**1. Introduction**

174 Chronic Kidney Disease

[53] Finnigan N., Chernick M. and Benz R. Nephrology, N-Acetylcysteine (NAC) May

[54] Fishbane S, Besarab A.Mechanism of increased mortality risk with erythropoietin treatment to higher hemoglobin targets. Clin J Am Soc Nephrol 2007;2:1274-1282

PO2587] ASN Renal week 2010 Abstracts

Improve Erythropoietin Resistant Anemia ( ERA) in hemodialysis patients[SA-

Chronic renal injury can be mediated by angiotensin II (Ang II) through hemodynamic and inflammatory mechanisms and attenuated by individual suppression of these mediators. Hypertension is usually associated with the development of vascular and renal fibrosis [3]. This pathophysiological process is characterized by structural changes in vasculature caused by increased synthesis and rearrangement of extracellular matrix proteins, such as the collagen type I [4]. Several studies support a major role for the renin-angiotensin system in the development of fibrosis [5, 6].

Hypertension injures blood vessels and thereby causes end-organ damage. The mechanisms are complicated and although they have been studied for decades in experimental animal models [7], they are only currently being elucidated. From the efforts of many investigators, we are now in the position of constructing a chain of events from the endothelium to the underlying matrix, to the vascular smooth muscle cells, and beyond to the adventitia, and surrounding tissues. The endothelial layer acts as a signal transduction interface for hemodynamic forces in the regulation of vascular tone and chronic structural remodeling of arteries [8]. Infiltration of the permeabilized endothelium by leukocytes sets the stage for an inflammatory cascade, involving cytokines, chemokines, growth factors, and matrix metalloproteinases. Altered integrin signaling, the production of tenacin, epidermal growth factor signaling, tyrosine phosphorylation, and activation of downstream pathways culminate in vascular smooth muscle cell proliferation [9]. Evidence is accumulating that matrix molecules provide an environment which decreases the rate of programmed cell death [10].

Molecular Mechanisms of Nephro-Protective

Action of HE-86 Liquid Extract in Experimental Chronic Renal Failure 177

session, two thirds of the left kidney were removed. One week after the first operation, the right kidney was removed. These procedures were performed under anaesthesia with sodium pentobarbital (The ShuGuang pharmaceutical factory in Shanghai). Two weeks after 5/6-nephrectomy, 24 rats were divided into pairs such that both rats in each pair exhibited almost the same levels of serum creatinine, blood urea nitrogen (BUN) (Table 1). One rat from each pair was assigned to (i) control uraemic group (n=12), the other to (ii) treatment uraemic group (n=12) which received HE-86, extract liquid which is effective composition isolated from Chinese medicine prescription, everyday at a dose of 0.75 g/100 g body weight for 8 weeks. For normal controls, rats underwent a sham operation consisting of laparotomy and manipulation of the renal pedicles but without damage to the kidney(n=12). The treatment group were administered by HE-86 infuse the stomach as pair-fed with the control uraemic rats, and the normal rats were fed ad libitum with standard solid chow

(BiKai Animal Lab. Company, Shanghai, China) containing 24.5% protein.

N BUN(mmol/L) Scr(μmol/L)

sham 12 7.510.75 19.004.00

control 12 16.170.99\* 49.506.53\*

treatment 12 16.182.42\* 49.239.36\*

for histopathological and gene expression studies.

analyser (Fullerton, CA, USA), respectively.

reference to bovine serum albumin (Sigma) standards.

every 2 weeks throughout the experimental time course.

**Renal Function Assessment and Blood Pressure Measurement** 

**2.2 Analytical procedures** 

Table 1. The variation of serum creatinine and blood urea nitrogen before treatment.

Blood pressure was measured before treatment and every two weeks after surgery. The levels of serum creatinine (Scr), Blood urea nitrogen (BUN), 24h urine protein excreation and urine TGF–β were determined at 4 or 8 weeks after starting the administration of HE-86, respectively. The remnant kidneys were removed after perfusion at the end of experiment

Serum creatinine (Scr) and Blood urea nitrogen (BUN) were measured using a Beckman Cx4

24h Urinary protein concentrations were determined by the Bradford method, adapted to a microtiter plate assay. Coomassie reagent (USB, Cleveland, OH) was added to the diluted urine samples. After 10 minutes, the absorbance at 595-nm wavelength was read on ELX800 microplate reader (Bio-Tek Instruments, VT). The protein concentrations were calculated by

Systolic blood pressure was recorded by tail plethysmography using the BP2000 blood pressure analysis system (Visitech Systems, Inc., Apex, NC) in conscious rats at baseline and

Hypertension is a major risk factor for renal and cardiac damage, however, the mechanisms are incompletely understood. Angiotensin (Ang) II, the key effector of the local and circulating renin-angiotensin system (RAS), plays a central role [11-12]. In addition to its vasoactive and growth-promoting action, Ang II stimulates circulating leukocytes and endothelial cells, thereby promoting inflammation and interstitial extracellular matrix accumulation [13-17]. Many inflammation-mediating genes are activated by the transcription nuclear factor-B (NF-B), which resides inactive and bound to the inhibitory protein I-B in the cytoplasm of T lymphocytes, monocytes, macrophages, endothelial cells, and smooth muscle cells [18-19]. Ang II stimulates NADPH oxidase, which generates reactive oxygen species (ROS) [20]. ROS may act as signal transduction messengers for several important transcription factors, including NF-B and AP-1 (activator protein-1) [21]. Recently, Ozes et al [22]showed that Akt/protein kinase B (Akt) is essential in tumor necrosis factor- (TNF-)–induced activation of NF-B. Takahashi et al, [23] as well as Ushio-Fukai et al, [24] have demonstrated Akt activation by Ang II, which may involve ROS. Akt-induced activation of NF-kB upregulates numerous genes, including interleukin (IL)-1, IL-6, IL-8, interferon-, TNF-, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and the chemokine MCP-1 (monocyte chemoattractant protein-1). Several reports [25-27] indicated that angiotensin converting enzyme (ACE) inhibition decreased NF-B in renal disease.

We have previously demonstrated that traditional Chinese medicine prescription documented in the ancient Chinese pharmacopoeia or monographs promoted blood circulation, decreased blood stasis, and improved renal function. They decreased urinary protein excretion ,balanced lipid metabolism and enhanced the effects of antioxidant in the treatment of patients with early and middle stage chronic renal failure [28-32].

It has been showen broad foreground to postpone progression of chronic renal dysfunction. But it is unclear that effective composition and mechanism of renal protection. Therefore, the study presented here was designed to test the hypothesis that HE-86 liquid extract, which is effective unite refined from above Chinese prescription, would prevent chronic renal failure rats induced by nephrectomized, in association with decreased expression of angiotensin II and AT- II receptors, further to suppress high expression of inflammatory and growth factors. In an attempt to obtain more effective renal protection, research design consisted of a group of Nx rats receiving a HE-86 liquid extract treatment comparing with chronic renal failure rats induced by subtotal nephretomized without treatment. At same time, in the present study, we also assess the influence of renal mass reduction (RMR) caused by subtotal (5/6) nephrectomy on gene expression for NF-B, TNF- and TGF-beta1 and evaluate the correlation between expression of these genes and activity of the intrarenal renin-angiotensin systems. The research result showed HE-86 played a critical role in improving renal disease and was a key mediator in delay process of vascular fibrosis, characterized by reduced lumen diameter and arterial wall thickening attributable to excessive deposition of extracellular matrix (ECM) through by the model study.

#### **2. Materials and methods**

#### **2.1 Experimental design**

Thirty-six of the normal kidney mass were removed from adult male Munich-Wistar rats (BiKai, Shanghai, China) weighing 200–210 g to make animal models of CRF. In a first

Hypertension is a major risk factor for renal and cardiac damage, however, the mechanisms are incompletely understood. Angiotensin (Ang) II, the key effector of the local and circulating renin-angiotensin system (RAS), plays a central role [11-12]. In addition to its vasoactive and growth-promoting action, Ang II stimulates circulating leukocytes and endothelial cells, thereby promoting inflammation and interstitial extracellular matrix accumulation [13-17]. Many inflammation-mediating genes are activated by the

and smooth muscle cells [18-19]. Ang II stimulates NADPH oxidase, which generates reactive oxygen species (ROS) [20]. ROS may act as signal transduction messengers for

Recently, Ozes et al [22]showed that Akt/protein kinase B (Akt) is essential in tumor

Ushio-Fukai et al, [24] have demonstrated Akt activation by Ang II, which may involve ROS. Akt-induced activation of NF-kB upregulates numerous genes, including interleukin

cell adhesion molecule-1 (VCAM-1), and the chemokine MCP-1 (monocyte chemoattractant protein-1). Several reports [25-27] indicated that angiotensin converting enzyme (ACE)

We have previously demonstrated that traditional Chinese medicine prescription documented in the ancient Chinese pharmacopoeia or monographs promoted blood circulation, decreased blood stasis, and improved renal function. They decreased urinary protein excretion ,balanced lipid metabolism and enhanced the effects of antioxidant in the

It has been showen broad foreground to postpone progression of chronic renal dysfunction. But it is unclear that effective composition and mechanism of renal protection. Therefore, the study presented here was designed to test the hypothesis that HE-86 liquid extract, which is effective unite refined from above Chinese prescription, would prevent chronic renal failure rats induced by nephrectomized, in association with decreased expression of angiotensin II and AT- II receptors, further to suppress high expression of inflammatory and growth factors. In an attempt to obtain more effective renal protection, research design consisted of a group of Nx rats receiving a HE-86 liquid extract treatment comparing with chronic renal failure rats induced by subtotal nephretomized without treatment. At same time, in the present study, we also assess the influence of renal mass reduction (RMR)

and evaluate the correlation between expression of these genes and activity of the intrarenal renin-angiotensin systems. The research result showed HE-86 played a critical role in improving renal disease and was a key mediator in delay process of vascular fibrosis, characterized by reduced lumen diameter and arterial wall thickening attributable to

Thirty-six of the normal kidney mass were removed from adult male Munich-Wistar rats (BiKai, Shanghai, China) weighing 200–210 g to make animal models of CRF. In a first

excessive deposition of extracellular matrix (ECM) through by the model study.

)–induced activation of NF-

B in renal disease.

caused by subtotal (5/6) nephrectomy on gene expression for NF-

treatment of patients with early and middle stage chronic renal failure [28-32].

B in the cytoplasm of T lymphocytes, monocytes, macrophages, endothelial cells,

B), which resides inactive and bound to the inhibitory

, intercellular adhesion molecule-1 (ICAM-1), vascular

B, TNF-

and TGF-beta1

B and AP-1 (activator protein-1) [21].

B. Takahashi et al, [23] as well as

transcription nuclear factor-

 (TNF-

(IL)-1, IL-6, IL-8, interferon-, TNF-

inhibition decreased NF-

**2. Materials and methods** 

**2.1 Experimental design** 

necrosis factor-

protein I-

B (NF-

several important transcription factors, including NF-

session, two thirds of the left kidney were removed. One week after the first operation, the right kidney was removed. These procedures were performed under anaesthesia with sodium pentobarbital (The ShuGuang pharmaceutical factory in Shanghai). Two weeks after 5/6-nephrectomy, 24 rats were divided into pairs such that both rats in each pair exhibited almost the same levels of serum creatinine, blood urea nitrogen (BUN) (Table 1). One rat from each pair was assigned to (i) control uraemic group (n=12), the other to (ii) treatment uraemic group (n=12) which received HE-86, extract liquid which is effective composition isolated from Chinese medicine prescription, everyday at a dose of 0.75 g/100 g body weight for 8 weeks. For normal controls, rats underwent a sham operation consisting of laparotomy and manipulation of the renal pedicles but without damage to the kidney(n=12). The treatment group were administered by HE-86 infuse the stomach as pair-fed with the control uraemic rats, and the normal rats were fed ad libitum with standard solid chow (BiKai Animal Lab. Company, Shanghai, China) containing 24.5% protein.


Table 1. The variation of serum creatinine and blood urea nitrogen before treatment.

Blood pressure was measured before treatment and every two weeks after surgery. The levels of serum creatinine (Scr), Blood urea nitrogen (BUN), 24h urine protein excreation and urine TGF–β were determined at 4 or 8 weeks after starting the administration of HE-86, respectively. The remnant kidneys were removed after perfusion at the end of experiment for histopathological and gene expression studies.
