increased the production of NO in obstructive kidneys via enhancement of endothelial NOS activity (Meng et al., 2007). In this study, we also evaluated the effect of AF on NO production. Figure 3 shows that the production of NO in obstructed kidney was slightly increased compared with the sham controls, but this was not statistically significant. Monotherapy with AS-IV or FA and the combination of the two constituents (AF) all markedly increased the production of NO in obstructed kidneys (P < 0.05).

AF inhibited TGF-b 1 -induced a-SMA and fibronectin expression in NRK-49F cells
During ureteral obstruction, interstitial fibroblasts are activated resulting in massive proliferation and excessive ECM accumulation. In this study, the expression of a-SMA and fibronectin protein in NRK-49F cells after stimulation with TGF-b1 was significantly higher than that in the cells treated with DMSO (P < 0.05). AF dramatically inhibited a-SMA and fibronectin overexpression compared with TGF-b1-treated cells (P < 0.05; Figures 4 and 5), but AS-IV or FA did not affect the expression of a-SMA and fibronectin after stimulation with TGF-b1. Furthermore, it was shown that AF inhibited the expression of a-SMA and fibronectin mRNA in NRK-49 cells treated with TGF-b1 (P < 0.05; Figures 6 and 7).

AF inhibited IL-1b-induced a-SMA and fibronectin expression and JNK phosphorylation in HK-2 cells
IL-1b stimulates the transition of renal tubular epithelial cells to myofibroblasts via a TGF-b1-dependent pathway (Fan et al., 2001). Our previous study showed that IL-1b induced HK-2 cells to express the myocyte biomarker a-SMA through the activation of JNK and P38 signalling pathways (Zhang et al., 2005). In this study, we evaluated the effects of AF on IL-1bstimulated HK-2 cells. The renal tubular epithelial cells displayed typical cobblestone morphology, but IL-1b induced profound morphological changes, characterized by shape elongation, dissociation from neighbouring cells and loss of their cobblestone monolayer pattern. Simultaneous incubation of AF with IL-1b significantly restored the morphology Table 3 Renal function and tubulointerstitial injury of obstructed kidney in rats  of the epithelial cells. As shown in Figures 8 and 9, IL-1b significantly increased a-SMA and fibronectin expression in HK-2 cells (P < 0.05). AF not only decreased the basal expression of a-SMA and fibronectin in HK-2 cells but also significantly inhibited the expression of a-SMA and fibronectin in IL-1b-induced cells (P < 0.05). Monotherapy with AS-IV or FA of HK-2 cells had no effect on the expression of a-SMA and fibronectin in either basal conditions or after IL-1b stimulation. It was also shown that AF inhibits the expression of a-SMA and fibronectin mRNA in HK-2 cells stimulated with IL-1b (P < 0.05; Figures 10 and 11). We further examined whether AF decreased EMT through the main MAPK signalling pathway, including JNK, extracellular signal-regulated kinases (ERK) and P38. IL-1b significantly increased JNK, ERK and P38 phosphorylation, but AF only significantly inhibited IL-1b-induced JNK phosphorylation (P < 0.05, Figure 12), with no effect on ERK or P38 phosphorylation (data not shown). Continued.

AF alleviated the expression of p-JNK in obstructed kidneys
Concomitant with the increased activity of JNK, the expression of p-JNK was confirmed in obstructed kidneys using immunofluorescence. Normally, p-JNK is localized to some tubular epithelial cells and occasional glomerular cells. UUO induced a marked increase in JNK phosphorylation with p-JNK staining prominent in dilated tubules, as well as in interstitial cells. AF alleviated the expression of p-JNK ( Figure 13).

Discussion and conclusions
In this study, we examined the renoprotective effects of AS-IV and FA, two active constituents of a mixture of Astragali

Figure 5
The expression of fibronectin in NRK-49F cells. AS-IV: astragaloside IV, FA: ferulic acid, AF: AS-IV and FA. AF decreased the expression of fibronectin in TGF-b1-treated cells (P < 0.05), but AS-IV or FA had no effect on fibronectin expression. Values are mean Ϯ SEM. *P < 0.05 versus DMSO-treated cells, #P < 0.05 versus TGF-b1-treated cells.

Figure 3
The production of NO in the obstructed kidneys. UUO: unilateral ureteral obstruction, AS-IV: astragaloside IV, FA: ferulic acid, AF-L: AS-IV and FA (low dosage). AS-IV and FA dramatically increased the production of NO in obstructed kidneys (P < 0.05). NO production was expressed as mmol·mg -1 protein of NO2in the tissue. Values are mean Ϯ SEM. *P < 0.05 versus UUO kidneys.

Figure 4
The expression of a-SMA in NRK-49F cells. AS-IV: astragaloside IV, FA: ferulic acid, AF: AS-IV and FA. AF decreased the expression of a-SMA in TGF-b1-treated cells (P < 0.05), but AS-IV or FA alone had not effect a-SMA expression. Values are mean Ϯ SEM. *P < 0.05 versus DMSOtreated cells, #P < 0.05 versus TGF-b1-treated cells.
Radix and Angelicae Sinensis Radix, which has been shown to inhibit the progression of renal fibrosis and improve renal function in several animal models of kidney diseases (Li et al., 1998;Wang et al., 2004;Zhang et al., 2006b;Meng et al., 2007). The result indicated that the combination of AS-IV and FA (AF) have renoprotective effects characterized by a decrease in serum creatinine and urea, and a reduction in tubulointerstitial fibrosis in obstructive nephropathy, even though neither AS-IV nor FA alone attenuated renal injury. Renal interstitial fibrosis is widely accepted as being closely associated with the deterioration of renal function because the tubulointerstitium comprises about 90% of the volume of the kidney (Bohle et al., 1987). The prevention of interstitial fibrosis can significantly slow the progression of kidney disease. Thus, the combination of AS-IV and FA might be beneficial to the injured kidney. UUO is recognized as an ideal experimental model of renal interstitial fibrosis. Ureteral obstruction leads to a significant reduction in renal blood flow, local ischaemia/ hypoxia and an inflammatory response, followed by renal interstitial myofibroblast proliferation and ECM accumulation in the renal interstitium (Chevalier et al., 2009). In the process of renal fibrosis, the overexpression of profibrotic cytokines (e.g. TGF-b 1, CTGF) and inflammatory factors (such as IL-b, TNF) contributes to kidney damage.
Notably, the proliferation of myofibroblasts in renal tubulointerstitium is the crucial event in the progression of renal fibrosis. The activation of resident fibroblasts results in a massive increase in the number of myofibroblasts during the early stage of obstructive nephropathy. From day 7 after ureteral obstruction, the transdifferentiation of epithelial and endothelial cells becomes the major source of myofibroblasts (Grande et al., 2009). TGF-b1 plays a critical role in the initiation and progression of renal fibrosis (Qi et al., 2008). In response to TGF-b1, resident fibroblasts can be activated (Suzuki et al., 2004), and tubular epithelial cells can transdifferentiate into myofibroblasts (Sebe et al., 2008). IL-1b
promotes tubulointerstitial fibrosis by impairing proximal tubule cells, inhibiting growth, a-SMA expression and fibronectin production, which are mediated by a TGF-b1dependent mechanism (Fan et al., 2001). In this study, we investigated the effects of AS-IV and FA on two of the main sources of myofibroblasts, fibroblasts and renal tubular epithelial cells. Although treatment with either AS-IV or FA alone had no effect, the combination of the two constituents (AF) significantly reduced the expression of a-SMA and fibronectin in activated fibroblasts and tubular epithelial cells. AF is thought to reduce the production of ECM resulting in the inhibition of myofibroblast proliferation.
The activation of MAPK signalling is an important pathway in TGF-b1-mediated renal fibrosis (Böttinger et al., 2002). The three phosphorylation cascades of MAPKs pathway, including ERK, JNK and p38 MAPK, participate in TGF-b1 signalling cascades. These kinases can be activated by growth factors and environmental stress leading to alterations in cell growth and other key cell functions, such as EMT (Rodrigues-Díez et al., 2008). Recently, JNK signalling was reported to be involved in TGF-b1-induced EMT in tubular epithelial cells (Mariasegaram et al., 2010). In other epithelial cell types, including tracheal epithelial cells and keratinocytes, TGF-b1 also induces EMT via JNK signalling (Alcorn et al., 2008;Santibañez et al., 2006). In this study, the inhibition of a-SMA and fibronectin by AF was found to be associated with the JNK pathway in the TGF-b1-treated NRK-49F and IL-b-treated HK-2 cells. Thus, the activation of JNK signalling may be an important pathway through which AF induces an antifibrotic effect in renal tubulointerstitial fibrosis.
AS-IV and FA have been found to inhibit the MAPK pathway in several studies by other researchers. Either AS-IV or FA was found to block the JNK pathway, but the doses used in these experiments were higher than those used in the present study. However, JNK activity is differentially regulated by various protein kinases, including MKK4 and MKK7 (Fleming et al., 2000), mixed lineage kinases (Hirai et al., 1997) and TGF-activated protein kinase 1 (Wang et al., 1997). JNK1 activity has also been shown to be up-regulated as a consequence of proteolytic cleavage of p21 WAF1/CIP1 (Shim et al., 1996), and Ca 2+ and reactive oxygen species (ROS) generation play an important role in the activation of the JNK pathway (Ham et al., 2006). AS-IV has been found to reduce the increase in ROS (Xu et al., 2008), as well as prevent the rise in intracellular [Ca 2+ ] (Hu et al., 2009). Also, FA was shown to elevate the protein content of the JNK1 inhibitor p21 WAF1/CIP1 (Hou et al., 2004). However, these effects of either AS-IV or FA alone are not enough to inhibit the activation of JNK at the doses used in our study, but when the actions of these two compounds are combined, a synergistic effect is produced that blocks the JNK pathway, induced by inhibition of JNK activation and cleavage.
Our study also demonstrated that AS-IV and FA (AF) increased NO production in the kidney. NO has been also shown to ameliorate renal injury in kidney diseases (Trachtman et al., 1995;Huang et al., 2000). AS-IV has been reported to enhance the activation of endothelial NO synthase (Zhang et al., 2007), and FA has been shown to decrease oxidative stress . Thus, AF not only increases eNOS activation but also protects NO by increasing the activity of scavenging ROS; these effects converge to increase NO production and so promote the remission of interstitial fibrosis.
Unfortunately, the positive role of AS-IV or FA alone on renal fibrosis was not observed at the doses of AS-IV or FA used in this study. Higher doses were not used because the range of concentrations of AS-IV or FA detected by HPLC method was limited by the concentrations of Astragali Radix and Angelicae Sinensis Radix available. Therefore, further studies needed to observe the effects of AS-IV and FA at different concentrations and to identify any other active components in Astragali Radix and Angelicae Sinensis Radix with renoprotective effects.
The mechanisms of renal fibrosis are associated with a series of dynamic pathological changes in the progression of renal injury. In this study, we demonstrated that AF possesses

Figure 12
The expression of JNK in HK-2 cells. AS-IV: astragaloside IV, FA: ferulic acid, AF: AS-IV and FA. AF decreased JNK phosphorylation in IL-1b-treated cells (P < 0.05). Values are mean Ϯ SEM. *P < 0.05 versus DMSO-treated cells, #P < 0.05 versus IL-1b-treated cells.  antifibrotic, which are characterized by a decrease in cellular infiltration, tubular atrophy and interstitial fibrosis, as well as inhibition of FN, a-SMA and TGF-b1 activity. Fibroblasts and tubular epithelial cells are two important sources of myofibroblasts; in vitro experiments were employed to explore the potential cellular mechanisms of AF, but the cellular experiments only demonstrated a small part of the complicated process. Thus, we cautiously conclude that AF can alleviate renal tubulointerstitial fibrosis, and this is associated with inhibition of tubular EMT and fibroblast activation, as well as an increase in NO production in the kidney.