REVERSAL OF EXPERIMENTAL MYOGLOBINURIC ACUTE RENAL FAILURE WITH BIOFLAVONOIDS FROM SEEDS OF GRAPE

Abstract

Rhabdomyolysis may account for about 10% of all cases of acute renal failure (ARF). This study was performed to explore the protective influence of proanthocyanidins from seeds of grape in an experimental model of myoglobinuric ARF. Rats were injected with 50% glycerol (8 mL/Kg, im) followed immediately and daily in the next three days by ip proanthocyanidins (20 mg/kg) or saline. After 96 h rats were sacrificed and kidney morphology, kidney cortex peptidase activities, and malondialdehyde (MDA) content were determined. A moderate renal failure was produced by glycerol injection with blood urea of 31.8 ± 11.0 vs. 7.68 ± 0.24 mmol/L, and serum creatinine of 153.6 ± 38.2 vs. 39.6 ± 9.0 μmol/L, in glycerol-induced ARF vs. control rats, respectively. Rats that received proanthocyanidins in addition to glycerol had significantly lower (p < 0.01) blood urea and serum creatinine levels compared to those receiving glycerol alone. These functional differences between the glycerol and glycerol plus proanthocianidins groups were also confirmed histologically. Kidney cortex dipeptidylpeptidase IV (DPP IV) activity was not significantly changed in glycerol-induced ARF, however, markedly increased after proanthocyanidins treatment. Kidney cortex malondialdehyde content was found significantly increased in glycerol-induced ARF over control level, and was markedly reduced by proanthocyanidin treatment. Taken together, these results provide strong evidence for the protective role of proanthocyanidins from seeds of grape in glycerol-induced ARF. The effect is probably due to the antioxidant activity of proanthocyanidins and to increased expression of kidney cortex DPP IV with effective degradation of TNF-α. This may provide therapeutic opportunities of preventing and/or treating myoglobinuric ARF in humans.

• Acute renal failure
• Acute tubular necrosis
• Myoglobinuria
• Reactive oxygen species
• Flavonoids
• Protection

INTRODUCTION

Rhabdomyolysis, non-traumatic and traumatic, may account for approximately 10–15% of all cases of acute renal failure (ARF) [[1]]. A great deal of knowledge on myoglobinuric ARF has been obtained inexperimental studies. The most widely used model of myoglobinuric ARF is produced by subcutaneous or intramuscular injection of hypertonic glycerol [[2]]. In glycerol-induced ARF there is an enhanced generation of hydrogen peroxide, and scavengers of reactive oxygen metabolites and iron chelators provide protection [3-5]. Amelioration of glycerol-induced (ARF) was seen in the rat pretreated with saline loading, which has the effect of depleting the renal store of renin. The rats passively immunized against angiotensin II showed no rise in blood urea and no mortality after exposure to glycerol, whereas rats receiving inactivated antiserum and glycerol developed raised blood urea levels and a certain mortality [[6]]. The inactivation of angiotensin II can be caused by many enzymes such as: aminopeptidase A (APA), and aminopeptidase N (APN) [7-8]. APA removes the N-terminal Asp from the octapeptide angiotensin II and inactivate much of its pressor activity.

Among the naturally-occurring constituents of plants which appear to possess antioxidant activity are polyphenolyc compounds such as flavonoids. There is currently much interest in the antioxidant role of flavonoids found in tea, wine, fruit and vegetables. Recently, protection against renal damage in mice treated with ferric nitrilotriacetate by alpha G-Rutin, a water-soluble antioxidant flavonoid, was described [[9]]. It was suggested that alpha G-Rutin works as an antioxidant in vivo either by scavanging reactive oxygen species or by chelating ferric ions and that this serves to prevent oxidative renal damage in mice treated with ferric nitrilotriacetate. Protective effect of proanthocyanidins isolated from seeds of grape on adriamycin-induced cardiotoxicity was also recently described [[10]].

The present study was performed to evaluate protective effects of proanthocyanidins isolated from seeds of grape on renal morphological changes, kidney function, activity of some kidney ectopeptidases (APA, APN and DPP IV), and oxydative damage in glycerol-induced experimental ARF.

METHODS

Isolation of Proanthocyanidins from Grape Seeds

Grape seeds were extracted with ethylacetate containing 10% of water, as described previously [[11]]. The HPLC of the purified extract revealed mainly proanthocyanidins of the lower molecular mass: catechin, epicatechin, dimers (procyanidins B1, B2, B3, B4), and trimer (procyanidin C1). The relative content of the particular flavan-3-ols was: catechin 35.7%,epicatechin 24.2%, procyanidins B1-B4 27.4%, procyanidin C1 12.7%.

Induction of ARF

All experiments were performed on male Wistar rats weighing between 150 and 200 g. For renal morphological and functional studies, four groups of 7–9 animals each, were used, dehydrated 18 h before induction of acute tubular necrosis. Rats were injected with 50% (v/v in sterile water) glycerol (8 mL/kg, im) followed immediately and daily in the next three days by an ip proanthocyanidin (20 mg/kg), or saline. Control group received saline only. Thereafter rats had a free access to water and standard chow diet. After 96 hours each rat was anesthetized with sodium barbital (60 mg/kg, ip), blood sample was taken from abdominal vein, and subsequently the kidney was removed for histological investigations and enzyme assays.

Sample Analysis

Blood samples were assayed for blood urea nitrogen (BUN) and creatinine with a Synchron CX-3 analyzer (Beckman, USA).

Histology

For histological examination (light microscopy) the multiple renal cortical slices were fixed in Brasil-Bouin fixative for 3 hours, postfixed overnight in 10% buffered formalin and embedded in paraffin. Another set of tissue specimens was fixed in 4% gluteraldehyde, dehydrated in alcohol and embedded in Epon 812.

Enzyme Assays

All enzyme activities were determined in homogenates of the rat kidney cortex.

Aminopeptidase N activity was measured in phosphate buffered saline, supplemented with 1 mM MgCl₂, pH 7.4, and using 1.5 mM alanine p-nitroanilide as substrate [[12]].

Angiotensinase A activity was determined from the hydrolysis of 10 mM L-glutamyl-p-nitroanilide incubated in 50 mM Tris-HCl buffer, pH 8.0,containing 130 mM NaCl and 10 mM CaCl₂ [[13]]. Dipeptidylpeptidase IV activity was measured in 50 mM Tris-HCl buffer, pH 7.8, contaning 130 mM NaCl and 1 mM MgCl₂, with 1.5 mM gly-pro-p-nitroanilide as substrate [[14]].

Incubation was carried out at 37°C for 3–10 min with gentle agitation, under zero-order kinetic conditions. Enzyme reaction was stopped with 0.1 mL of 10% (w/v) trichloroacetic acid. The amount of p-nitroanilide formed was measured by reading at an OD of 405 nm. Enzyme activity was expressed as nmol of p-nitroanilide formed per min and per mg of protein.

Lipid Peroxidation

Malondialdehyde content, a measure of lipid peroxidation, was determined in a 10% (v/v) homogenate of kidney cortex in sodium phosphate buffer (pH 7.4), by using thiobarbituric acid assay [[4]]. Renal cortical malondialdehyde content was expressed as nanomoles of malondialdehyde per mg of protein.

Protein Determination

After appropriate digestion with 1 M NaOH protein was measured according to the method of Lowry et al. [[15]].

Statistical Analysis

All values are given as means ± S.E.M. Data were analyzed by one-way analysis of variance (ANOVA) followed, where appropriate with Student's t-test. Significance was judged by a p < 0.05.

RESULTS

Kidney Function

The kidney function data obtained are listed in table 1. Ninety-eight h after glycerol injection a moderate ARF was documented after blood urea of 31.8 ± 11.0 vs. 7.68 ± 0.24 mmol/L, and serum creatinine of 153.6 ± 38.2 vs. 39.6 ± 0.0 μmol/L in glycerol-induced ARF vs. control rats, respectively. Rats that received proanthocyanidin in addition to glycerol had significantly lower (p < 0.01) blood urea and serum creatinine levels than those receiving glycerol alone.

Table 1. Effect of bioflavonoids on kidney fucntion in glycerol-induced acute renal failure

Group	N	Blood urea mmol/L	Serum creatinine μmol/L
1. Control	7	7.68 ± 0.24	39.6 ± 9.0
2. Glycerol-induced ARF	8	31.8 ± 11.0^a	153.6 ± 38.2^a
3. Proanthocyanidins	9	6.22 ± 1.11	45.5 ± 9.5
4. Glycerol-induced ARF + Proanthocyanidins	8	13.0 ± 4.84^b	82.9 ± 18.7^b

^a vs. control and proanthocyanidins p < 0.001

^b vs. other groups p < 0.01

Histology

Glycerol-treated rats

Morphologic changes were registered primarily in proximal tubules in the subcapsular region of renal cortex (Fig. 1). Many of the proximal convoluted tubules showed severe tubular necrosis and tubulorhexis. The microvilli were often clumped and of variable length. Swelling of mesangial spaces and mesangial cells in glomeruli was evident, however, many of them appeared to be normal. In the boundary zone between cortex and medulla, the tubules were lined by epithelial cells with vacuolar and hydropic change. They lumen often contained granular material and exfoliated epithelial cells. Tubular epithelium was often flattened, and some tubules were dilated and contained pigment casts. Only focal areas of necrosis were present in this region. In the interstitum, focal mononuclear infiltration was observed. Within inner medullary collecting duct, close to the papillary tip, the presence of pigment casts was a prominent finding.

Figure 1. Renal morphology in rats treated with glycerol. Many of the cortical proximal tubules showed severe tubular necrosis with detachement of necrotic cells in the lumen. Necrosis of the tubular basement membrane (tubulorhexis) was present also. In the lumen of few proximal tubules myoglobin casts were present. Glomeruli showed only segmental sweling of the mesangial spaces. (semi-thin section, toluidin-basic fuchsin, x1000)

Glycerol- and proathocyanidin-treated rats

In subcapsular region of the renal cortex epithelial cells of proximal tubules showed focal necrosis and detachment in the lumen, with vacuolar and hydropic cell degeneration (Fig. 2). However, there were no tubulorexis, and no glomerular changes, observed in rats treated with glycerol only. In the corticomedullary zone tubules were often dilated, containing exfoliated cells and granular eosinophilic material. The interstitium showed only sparce mononuclear cell infiltration.

Figure 2. Renal morphology in rats treated with glycerol and proanthocyanidins. Many cortical proximal tubule epithelial cells showed focal necrosis and detachement in the lumen, and vacuolar and hydropic cell degeneration. The microvilli were often clumped. There were not tubulorhexis and glomerular changes. (semi-thin section, toluidin-basic fuchsin stain, x1000)

Proanthocyanidin-treated rats

In this group there were no morphologic changes in cortex and medulla, and renal histology was indistinguishable from control.

Kidney Peptidases

Kidney peptidase activities, APA, APN and DPP IV, although slightly decreased, were not significantly changed in glycerol-induced ARF (table 2). In rats with glycerol-induced ARF pretreated with BP1 significantly lower activity (p < 0.051 vs. control and proanthocyanidin) of APA and APN was demonstrated, however, DPP IV activity was significantly increased (p < 0.001 vs. glycerol-induced ARF).

Table 2. Effect of bioflavonoids on kidney cortex peptidase activities

Group	Aminopeptidase A nmol mg^−1 h^−1	Aminopeptidase N nmol mg^−1h^−1	Dipeptidylpeptidase IV nmol mg^−1h^−1
1. Control	14.0 ± 5.2	19.3 ± 4.8	19.8 ± 2.2
2. Glycerol-induced ARF	10.2 ± 2.6	15.4 ± 3.1	17.3 ± 2.0
3. Proanthocyanidins	14.2 ± 5.5	17.7 ± 2.7	20.6 ± 1.5^b
4. Glycerol-induced ARF + Proanthocyanidis	8.9 ± 2.6^a	13.5 ± 3.4^a	22.4 ± 3.0^c

^avs. control and proanthocyanidins p < 0.05

^bvs. glycerol-induced ARF p < 0.005

^cvs. glycerol-induced ARF p < 0.001

Lipid Peroxidation

Kidney cortex malondialdehyde content was significantly increased (p < 0.001) in glycerol-induced acute renal failure compared to untreated and proanthocyanidins treated rats (Table 3). Malondialdehyde content in rats treated with both glycerol and proanthocyanidins was reduced in comparison with glycerol alone, however, it was still over the level in untreated and proanthocyanidins treated rats.

Table 3. Effect of bioflavonoids on kidney cortex malondialdehyde content in glycerol-induced acute renal failure

Group	N	Malondialdehyde nmol/mg protein
1. Control	7	10.52 ± 2.23
2. Glycerol-induced ARF	8	17.35 ± 1.65^a
3. Proanthocyanidins	9	10.21 ± 0.77
4. Glycerol-induced ARF + Proanthocyanidins	8	14.23 ± 1.637-8^b,c

^avs. control and proanthocyanidins p < 0.001

^bvs. proanthocyanidins p < 0.01

^cvs. control and glycerol-induced ARF p < 0.05

DISCUSSION

The present study examines the effects of proanthocyanidins, extracted from seeds of grape, on glycerol-induced ARF. Proanthocyanidins afforded a significant morphological improvement, as well as an amelioration in kidney function as evidenced by significantly lower blood urea and serum creatinine levels in rats that received proanthocyanidins in addition to glycerol.

Medullary vasoconstriction, exacerbating intrarenal regional hypoxia, may play a role in myoglobin induced ARF [[16]]. Severity of acute tubular necrosis may be greatly increased by the combination of a relatively minor level of myoglobinuria with renal ischemia [[17]]. The deterioration in kidney function represents the combined effects of cortical damage, medullary hypoxic injury, and tubular obstruction [[16]]. The presence of large numbers of distal nephron hem pigment casts has been noted in every study of myoglobinuric ARF, including this one. Cast formation with marked dilation of the collecting ducts and focal tubular necrosis and rupture at the outer medullary region probably play a major role in the deterioration of kidney function [[16]].

Kidney cortex peptidases were not significantly changed upon glycerol treatment, which is consistent with focal subcapsular proximal collecting tubules necrosis and dilatation of cortical collecting ducts [[16]]. This is a quite different finding from uranyl nitrate-, mercuric chloride-, and gentamicin- induced ARF, exhibiting a direct nephrotoxic effect, and producing a marked decrease of kidney ectopeptidase activity [18-19]. However, proanthocyanidins in addition to glycerol produced a significant decrease of APA and APN activity, and an increase in DPP IV activity. Decrease of kidney cortex APA and APN upon addition of proanthocyanidins to glycerol-treated rats could have some important modulatory effect through inactivation of angiotensin II [[8]], further increasing the level of angiotensin II, with important consequences upon renal function and metabolism. The real meaning of this APA decrease in glycerol-induced ARF treated with proanthcyanidins cannot be explained at present, however, this could be a negative effect of proanthcyanidins treatment.

Severe muscle injury can activate endotoxin cytokine cascade, and TNF-α could have a direct role in pathogenesis of glycerol-induced ARF in rats. As TNF-α antiserum given prior to glycerol injection had significantly protected kidney function [[20]]. DPP IV could effectively degrade TNF-α [[21]], and thus increased expression of DPP IV in rats treated with proanthocyanidins in addition to glycerol could have a protective effect.These regulatory changes demonstrated in homogenates of the kidney cortex, probably do have marked expression at the particular stuctures of the kidney, glomeruli and some tubule segments, which deserves a further study.

Protective effect of proanthocyanidins in glycerol-induced ARF was also due to its antioxidant activity. The biochemical background for the antioxidative effect of flavonoids is inhibition of lipid peroxidation. Evidence is presented for enhanced generation of hydrogen peroxide in glycerol-induced ARF [[4]]. In 1988 Paller has noted that treatment with an iron chelator, deferoxamine (DFO), mitigated renal injury in iv hemoglobin-induced nephrotoxicity, glycerol-induced ARF, and a combined renal ischemic/hemoglobinuric insult [[22]]. Shah and Walker demonstrated that DFO and hydroxyl radiacal scavengers each protected against the glycerol ARF model [[3]]. Both of these studies reported a heme-induced increase in lipid peroxidation, as assessed by malondialdehyde generation. Manipulation in the rat's glutathione status altered the expression of heme-mediated injury. When rats were supplemented with glutathione they were partially protected against glycerol-induced ARF [[23]]. In the present study we have demonstrated decreased kidney cortex malondialdehyde in glycerol-induced ARF treated by proanthtocyanidins. Owing to the polyphenol structure of flavonoids, this inhibition can be brought about either by chelating of iron or by scavenging of free radicals with the formation of less reactive flavonoid aroxyl radicals.

Up-to date mostly the effects of flavonoids from wine were studied [[24]]. Epidemiological studies have revealed that Mediterranean diet, with flavonoids-rich food including the red wine, correlated with the increased longevity and decreased incidence of cardiovascular disease [[25]]. Flavonoids have been shown to protect low-density lipoproteins from oxidation (prevent atherosclerotic plaque formation), prevent platelet aggregation (antithrombic effect), and prevent cardiovascular smooth muscle relaxation (antihypertensive effect). In addition flavonoids have been shown to have antiviral and carcinostatic properties. Flavonoid intake in some circumstances may be involved in the cancer process, resulting in lowered risk [[26]]. Flavonoids in regulary consumed foods may reduce the risk of death from coronary heart disease in elderly men [[27]]. The habitual intake of flavonoids and their major source (tea) may protect against stroke [[28]]. Evidence is presented in this work that bioflavonoids from seeds of grape exhibit morphological and functional protection to the myoglobinuric ARF in the rats. Further studies may provide therapeutic opportunities of preventing or treating myoglobinuric ARF in humans.

ACKNOWLEDGMENT

Proanthocyanidins extracted from seeds of grape were kindly supplied by Professor B. Pekic, form the Faculty of Technology, Novi Sad, Yugoslavia, who prematurely passed away in 1998. This paper is dedicated to the late Professor Branislav Pekic.