Cisplatin-induced kidney injury in the rat: l-carnitine modulates the relationship between MMP-9 and TIMP-3

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Abstract

Renal interstitial fibrosis is a major complication of cisplatin treatment, due to the increased accumulation of extracellular matrix (ECM) proteins whose remodeling is important for the development of normal tissues; indeed, its malfunction might play a role in the etiology of various diseases.

Biopharmacological evaluations suggest that l-carnitine can prevent cardiac metabolic damage caused by doxorubicin, as well as can inhibit cisplatin-induced injury in the kidney and in the small intestine, without any interference with the drug's antitumoral properties. Since the glomerular basement membrane and the mesangial matrix constitute the ECM of the renal glomerulus, we examined the localization and expression of MMP-9 and TIMP-3 in normal rat kidney and the changes in their expression over a period of time by treatment with cisplatin, with and without l-carnitine. MMP-9 immunoreaction in cisplatin-treated rat kidney tissue suggests an involution of the basal membrane, an alteration of ECM components and low glomerular function, due to the increased thickness of the mesangium. Our results suggest that the matrix remodeling by MMP-9 and TIMP-3, in the later stages, can play an important role in the development of glomerular sclerosis and interstitial fibrosis after cisplatin treatment. It can also be postulated that l-carnitine protects from cisplatin injury, by modulating the relationship between MMP-9 and TIMP-3.

Introduction

Cisplatin (cis-diammine-dichloro-platinum) is an inorganic platinum compound with broad-spectrum antineoplastic activity against different types of human tumors. Nevertheless, both clinical and experimental studies report a dose-limiting nephrotoxicity, which restricts its clinical use in cancer chemotherapy because of its substantial side effects.

Renal interstitial fibrosis is a major complication of cisplatin treatment, due to the increased accumulation of extracellular matrix (ECM) proteins (Nazneen et al., 2002). Although several processes have been suggested to account for cisplatin-induced nephrotoxicity, the precise mechanisms underlying its pathogenesis are not yet completely understood.

Remodeling of the ECM is an important step in the development of normal tissues: therefore, its malfunction might cause different diseases. It has been suggested that matrix metalloproteinase (MMP) might play an important role in the EMC turnover, especially in the glomerulus. MMPs are a family of zinc-dependent endopeptidases that degrade structural components of the ECM (Zhao et al., 2004). MMP-9, particularly, is involved in the normal turnover of the kidney ECM (Kuroda et al., 2004) for its gelatinolytic activity as well as its degradative effect on type IV collagen. The complex regulation of these enzymes takes place at three levels: transcriptional regulation of the genes, activation of secreted proenzymes and regulation via specific inhibitors, (i.e. tissue inhibitors of metalloproteinases, TIMPs).

Since the first description of MMPs, their important role in ECM remodeling has been extensively investigated. For example, Limb et al. showed for the first time that MMP-1 has extracellular functions, and that itis also strongly associated with the mitochondria and nuclei and accumulates within the cells in the mitotic phase of the cell cycle (Limb et al., 2005). These observations strongly suggest that the intracellular association of MMP-1 with the mitochondria and nuclei confers resistance to apoptosis and may account for the well-known association of this enzyme with tumor cell survival and spread.

The Food and Drug Administration approved l-carnitine (beta-hydroxy-trimethylaminobutyric acid) in 1986 as an orphan drug for the treatment of primary l-carnitine deficiency. The drug can also be useful in the management of patients with conditions known to produce secondary l-carnitine deficiency, such as renal disease, cardiomyopathies, and ischemic cardiovascular disease. In addition, biopharmacological evaluations suggest that l-carnitine can be used to prevent doxorubicin-induced cardiac metabolic damage without interfering with its antitumoral activities (Sayed-Ahmed et al., 1999), as well as to inhibit cisplatin-induced injury of the kidney and small intestine (Chang et al., 2002).

Since the glomerular basement membrane and the mesangial matrix constitute the ECM of the renal glomerulus, we examined the localization and expression of MMP-9 and TIMP-3 in normal rat kidney. We also report the changes in their expression in a time-dependent cisplatin-induced nephrotoxicity model with and without l-carnitine treatment.

Section snippets

Animals

The study was conducted on Sprague–Dawley male rats (to avoid possible modifications of l-carnitine activity due to the hormonal cycle) (Constantin-Teodosiu et al., 2002), aged about 4 months, weighting 194.90±2.03 g, obtained from Stefano Morini (S. Polo D’Enza, Reggio Emilia, Italy). The number of rats required exceeded those necessary for the experimental tests. After their arrival, the animals underwent a veterinary check to verify their conditions of health and their weight, after which

Histological results

In contrast with the kidney samples obtained from control rats, sections of kidney which were treated with cisplatin showed micro- and macro-haemorrhages, intratubular cylindrical casts and local cellular alterations in the glomerular and tubular cell. In the sections treated with hematoxylin-eosin, with the Masson's trichrome stain and PAS reaction, areas of necrosis, pycnosis and hydropic degeneration and tubular lamina alterations were also detected. After 15 days of treatment with cisplatin

Discussion

This study was performed to elucidate the mechanism(s) of cisplatin nephrotoxicity and to establish whether l-carnitine treatment is capable of protecting the kidney from cisplatin-induced damage by directly inhibiting its onset or by interfering with molecular interactions, which are involved in the nephrotoxicity.

Under physiological conditions, the synthesis and degradation of the ECM is balanced by a fine regulation operated by the proteolytic matrix metalloproteinases (MMPs) and their

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