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The American Journal of Chinese Medicine, Vol. 33, No. 3, 491500

2005 World Scientific Publishing Company

Institute for Advanced Research in Asian Science and Medicine

491

The Nephroprotective Effects of the Herbal

Medicine Preparation, WH30+, on the

Chemical-Induced Acute and Chronic

Renal Failure in Rats

Heidi H.Y. Ngai, Wai-Hung Sit and Jennifer M.F. Wan

Division of Food and Nutritional Sciences, Department of Zoology

The University of Hong Kong, Hong Kong SAR, P.R. China

Abstract: In this study, we evaluated the renal protective effects of a Chinese herbal preparation

WH30+ in male Wistar rats with glycerol-induced acute renal failure and adenine-induced

chronic renal failure. WH30+ is a Chinese herb preparation composed ofRheum Palmatum,

Salvia Miltiorrhiza, Cordyceps Sinensis,Leonurus Sibiricus,Epihedium Macranthum,Radix

Astragali, andRadix Codonopsis Pilosulae, which has been used to treat kidney deficiency in

human. An acute renal failure and chronic renal failure rat model were introduced by glycerol

injection (i.m.) and fed with adenine-excessive diet, respectively. WH30+ was administered to

rats at the dose of 50 mg/kg/day from 10 days before the diseases were induced until the rats

were sacrificed. A reduction in body weight (p < 0.01) was observed in rats with chronic renal

failure, but there was no difference between treatment groups. However, the body weight of

rats with acute renal failure without treatment was significantly lower than those treated with

WH30+ (p < 0.05). Overall, serum creatinine and urea nitrogen were elevated significantly

(p < 0.01) in renal failure rats compared to control. Treatment with WH30+ improved both

serum creatinine and urea nitrogen slightly in both models. The WH30+-treated rats with

acute renal failure had significantly (p < 0.05) greater creatinine clearance than those without

treatment. The results of the study show that WH30+ is more effective in the prevention of

acute renal failure than chronic renal failure.

Keywords: Chinese Herb Preparation; WH30+; Rheum Palmatum; Salvia Miltiorrhiza;

Cordyceps Sinensis; Adenine; Glycerol; Chronic Renal Failure; Acute Renal

Failure.

Correspondence to: Dr. Jennifer Wan, Department of Zoology, Kadoorie Biological Sciences Building, The

University of Hong Kong, Hong Kong SAR, P.R. China. Tel: (+852) 2299-0838, Fax: (+852) 2559-9114, E-mail:

[email protected]

by203.24.50.25on02/13/13.Forpersonaluseonly.

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H.H.Y. NGAI et al.492

Introduction

Chinese medicinal herbs have been used by the Chinese population since ancient times.

This study suggests that some herbs may be a potential source of pharmaceutical remedies

for renal disease prevention. The WH30+ used in this study is a commercial name for

the extract of Chinese herbal preparation composed of seven medicinal herbs, including

Rheum Palmatum, Salvia Miltiorrhiza, Cordyceps Sinensis,Leonurus Sibiricus,Epihedium

Macranthum,Radix Astragali, andRadix Codonopsis Pilosulae. When studied individually,

these ingredients have been shown to be useful in improving general health, particularly

kidney functions (Cai et al., 2001; Chen and Kwan, 2001; Ji et al., 2003; Yokozawa et al.,

1997; Zhang and el Nahas, 1996). However, there is no sufficient evidence to explain the

nephroprotective effects of these herbs in combined formulation. In the present study, we

aimed to evaluate the effects ofWH30+

on (1) the prevention of acute renal failure (AFR)and (2) the disease progression of chronic renal failure (CRF). An acute renal failure and

chronic renal failure model were introduced in rats by glycerol injection intramuscularly

and feeding with adenine-excessive diet, respectively. The model of glycerol-induced renal

failure shares many similarities to human myoglobinuric acute tubular necrosis associated

with muscle trauma and massive hemolysis (Holt and Moore, 2000). The model of adenine-

induced renal failure produces metabolic abnormalities which suppress the excretion of

nitrogen compounds by renal tubular occlusion resembling chronic renal failure in humans

(Yokozawa et al., 1986). This is the first report to demonstrate that WH30+ possesses

certain preventive effects on acute renal failure.

Materials and Methods

Preparation of WH30+ Extract

The WH30+ preparation (obtained from the Herbs Products Ltd, Hong Kong) was dissolved

in distilled water at concentration of 0.07% (700 mg/l). Daily water intake of rats was

monitored and concentration of treatment extract was adjusted accordingly. All extracts

were prepared fresh at the day of feeding. The recommended daily dose of WH30+ to a

human adult is 2100 mg; therefore, dose of 50 mg/kg to rats was calculated by assuming

the average body weight of a normal adult as 42 kg.

Animals and Treatments

Male Wistar rats initially weighing 290390 g were used in the study. All animals were

obtained from the Animal Unit, The University of Hong Kong. Rats were housed in

environmentally controlled conditions with a 12-hour light/dark cycle. All animals had

free access to standard rodent pellet food and water ad libitum. Animals in treatment

groups were administered with the Chinese herbal extract WH30+ at the dose of

50 mg/kg/day 10 days before the diseases were induced and continued until the rats weresacrificed. A total of 42 rats were randomly divided into five groups: (1) Control (Ctr)


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493NEPHROPROTECTIVE EFFECTS OF HERBAL MEDICINE

(n = 7) was given normal rat chow and drinking water throughout the experiment;

(2) rats with ARF (Gly) (n = 7) were injected 10 ml/kg glycerol (50% v/v in sterile saline)

intramuscularly after a 24-hour period of dehydration at day 10 of experiment; (3) ratswith ARF and WH30+ (Gly-WH) (n = 8) were injected glycerol as above and received

WH30+ throughout the experiment; (4) rats with CRF (Ade) (n = 10) were given adenine-

excessive diet (0.04%) for 28 days at day 10 of the experiment; and (5) rats with CRF and

WH30+ (Ade-WH) (n = 10) were given adenine-excessive diet as above and pre-treated

with WH30+ in drinking water until the end of the experiment. Rats in the control and

adenine-treated (Ade and Ade-WH) groups were sacrificed on the 38th experimental day,

while rats in the ARF (Gly and Gly-WH) groups were sacrificed on the 12th day, a day

after glycerol injection.

Determination of Serum Creatinine and Urea Nitrogen Levels and

Glomerular Filtration Rate

The blood was collected between 10:00 am to 12:00 pm. Each blood sample was centrifuged,

and the serum was stored at 40C until measurement. Serum creatinine (SCr) and blood

urea nitrogen (BUN) were measured by a standard spectrophotometric method. Samples

were run in duplicate in a single assay. Glomerular filtration rate (GFR) was determined

by measuring the renal clearance of creatinine over a 24-hour period and the following

formula was used:

CCr (ml/min) = UCr V SCr1

where UCr = urine creatinine (M), V = volume of urine (ml/min), and SCr = serum

creatinine (M).

Histological Analysis

The removed kidneys were fixed overnight in Dubosq-Brazil, dehydrated in alcohol, and

embedded in paraffin. Kidney samples were sectioned with 4 m intervals and the sections

were stained with hematoxylin and eosin (H&E). The histological profile of 200 glomeruli

randomly selected per rat was recorded using a Leica Qwin Image Analyzer (Cambridge,UK). The extent of glomerular damage was expressed as the percentage of glomeruli

presenting sclerotic lesions. All renal biopsies were analyzed by the same examiner who

was unaware of the nature of the experimental groups.

Statistical Analysis

Survival data are presented in the form of survival curves, and they were analyzed using a

log rank test. All other values are expressed as mean standard error. Paired and unpaired

Students t test was used to analyze the statistical difference between treatment and control

groups. The level of difference at p < 0.05 was considered significant.


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Results

Body Weight Change

The body weight of both the disease groups was significantly lower than that of the

control. Only little difference was observed between the Ade and Ade-WH group in all

measurements. However, body weight gain was improved with WH30+ treatment in the

Gly-WH group compared to the Gly group (p < 0.05) as shown in Fig. 1. Daily water intake

of all rats was measured and it appeared to be constant and approximately at 3050 ml

daily. Edema was determined by measuring the serum albumin and there was no difference

between groups (data not shown). This finding indicates that severe edema was not present

in disease groups and thus the body weight measured reflects the true body weight lost.

Survival Rate

As shown in Fig. 2, the survival rate of rats (57%, 4 of 7) receiving WH30+ treatment with

glycerol-induced acute renal failure was significantly greater (p < 0.05) than that of the rats

without WH30+ treatment (14%, 1 of 7). They died 3 days after injection of glycerol.

Biochemical Analysis

On day 0, the baseline levels of serum creatinine and urea nitrogen were similar among

groups. After the induction of renal failures, the levels of both SCr and BUN were increasedsignificantly (p < 0.01). As shown in Table 1, after induction of ARF by glycerol at day

12, serum levels of creatinine and urea nitrogen in WH30+-treated rats were considerably

Figure 1. Body weight change of rats in different treatment groups. The difference was made between days 0

and 12 in Gly and Gly-WH treated rats, days 0 and 38 of Ctr, Ade and Ade-WH treated rats. Ctr: Control group

(n = 7), Gly: glycerol-treated group (n = 7), Gly-WH: glycerol with WH30+-treated group (n = 8), Ade: adenine-

treated group (n = 10), and Ade-WH: adenine with WH30+

-treated group (n = 10).**

p < 0.01 versus control,#p < 0.05 versus glycerol-treated group.

-60

-35

-10

15

40

65

90

Bo

dy

We

ig

ht

Change

(g

)

#

** **

Ade Ade-WH

Ctr Gly Gly-WH


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Figure 2. Survival rate of rats with acute renal failure after injection of glycerol. Gly-WH: Glycerol with WH30+-

treated group and Gly: glycerol-treated group. *p < 0.05 between treatment groups.

0

20

40

60

80

100

0 1 2 3

*

Day

Surv

iva

l

Rate

(

%) Gly

Gly-WH

Table 1 . Levels of Serum Creatinine (SCr) and Blood Urea Nitrogen (BUN)

of Rats with Acute Renal Failure at Day 12

Treatment Group SCr (mg/dl) BUN (mg/dl)

Ctr 2.18 0.18 35.77 1.96

Gly 5.22 0.58* 367.31 15.26

Gly-WH 4.29 0.61* 339.29 22.82

Data are expressed as mean SEM. Ctr: Control group (n = 7), Gly: glycerol-

treated group (n = 7), and Gly-WH: glycerol with WH30+-treated group (n = 8).

Table 2. Levels of Serum Creatinine (SCr, in mg/dl) (A) and Blood Urea Nitrogen (BUN, in mg/dl) (B) of

Rats with Chronic Renal Failure at Different Experimental Days

(A)

Day

0 30 38

Ctr 1.93 0.25 1.62 0.29 1.72 0.05

Ade 2.24 0.24 4.50 1.30* 5.29 0.77*

Ade-WH 2.16 0.30 5.68 1.07* 6.43 0.46*

(B)

Day

0 20 30 38

Ctr 33.27 1.54 38.71 4.02 40.86 6.12 41.57 4.14

Ade 35.78 1.33 151.09 8.62 262.89 35.62 367.81 34.19

Ade-WH 32.73 2.65 131.15 13.32 264.90 12.73 364.24 36.74

Data are expressed as mean SEM. Ctr: Control group (n = 7), Ade: adenine-treated group (n = 10), and Ade-WH:adenine with WH30+-treated group (n = 10). *,p < 0.05, 0.01 versus control.

Treatment

Group

Treatment

Group


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Figure 3. The glomerular filtration rate (ml/min) in each treatment group. Ctr: Control group (n = 7), Gly:

glycerol-treated group (n = 7), and Gly-WH: glycerol with WH30+-treated group (n = 8). **p < 0.01 versus control,#

p < 0.05 versus glycerol-treated group without WH30+

.

0

0.4

0.8

1.2

1.6

Glomeru

lar

Filtrat

ion

Ra

te

(m

l/m

in

)

**

** #

Ctr Gly Gly-WH

lower than those without WH30+ treatment. For the rats with adenine-induced CRF, the

levels of SCr and BUN on days 20, 30 and 38 were significantly increased compared with

that at day 0 and control (Table 2A and 2B). However, there were no significant differences

between treatment groups. In the ARF group, glomerular filtration rate was significantly

improved (p < 0.05) in rats treated with WH30+ (0.15 0.04 and 0.04 0.02 ml/min)

(Fig. 3).

Structural Damage in the Kidneys

The morphological changes in rats with glycerol-induced renal failure were registered

primarily on the proximal tubules in the subcapsular region of the renal cortex. Many

of the proximal convoluted tubules showed severe tubular necrosis and tubulorhexis

(Fig. 4B). In some glomeruli, there was swelling of the mesangial spaces and mesangial

cells (Fig. 4C), however, many of them appeared to be normal in WH30+ treated rats.

Crystal deposition in glomeruli, tubules and interstitium were observed in rats with

adenine-induced CRF. The extent of renal damage and crystal deposition was more severe

in Ade group compared to the Ade-WH group (Figs. 4C and 4D). There was 10.75% ofglomerulosclerosis found in the kidney section of rats in the Ade-WH group, while there

was 21.83% glomerulosclerosis in the Ade group (p < 0.05).

Discussion

The results from the present study reveal that WH30+ improves survival rate and to some

extent prevents renal damage after induced acute renal failure. However the preventive

effect ofWH30+ seems to be less effective in chronic renal failure in a rat model.

Glycerol-induced renal injury is a well-established model of acute renal failure in

rat (Holt and Moore, 2000). This model shares many similarities to human acute tubular


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necrosis associated with muscle trauma and massive hemolysis. Intramuscular injection of

glycerol causes rhabdomyolysis and subsequent deposition of myoglobin in the kidneys

which in turn leads to renal vasoconstriction and acute tubular necrosis (Zager, 1996).

Morphologically, there is extensive renal tubular cell necrosis and plugging of tubules with

casts and heme crystals. There was lesser cast formation observed in the rats treated with

WH30+, which indicates the improvement of renal morphological changes during acute

renal failure. In fact, strategies to prevent acute renal failure practically aim at restoring

renal perfusion and minimizing renal tubular epithelial cell injury. When rats suffered from

glycerol-induced ARF, their renal blood flow, glomerular filtration rate, and urine output

are reduced. Much of these renal damages are believed to result from ischemia.

Savic et al. (2002) showed that pentoxifylline, a methylxantine, decreased the

BUN and SCr of glycerol-treated rats significantly (p < 0.001). They suggested that

the protective effects of pentoxifylline may be due to enhanced local blood flow and

inhibition of kidney extracellular matrix synthesis and myofibroblastic differentiation.

In our study, administration ofWH30+ prior to glycerol injection also improves survival

and prevents total renal failure. This may be due to the fact that the main ingredient of

WH30+, Rhuem Palmatum, helps maintain renal blood flow and limit ischemic damage.

The study of Yokozawa et al. (1991) demonstrated that tannins in Rhuem Palmatum

reduce levels of uremic toxins and improve glomerular filtration and blood flow to the

kidneys in experimental animals. Moreover, glomerulosclerosis has also been shown to be

reduced after aqueous extract ofRheum Palmatum was administered to rats that underwent

subtotal nephrectomy compared to those given only plain water (Zhang and el Nahas,

1996). Another study in rats with diabetic nephropathy found that Rheum Palmatum

extract speeded nitrogen excretion and alleviated hyperlipidemia compared to control rats(Yokozawa et al., 1997). Though more work is needed to determine the exact mechanisms

Figure 4. Hematoxylin and eosin stain of renal section. (A) Normal healthy rat, no lesions are visible ( 40).

(B) Glycerol-treated group, tubular necrosis ( 40). (C) Glycerol with WH30+-treated group, swelling glomerulus

( 40). (D) Adenine-treated group, severe glomerulosclerosis ( 20). (E) Adenine with WH30+-treated group,

scattered glomerulosclerosis ( 10).

A B C

D E


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ofRheum Palmatum extracts on the kidney, the existing data strongly suggest that Rheum

Palmatum, an ingredient in WH30+, exerts renal protective effects on kidney lesions by

glycerol injection.In addition to Rheum Palmatum, other ingredients in WH30+ may also work

synergetically to protect the kidneys from ischemic damage. As demonstrated by the

study of Ji et al. (2003), Cordyceps Sinensis increased the activities of antioxidant defense

enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase

(GSH-Px), and glutathione S-transferase (GST) in the liver of rats. The findings indicate

that Cordyceps Sinensis can scavenge various free radicals effectively from different sites

of antioxidant systems through enhancing the activities of the antioxidant enzymes in rats

including the kidneys. Also ingredients like Leonurus (Chen and Kwan, 2001) andRadix

Astragali (Cai et al., 2001) were shown to have vasorelaxation effects and protect kidney

against ischemic insult and accelerate both functional and histological recovery after acuterenal ischemia or reperfusion injury.

Surprisingly, in spite of the main ingredientRheum Palmatum has long held an esteemed

place in traditional Chinese herbalism as part of treatment protocols for patients with

CRF, little difference was observed between the groups who received WH30+ treatment

and no treatment of rats with adenine-induced CRF. In fact, Rhuem Palmatum has been

investigated in preliminary clinical trials in China and shown to have beneficial effects

on symptoms, such as blood urea nitrogen and serum creatinine levels in CRF patients

(Yarnell, 2002).

The model of adenine-induced chronic renal failure is developed by Yokozawa et al.

(1986) in which long-term feeding of adenine to rats produced metabolic abnormalities

resembling chronic renal failure in humans. In mammalian metabolism, when it is present

in excess, adenine becomes a significant substrate for xanthine dehydrogenase (XDH),

which can oxidize adenine to 2,8-dihydroxyadenine (DHA) (Stockelman et al., 1998).

Because adenine and DHA have very low solubilities, they lead to precipitation in the

tubules of the kidney.

Adachi et al. (1998) showed that adenine-rich diets increase blood urea nitrogen and

serum creatinine by decreasing the urinary excretion of these substances, because excretion

of nitrogen compounds is suppressed by renal occlusion due to DHA. Deng et al. (1998)

also pointed out that the disease progression of adenine-induced chronic renal failure is

time dependent the longer the feeding time, the more severe the disease. The unexpected

results ofWH30+ in this study may be due to the fact that the kidneys were too damaged

by the adenine diet for 38 days.

In SD rats, Deng et al. (1999) showed that a Chinese medicine Tongfugushen improves

the symptoms of adenine-induced chronic renal failure by decreasing the BUN and SCr

levels significantly (p < 0.01). The main ingredients of Tongfugushen are similar to the

WH30+. However, they sacrificed the rats at 14 days after induction of the adenine diet.

In addition, the dosage of medicinal herbs used may also be an important factor. In the

study of Deng et al. (1999), Tongfugushen at a dose of 13 g/kg was used, whereas in our

study only WH30+

at 50 mg/kg body weight was used. In another study from Wang andOura (1994), they showed that a dry extract of Chinese medicinal herb Herba Ephedra


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at a daily dose of 30 mg also significantly decreased the levels of BUN and SCr of rats

with adenine-induced chronic renal failure 24 days after feeding on the herb. Therefore, a

larger dose might be needed for WH30+

to exert its renal protective effect against adenine-induced chronic renal failure.

However, it is clear from the histological examinations that WH30+ significantly

ameliorated the kidney damage in rats with adenine excessive diet by reduced formation

of glomerulosclerosis in the renal tissues. Whether the preventive effect is related to the

increased renal blood flow by WH30+ remains to be determined.

In summary, the findings of this study indicate that the Chinese medicinal preparation

WH30+ may have protective effects against renal injury in rats with chemical-induced renal

failure. Future in vivo-guided chemical fractionation method is needed to identify which

component(s) in the ingredient are responsible for the observed effects.

Acknowledgments

This work is supported by the Hong Kong Association for Health Care, Hong Kong, and

CRGC grants, The University of Hong Kong.

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