|Year : 2016 | Volume
| Issue : 3 | Page : 84-91
The Protective Effect of Danhong on Tubulointerstitial Fibrosis is Associated with Suppression of Angiotensin II and Transforming Growth Factor β in the Kidney
Cheng-Jun Wang, Ying-Jing Shen, Kai Chen, Xiao-Rong Bao
Department of Nephrology, Jinshan Hospital, Fudan University, Shanghai, China
|Date of Web Publication||4-Aug-2016|
1508, Longhang Road, Jinshan, Shanghai
Source of Support: None, Conflict of Interest: None
Background and Objectives: Tubulointerstitial fibrosis (TIF), the common pathway of chronic kidney disease (CKD) to end-stage kidney disease, is sorely lacking of effective drugs in prevention, even synthetic chemical drugs have made significant progress. Therefore, this study was to investigate the effects of Danhong (active agents of Salvia miltiorrhiza and safflower Carthamus) on TIF in early CKD with unilateral nephrectomy animal model. Materials and Methods: Forty Sprague-Dawley rats were randomly divided into four groups (each n = 10): A, control; B, CKD; C, CKD + Danhong; and D, CKD + Losartan. In Group A and B, rats were treated with normal saline for 12 weeks. In Group C and D, rats were treated with Danhong (8 mL/kg/day) and losartan (20 mg/kg/day), respectively. The concentrations of angiotensin II (Ang II) and transforming growth factor (TGF-β) in kidney were measured by immunohistochemistry technique and enzyme-linked immunosorbent assay. The relationship among the indicators relating to TIF was analyzed. Data were collected and analyzed using SPSS software. Results: The concentrations of Ang II and TGF-β in renal cortex homogenate, TGF-β1 positive area in renal cortex tissue, and semi-quantitative nephropathological scores of TIF were dramatically lower in CKD rats treated with Danhong, compare with those without Danhong management. Conclusions: Danhong has a protective effect on TIF in CKD rats, similar to losartan, may through a series of approach including suppression of Ang II and TGF-β in kidney.
Keywords: Angiotensin II, Danhong, transforming growth factor β, tubulointerstitial fibrosis
|How to cite this article:|
Wang CJ, Shen YJ, Chen K, Bao XR. The Protective Effect of Danhong on Tubulointerstitial Fibrosis is Associated with Suppression of Angiotensin II and Transforming Growth Factor β in the Kidney. J Integr Nephrol Androl 2016;3:84-91
|How to cite this URL:|
Wang CJ, Shen YJ, Chen K, Bao XR. The Protective Effect of Danhong on Tubulointerstitial Fibrosis is Associated with Suppression of Angiotensin II and Transforming Growth Factor β in the Kidney. J Integr Nephrol Androl [serial online] 2016 [cited 2021 Aug 3];3:84-91. Available from: http://www.journal-ina.com/text.asp?2016/3/3/84/187792
| Introduction|| |
Previous studies suggested that tubulointerstitial fibrosis (TIF), characterized by monocyte and macrophage infiltration, tubular atrophy and fibroblast proliferation/differentiation, extracellular matrix accumulation, and ultimate TIF, is the most significant consequences of chronic kidney disease (CKD) that leads eventually to end-stage kidney disease. , Hence, there is widespread interest to understand the pathological mechanism of TIF and to find drugs effective in retarding or ameliorating CKD progression. Many studies indicate that overactivation of renin-angiotensin-aldosterone system (RAAS) and transforming growth factor β (TGF-β) may play a crucial role in this progression. ,,, To alter this intricate status, there were increasing studies focus on natural agents such as Danhong, an extraction of Chinese herbal medicine compound preparations, containing active ingredients of Salvia miltiorrhiza and safflower Carthamus,  which are extensively used and proven effective in the treatment of cardiovascular, hepatic and lung disease based on their wide effects of antifibrosis via simultaneously hit different targets ,,, including improving endothelial function,  suppressing RAAS,  and antifibrosis.  Nevertheless, whether it can relieve the CKD or not remains unclear. Therefore, this study was to investigate the effects of Danhong on TIF in early CKD with unilateral nephrectomy animal model.
| Materials and methods|| |
Reagents and antibodies
Danhong, losartan potassium (used as positive control), , and normal saline were purchased from Buchang Pharmaceutical Co., Ltd. (Heze, Shandong, China), Yangtze River Pharmaceutical Co., Ltd. (Dujiangyan, Sichuan, China), and Baxter Healthcare Co., Ltd (Shanghai, China), respectively. The enzyme-linked immunosorbent assay (ELISA) kits for angiotensin II (Ang II) and TGF-β were purchased from Nanjing Jiancheng Biology Engineering Institute (Nanjing, Jiangsu, China). The staining kits of glycogen, silver hexamine, and fibrous connective tissue were provided by Lok Cheung Medical Reagent Technology Co., Ltd (Shanghai, China). TGF-β1 antibody was provided by Santa Cruz Biotechnology Incorporation (Santa Cruz, California, USA). Goat antirabbit IgG-HRP labeled the second antibody was provided by Shanghai Pepro Biological Technology Co., Ltd. (Shanghai, China).
The establishment of early chronic kidney disease animal model and participants group
Forty male Sprague-Dawley rats, weighing 160-180 g and 6 weeks old, obtained from Experimental Animal Center of Public Health Clinical Center affiliated to Fudan University (Shanghai, China), were randomly assigned to four groups (each n = 10): Group A (control group, sham surgery), Group B (CKD group, unilateral nephrectomy), Group C (CKD + Danhong group), and Group D (CKD + losartan group), and housed in standard plastic cages in temperature (22°C) controlled room with ad libitum freely access to standard rat chow and water during the entire study.
Anesthesia was achieved by the intraperitoneal administration of 5% ketamine injection (100 mg/kg body weight). Then, the surgical region was shaved, and the shin was cleaned with 75% alcohol. Instantly, the right kidney was exposed through a lumbar longitudinal incision under the right costal arch. Subsequently, right subcapsular nephrectomy was performed, while right kidney in Group A was exposed and gently manipulated but left intact. Finally, operation field was cleared, and the incision was closed. After 1 week of normal feeding, the rats in four groups were in good condition. Afterward, rats in Group C were injected with Danhong (8 mL/kg body weight, intraperitoneal administration) daily, and those in Group D were administered orally with losartan potassium suspension (20 mg/8 mL, 20 mg/kg body weight, gavage), ,, whereas rats in Group A or B only treated with same volume normal saline by gastric lavage daily before sacrifice. During the experiment, the administered dose was adjusted based on body weight, which was measured weekly. 
Sample collection and indexes detection
All rats were sacrificed 12 weeks after surgery, and the remnant kidneys were resected and decapsulated for histological and molecular immunohistochemical studies. Biochemistry study in urine and blood was done using Beckman CX 9 Biochemical Analyzer (Shanghai, China).
Twenty-four hours before surgery and sacrifice, all rats were housed in metabolic cages. Urine was collected for detection of urinary protein (with the Coomassie Brilliant Blue method); blood was collected from the heart in surgery into nonheparinized tubes and serum was then obtained via centrifugation and stored at −70°C for detection of serum creatinine, urea nitrogen, uric acid, cystatin C, and albumin.
The 24 h urinary albumin excretion rate (UAER) and creatinine clearance rate (Ccr) were also calculated with the following formula, respectively:
UAER (mg/24 h) = urinary albumin (mg/ mL) × 24 h urine volume (mL)
Ccr (mL/min/kg) = (urinary creatinine [μmol/L] × urinary volume [mL]/serum creatinine [μmol/L])/1440 (min)/(body weight [g]/1000) 
After weighing of the remnant kidneys, kidney coefficient and kidney coefficient ratio were also calculated with the following formula, respectively:
Kidney coefficient = kidney weight/body weight
Kidney coefficient ratio = kidney coefficient after experiment/kidney coefficient before experiment.
Renal cortex homogenate
Renal cortex homogenate was obtained for detection of Ang II and TGF-β (with ELISA).
An 8 mm 3 wedge was taken from the cortex of each kidney at sacrifice then immediately immersion-fixed in 10% neutral buffered formalin. After routine processing, paraffin-embedded sections were cut into slice (4 μm thickly) and stained with hematoxylin and eosin, periodic acid-Schiff (PAS), and Masson's trichrome. Pathological changes were observed under light microscope in a masked fashion at a magnification of ×200 light microscopy by 2 pathologists blind to all groups. Assessment of injuries of glomerulus, glomerular basement membrane (GBM), and tubular interstitium was performed with semi-quantitative scores described below. ,
Glomerular injury was assessed by examining 15 glomeruli in PAS-stained sections. Each glomerulus was graded as either normal (0), mildly glomerular damage or sclerotic (1, lesion including the mesangial matrix and/or hyalinosis with focal adhesion occupying <25% of glomerular tuft), moderately sclerotic (2, lesion occupying 25-49% of glomerular tuft), severely sclerotic (3, lesion occupying 50-75% of glomerular tuft), or globally sclerotic (4, lesion occupying 76-100% of glomerular tuft).
GBM injury was assessed by examining 10 glomeruli in periodic acid silver methenamine-stained sections. Each glomerulus was graded as either normal (0), mildly GBM damage (1, generally normal glomerulus, slightly dilated glomerular capillary loops, stiffness, vacuoles in GBM, and tiny deposit under epithelial cells), moderately GBM damage (2, diffuse thickening GBM, spikes, and granular eosinophilic deposit around spikes under epithelial cells), or severely GBM damage (3, more severe thickening of irregular and spongy GBM, mesangial proliferation and sclerosis, or segmental or global glomerulosclerosis).
Tubulointerstitial injury was assessed by examining 15 fields of tubulointerstitial area in the cortex in Masson-stained sections. Each field was graded as either normal (0), mildly tubulointerstitial injury (1, the area of interstitial inflammation and fibrosis, tubular atrophy, and dilation with cast formation occupying <25% of the field), moderately damage (2, lesion area lesion occupying 25-49% of the field), or severely damage (3, lesion occupying 50-100% of the field).
The mean scores of the three kinds of (glomerulus, GBM, and tubulointerstitium) injury were, respectively, calculated by averaging the scores assigned to all glomeruli, GBM, or tubular fields. The total score was the sum of previous three (glomerular, GBM, and tubulointerstitial injury) scores.
TGF-β1 in kidney tissue was detected with immunohistochemistry technique. Ten random and nonoverlapping fields without glomerulus on each slide were selected to detect the values of the integral optical density and the total area, and the expression intensity was calculated as the percentage of the integral optical density to the total area which was performed by JEDA 801D pathology microscope image analysis system version 1.0.
All of the statistical analyses were performed with Statistical Package for Social Sciences, version 11.5 (SPSS Inc., Chicago, IL, USA). In brief, continuous data with normal distribution and homogeneity of variance determined by Shapiro-Wilk test were expressed as mean ± standard deviation (x- ± s), those with abnormal distribution or variance homogeneity were logarithmically transformed before analysis and treated as normally distributed if met the above conditions. Multigroup data were compared using single factor analysis of variance (one-way). All multiple testing was corrected using Bonferroni correction. Single factor linear correlation analysis was used to explore the associations between examined continuous variables with parametric distribution if the plot showed a linear relationship. P < 0.05 was considered statistical significance.
| Results|| |
Early chronic kidney disease animal model and renal function
There was no obvious pathology change observed in Group A [Figure 1]a-d and no glomerulosclerosis, no necrosis of epithelial cells in Bowman's capsule or tubular or small vessel lesions in tubular interstitium, rare obvious fibrosis, but mild glomerular mesangial cell proliferation, mesangial matrix increasing, mesangial area broadening with or without focal adhesion and hyaline degeneration, accidentally squeezed capillary, thickening of GBM, tubular dilatation, and sometimes protein cast in it, accidental inflammatory cells infiltration in the tubular interstitium in Group B [Figure 1]e-h. Lesions in Group C [Figure 1]i-l and D [Figure 1]m-p even mild than those in Group A [Figure 1]. Meanwhile, there was no difference in urine volume, plasma albumin, serum creatinine, and uric acid but UAER and cystatin C among the four groups [Table 1]. Besides, kidney weight after experiment, kidney coefficient after experiment, kidney weight ratio, and kidney coefficient ratio in unilateral nephrectomy rats were higher than those in kidneys undisturbed rats. All results above were in accordance with the characteristics of early CKD [P < 0.05, respectively, [Table 1].
|Figure 1: Nephropathological pictures with hematoxylin and eosin, periodic acid-Schiff, periodic acid silver methenamine, and Masson stain of Groups A, B, C, and D, respectively. (a-d) There was no obvious pathology change observed in Group A (u200). (e-h) The kidneys of Group B showed glomerular mesangial cells proliferation, mesangial area broadening (e-g); mesangial matrix increasing with focal adhesion and hyaline degeneration, capillary cavity squeezed accidentally (f), glomerular basement membrane thickening diffusely (g); tubular epithelial cells well organized in general, while tubular dilatation (e, f, and g) and sometimes protein cast in it (f and h), and many inflammatory cells infiltration in the tubule-interstitium (e and h), even obvious fibrosis (h) (e200). (i-l) The kidneys of Group C showed mild glomerular mesangial cells proliferation, mesangial area broadening (i-k), mesangial matrix increasing without focal adhesion or hyaline degeneration, capillary cavity squeezed accidentally (j), and glomerular basement membrane thickening (k); tubular epithelial cells well organized, mild tubular dilatation without protein cast, and accidental inflammatory cells infiltration in the tubule-interstitium (i and l), rare obvious fibrosis (h) (h200). (m-p) The kidneys of Group D showed similar lesions to Group C in general|
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|Table 1: Kidney weight, kidney coefficient, and renal function of rats in four groups |
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Semi-quantitative histological scores of kidney lesion
Furthermore, to evaluate the kidney lesion in each group, semi-quantitative histological scores of glomerular sclerosis, GBM, TIF, and the total score were estimated. The result showed that all scores were significantly higher in unilateral nephrectomy rats (Group B, C, and D) than those in kidney undisturbed rats (Group A). While all these scores in both Group C and D were lower than those in Group B [P < 0.05, respectively, [Table 2].
Indicators relating to endothelial function and tubulointerstitial fibrosis
Ang II and TGF-β in renal cortex homogenate and TGF-β1 positive area in renal cortex tissue as possible middle markers of TIF progress were reported in and [Figure 2] and [Figure 3]. The concentrations of Ang II and TGF-β in renal cortex homogenate and TGF-β1 positive area in renal cortex tissue were dramatically higher in Group B, C, and D than those in Group A. Contrastively, levels of these indicators considerably lower in both Group C and D, compare with those in Group B and Group A.
|Figure 2: Indicators relating to endothelial function and tubulointerstitial fibrosis of rats in four groups. Ang II = Angiotensin II; TGF-ƒÀ = Transforming growth factor|
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|Figure 3: Transforming growth factor 1 showed with immunohistochemistry. The kidneys of Group B (R) showed more positive signal (tan area) than other three groups|
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Correlation between indicators relating to kidney lesion, renin-angiotensin-aldosterone system (RASS), and tubulointerstitial fibrosis
The total score had positive correlations with the concentrations of Ang II and TGF-β in renal cortex homogenate, and TGF-β1 positive area in renal cortex tissue [P < 0.05, respectively, [Table 3].
|Table 3: Single factor linear correlation analysis showing variables independently associated with total nephropathological score |
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| Discussion|| |
It is well known that the kidney's particular susceptibility to fibrosis and TIF plays an important role in the aggravating of CKD. Meanwhile, overactivation of RAAS and overproduction of TGF-β, which is prevalent in CKD patients, will lead to TIF and kidney function impairment. ,,, A possible explanation may be the biologically complex interactions between the RAAS and TGF-β: (1) Alterations in glomerular hemodynamics can activate both the RAAS and TGF-β. (2) In renal tubulointerstitial injury and tissue hypoperfusion, TGF-β is released from juxtaglomerular cells and may act synergistically with Ang II to accentuate vasoconstriction. (3) Components of the RAAS simultaneously stimulate the production of TGF-β and plasminogen activator inhibitor, then stimulate matrix protein synthesis, inhibition of matrix degradation, and enhanced integrin expression that facilitates matrix assembly, leading to matrix accumulation and renal failure. When injury is recurrent or continual, this interplay between the renin-angiotensin system (RAS) and TGF-β causes continual activation that may result in chronic hypertension and progressive tissue fibrosis leading to organ failure. Hence, interaction of the RAAS and TGF-β has important clinical implications. Such an agent or combination may be required if progressive fibrotic diseases are to be truly prevented, instead of just delayed. Current research of synthetic chemical drugs has made significant progress. Even subdepressor dose of angiotensin Type-1 receptor blocker can significantly inhibit fibrosis. Whereas increasing the dose of any drug above their usually depressor dose can only reduce production of TGF-β about 50%. Ang II blockade alone is not sufficient to halt renal fibrosis. Therefore, traditional Chinese herbs, especially Danhong and their active ingredients, for their extensive pharmacological action, become considerably compelling. Consequently, our research is to investigate the effects of Danhong on TIF in early CKD animal model.
Our study showed that compensatory hypertrophy of the remnant kidney accompanied by pathological changes that lead to reduced renal function, such as increased levels of urinary protein and cystatin C, was well recognized. This phenomenon was in accordance with the characteristics of early CKD. The results above indicated that an early CKD animal model with mild kidney lesion was successfully established. Furthermore, the severity of histological lesions, all histological scores, levels of urinary protein, and serum cystatin C were significantly higher in CKD rats than those in kidney undisturbed rats. This adverse status could be improved by treatment with Danhong, and the nephroprotective effects were similar to losartan.
Danhong is a compound preparation of traditional Chinese medicine S. miltiorrhiza and Carthamus tinctorius and has been widely applied in treating coronary heart diseases and ischemic encephalopathy in clinic. Our data present that the expressions of Ang II and TGF-β in renal cortex homogenate and TGF-β1 positive area in renal cortex tissue were dramatically higher in Group B, C, and D than those in Group A. Contrastively, these indicators were considerably lower in both Group C and D, compare with those in Group B. Previous study showed that colocalization of renin and TGF-β in the hypertrophic juxtaglomerular apparatus provides the basis of anatomy for significant co-regulation between the RAS and TGF-β.  Even when sclerosis is not present in early CKD, it was seen that marked elevations of angiotensinogen mRNA colocalized with TGF-β mRNA in glomerular endothelium. Furthermore, study confirmed that TGF-β mRNA was colocalized with fibrotic matrix components laminin and fibronectin.  Hence, fibrogenic effects attributed to Ang II are actually mediated by TGF-β, which stimulates the contraction of vascular smooth muscle cells and glomerular mesangial cells, then chemoattractant renal interstitial monocyte/macrophages that debride the lesion and fibroblasts that begin matrix synthesis. When injury is recurrent or continual, proliferations of monocyte/macrophages and fibroblasts as well as fibrosis will continuous progress. Furthermore, evidence indicates that the RAS and TGF-β coregulate each other's expression. Thus, this interaction in the kidney may keep both systems activated long after injury response should have been terminated. This sustained activity would lead to progressive fibrosis. In our study, line correlation analysis showed that total nephron-pathological score was positively correlated with Ang II and TGF-β in renal cortex homogenate and TGF-β1 in renal cortex tissue. As a result, we thought that a significant amelioration of TIF contributed to Danhong, which suppressed activation of RAAS and expression of TGF-β. This is accordance with previous studies that showed Danhong and its active ingredients have a significant downregulate the levels of renin and Ang II as well as suppress of TGF-β. ,,,,
Moreover, endothelium as an important endocrine organ can secrete many active substances, including Ang II, and plays important roles in the cardiovascular system. Meanwhile, the kidney could be regarded as an important part of the vascular system and a critical link in the process of regulating vessel function because of its large number of capillary loops and the juxtaglomerular apparatus with special features. Fluid shear stress has been shown to activate both the RAS and TGF-β in glomerular endothelium. ,, Ang II may promote oxidative stress damage via activate NADPH oxidase of vascular smooth muscle cells, increase synthesis of the reactive oxygen species (ROS). TGF-β1 may promote expression of many cytokines including vascular endothelial growth factor in glomerular mesangial cells. Previous studies have demonstrated that the active ingredients of Danhong such as salvianolic acid A, salvianolic acid B, tanshinone IIA, hydroxysafflor yellow A, and safflor yellow B can alleviate Ang II-induced endothelium dysfunction via suppressing the expression of phospho-STAT5 and phospho-Akt (473),  and upregulating Bcl-2 expression, thereby attenuating the production of ROS, , enhancing SOD activity and decrease the MDA in anoxic cells,  thereby alleviating oxidative stress,  reducing calcium overload, keeping mitochondrial structure and function, and inhibiting apoptosis in turn,  then relieving inflammatory cell infiltration, medial smooth muscle cell proliferation, and migration,  preventing fibroblast proliferation and matrix accumulation. , Therefore, we thought that Danhong protected against TIF may also base on their wide protective effects of endothelium via simultaneously hit different targets.
| Conclusions|| |
An early CKD animal model was successfully established in our study by unilateral nephrectomy. With the model, we found that Danhong has a protective effect on TIF in CKD rats, similar to losartan. This effect may be associated with suppression of Ang II and TGF-β in the kidney tissues. Such unique property makes Danhong excellent candidates for prevention and treatment of TIF. Moreover, this study may provide useful insight in understanding the pharmacological efficacy of Chinese herbal medicine.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]