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Effect of Triethylenepentarninehexaacetic Acid on the Renal Damage in Cadmium-Treated Syrian Hamsters TOSHIAKISHIBASAKI,*Q.-Y. Xu, IWAOOHNO, FUMIO ISHIMOTO, AND O S A M U SAKAI

Second Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, Japan Received April 25, 1994; Revised June 9, 1994; Accepted August 3, 1994

ABSTRACT Cadmium (Cd)-induced nephropathy was treated by triethylenepentaminehexaacetic acid (TTHA) in male Syrian hamsters. Hamsters injected three times a week with 3 m g / k g body wt CdC12 showed proteinuria, urinary N-acetyl-~-D-inglucosaminidase (NAG), and fractional excretion of sodium (FENa) when compared to saline-injected control. Cd-treated hamsters injected ip with TTHA 10 m g / k g body wt five times a week showed reduction of renal damage, including reductions in urinary protein (from 6.7 + 2.2 to 4.3 _+ 0.5 m g / d ) and NAG (0.17 + 0.06 to 0.04 _+ 0.02 U/d). Urinary excretion of Cd was significantly increased (from 87 + 51.3 to 3052 _+1485 mg/L) by TTHA administration. Cd concentration in renal cortical tissue was slightly reduced (26.4 _+ 3.0 to 21.8 _+ 2.7 mg/g. protein). Excretion of malondialdehyde (MDA) was increased only in Cd-injected hamsters (to 2.1 _+ 1.6 nM/L), and elevated MDA in renal cortical tissue was not reduced by the administration of TTHA (1041 _+ 105 vs 1104 _+ 358 n M / g protein). Glutathione (GSH) concentration in the renal cortex was significantly elevated after Cd administration and further increased after TTHA administration (5.5 + 2.1 to 9.8 _+ 2.0 ~tg/50 mg protein). There were no marked effects on creatinine clearance (Ccr) and hematocrit. Moreover, renal morphological changes were improved significantly by treatment with TTHA. We demonstrated the efficacy of TTHA in the treatment of Cdinduced nephropathy in hamsters. Although the precise mechanism *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research

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of the TTHA effects on Cd-induced nephropathy has not been elucidated, it might involve GSH reducing the elevated MDA concentration in renal tissue. Index Entries: Cadmium-induced nephropathy; Syrian hamster; triethylenepentaminehexaacetic acid (TTHA); N-acetyl-~-D-glucosaminidase (NAG); malondialdehyde (MDA); glutathione (GSH).

INTRODUCTION Cadmium (Cd) is recognized as a highly nephrotoxic heavy metal that sometimes induces irreversible damage in humans and animals (1). In general, proximal tubular damage is revealed by increased proteinuria or urinary lysosomal enzymes, such as N-acetyl-~-D-glucosaminidase (NAG), alanine aminopeptidase (AAP), and so forth. Although there are many animal models for evaluating the mechanism or treatment of Cdinduced nephrotoxicity (2,3), Rehm et al. stressed the suitability of Syrian hamsters as a model system (4). Furthermore, we have suggested that this model may be used to evaluate proximal renal tubular function, since marked morphological changes in the kidney appear after a single injection of CdC12 3 m g / k g body wt (5). However, the precise onset mechanisms of nephrotoxicity owing to Cd remain unclear. In Cd nephropathy in rats, it was suggested that increased malondialdehyde (MDA), the final product of lipid peroxidation, in the renal cortex may play a role in inducing renal damage. In the treatment of Cd-induced nephrotoxicity, we have reported the efficacy of polyaspartic acid, polyamino acid, or pentoxifylline in preventing cerebral accidents (6). Although chelating agents are an ideal treatment for Cd toxicity in humans and animals, clinical use has been limited because of potential adverse effects of the chelators (7,8). Triethylenepentaminehexaacetic acid (TTHA), one of these chelating agents, is thought to be the most potent chelator for heavy metal poisoning. We tried using TTHA for the treatment of Cd-injected hamsters in order to evaluate its efficacy and precise mechanisms.

MATERIALS AND METHODS Animals Twenty-three male Syrian hamsters (110-130 g body wt), purchased from Sankyo Lab. (Shizuoka), were used for the present study. These hamsters were given free access to normal chow and tap water in regular cages in an air-conditioned room. The hamsters were divided into the following four groups: seven Cd-treated; five TTHA-treated; five Cd plus TTHA-treated; and six saline-treated controls.

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Experimental Design CdC12 and TTHA, purchased from Sigma Co. (St Louis, MO), were dissolved in 1 mL of saline for injection. CdC12 (3 m g / k g body wt) was injected sc on days 1, 3, and 5, and TTHA (10 m g / k g body wt) was injected ip daily for 5 d. Body weight was checked on the initial and final days of the experiment. At the final observation, each hamster was placed in a metabolic cage (Sugiyama Co., Tokyo) for 24 h for total urine collection into an ice-cold container to prevent the degradation of urinary enzymes. Furthermore, the glomerular filtration rate was determined by creatinine clearance. Hamsters were then bled and nephrectomized under ether anesthesia for blood chemical analysis and renal morphological study. Urinary protein and NAG were determined by Tonein's and MCP-NAG methods, respectively (9,10).

MDA and GSH Determinations The mechanism of renal damage was investigated by the determination of MDA, the final product of lipid peroxidation, in the urine and renal cortex at the final observation on day 6. MDA was determined by the method of Ohkawa et al. (11). GSH was measured using Owens & Belcher (12) to evaluate the scavengering effect against lipid peroxidation in the renal cortex.

Cd Determination Cd concentrations in the urine and renal cortex were measured at the final observation on day 6 to evaluate the effect on renal damage. Cd was assayed by a flame photometer. The concentration of Cd in the kidney was expressed as m g / g wet tissue weight.

O t h e r Parameters To evaluate renal function, 24-h creatinine clearance (Ccr) was measured in hamsters in the metabolic cages, and sodium (Na) in urine and serum were determined to establish fractional excretion of Na (FENa). Serum creatinine and electrolytes were determined by an autoanalyzer, respectively.

Morphological Analysis At the final observation on day 13, renal morphology was examined by means of an optical microscope. Renal tissue specimens were stained with hematoxylin-eosin (HE), Periodic-Acid-Schiff (PAS) and Masson staining methods. Furthermore, renal morphology in some of the hamsters was examined by electron microscopy.

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Statistical Analysis Data were expressed as mean _+standard deviation (SD). The results obtained were analyzed using Student's t-test, and the significance level was set at p < 0.05.

RESULTS Table 1 shows the laboratory profiles for the present study. Although body weight was changed slightly during this brief experiment, there were no significant differences between the Cd-injected group and the other three groups. Only Cd-injected hamsters showed a significant reduction in daily urine volume, and this phenomenon had been reported previously in hamsters. Urine protein and 24-h NAG showed significant increases after Cd administration and improved after TTHA administration. Ccr and FENa were reduced by Cd administration and showed no improvement after TTHA treatment. There were no differences among these four groups in red blood cell (RBC) counts and liver function (data not shown), indicating that there was no bone marrow suppression or liver damage due to Cd or TTHA. To clarify the mechanisms by which TTHA reduced proteinuria and NAG excretion, MDA and Cd concentrations in urine and renal cortical tissue were determined (Table 2). Mean urinary excretion of Cd was 87 _+ 51.3 m g / L in the Cd-injected group, and this value was markedly enhanced by the addition of TTHA (3052 _+ 1485 mg/L). In contrast, the concentration of Cd in the renal cortical tissue showed no difference between Cd-injected and Cd plus TTHA-treated hamsters. Furthermore, GSH concentrations in the renal tissue of Cd-treated hamsters were higher than those in controls (5.5 +_ 2.1 vs 1.6 + 0.7 Bg/50 mg. protein). However, the addition of TTHA further increased the level of GSH (9.8 _+ 2.0 ~tg/50 mg. protein). Cd increased the concentration of MDA in renal cortical tissue, but the elevation in MDA excretion owing to Cd was reduced by the addition of TTHA. Figure 1 shows the morphological appearance of the kidney. Proximal tubules in Cd-administered hamsters showed marked degeneration and necrosis in spite of only a slight reduction in glomeruli. TTHA modified the epithelial changes caused by Cd administration. These morphological changes were confirmed by electromicroscopic findings (data not shown).

DISCUSSION Our model of Cd-induced nephrotoxicity in hamsters is evaluating renal proximal tubular damage both functionally phologically (5,13). In spite of the brief duration of the present hamsters showed renal damage owing to Cd administration,

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Fig. 1. Renal light microscopical changes. Proximal tubules in Cd-administered hamsters showed marked degeneration and necrosis in spite of only a slight reduction in glomeruli (A). TTHA modified the epithelial changes caused by Cd administration (B).

increased urinary excretion of protein and NAG. Thus, Syrian hamsters appear to be an excellent model for studying Cd-induced nephrotoxicity. Moreover, administration of TTHA, a chelating agent, appears to improve renal dysfunction in this model. Furthermore, TTHA reduced the Cd content in renal cortical tissue by enhancing Cd excretion in the urine.

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Although there are many types of chelating agents for the treatment of heavy metal toxicity, few have proven to be appropriate for clinical use (14), because these agents may have severe side effects, such as bone marrow suppression, or liver or kidney damage, and can have lethal effects even at low doses. Chelators include such well-known agents as TTHA, hydroxyethylenediaminetriacetic acid (HEDTA), ethylenediaminetetraacetic acid (EDTA), cyclohexanediaminetetraacetic acid (CDTA), and diethylenetriaminepentaacetic acid (DTPA). TTHA has the mildest adverse effects and chelating potency compared to the others (7). In the present study, there was no evidence of cytotoxicity or liver damage in hamsters. Furthermore, renal damage was not induced by TTHA administration. These results suggest the possibility of clinical use of TTHA in the treatment of humans. However, there are still many problems to be resolved in the clinical use of these chelating agents. The use of chelating agents must be limited to avoid marked potential adverse effects. In the present study, we also used another chelating agent, EDTA (10 m g / k g body wt), but most hamsters treated with this agent died because of marked toxicity (data not shown). EDTA is usually utilized in vitro and cannot be used clinically. Agents that induce renal proximal tubular damage, such as aminoglycosides or penicillin antibiotics, usually cause polyuria and elevated FENa, which mean the precise proximal tubular function (15). In rats injected with Cd, renal proximal tubular damage could not be detected by means of polyuria or increased FENa and urinary enzymes (NAG or AAP) (5). The mechanisms leading to reduced urine volume or FENa are unknown, but other evidence for Cd toxic effects on tubular handling might exist. The different results seen in Cd-administered hamsters may be related to the observed histological changes, but further study is needed to understand the precise mechanisms. Although animal models, such as mice, rats, or rabbits, are frequently used for the study of Cd nephrotoxicity, this is the first report evaluating a chelating agent for Cdinduced nephrotoxicity in hamsters. These animals provided an excellent means of evaluating Cd-induced nephrotoxicity and the effect of chelating agents because of stable responses with respect to renal morphological and chemical changes. Although there are no reports on the influence of lipid peroxidation in Cd nephrotoxicity in animal models, previous data showed that renal proximal tubule cell death was induced by lipid peroxidation (16) producing halokene cysteine conjugates, an oxidant substance. This evidence suggests that cellular damage in proximal tubules might be induced by the activation of lipid peroxidation. In the present study, the MDA concentration in Cd-treated hamsters was elevated in the renal cortex. Cd may have induced renal tubular damage by accelerating lipid peroxidation, this hypothesis was supported by the simultaneous activation of GSH production in the renal cortex, which is a major scavenger of MDA.

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Thus, the stimulation of GSH may have enhanced repair of the renal tubular damage caused by MDA activation in renal cortical tissue. Electronmicroscopic analysis in a hamster model by Rehm et al. and in our study showed marked changes shortly after the injection of C d and provided an excellent means of evaluating Cd toxicity. In summary, TTHA showed excellent efficacy in chelating Cd from the kidney in hamsters. However, the mechanism of action of Cd-chelating agents in renal tissue and the potential for clinical usage must be investigated further.

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