Elsevier

Toxicology

Volume 165, Issues 2–3, 28 August 2001, Pages 205-216
Toxicology

Subchronic cadmium treatment affects the abundance and arrangement of cytoskeletal proteins in rat renal proximal tubule cells

https://doi.org/10.1016/S0300-483X(01)00450-4Get rights and content

Abstract

Disfunction of proximal tubules (PT) in cadmium (Cd) nephrotoxicity in mammals results from the diminished functional capacity of brush-border membrane (BBM) caused by (a) direct inhibition of BBM transporters by Cd, (b) shortening and loss of microvilli, and (c) loss of specific BBM transporters. The loss of transporters may partially result from impaired intracellular vesicle recycling due to loss or/and inhibition of vacuolar H+-ATPase in the PT cell organelles. Cytoskeleton plays an important role in vesicle-mediated recycling and processing of BBM transporters in PT cells. Experiments in vitro have indicated that Cd may affect the state of polymerization of some cytoskeletal proteins. In this work we studied the in vivo effect of CdCl2-treatment in rats (2 mg Cd/kg b. m., s.c., daily for 14 days) upon abundance and arrangement of actin filaments, actin-bundling protein villin, and microtubules (MT) in PT cells. Cd-treatment elicited a dramatic accumulation of Cd in the kidney cortex (200 μg/g tissue wet mass after 14 days) and a strongly increased abundance of metallothionein in PT cells. As revealed by immunocytochemistry in tissue cryosections, the staining intensity of actin and villin in PT cells of Cd-treated rats was generally decreased, without a marked change in their intracellular distribution, whereas MT became largely irregular, diminished in most cells, and lost in many cells. However, the immunoblots revealed an increased content of villin and α-tubulin in cortical tissue homogenates from Cd-treated rats, thus indicating an impaired bundling of actin and greatly depolymerized MT in cells intoxicated with Cd. The partial loss of apical actin and villin in PT cells of Cd-treated rats may reflect (or cause) shortening and loss of microvilli, whereas derangement and depolymerization of MT may contribute to the impairment of intracellular recycling of BBM proteins, and lead to the loss of BBM transporters.

Introduction

Nephropathy due to chronic exposure to cadmium (Cd) in man and experimental animals is manifested by reabsorptive and secretory disfunctions of renal tubules. The main symptoms of tubular damage, that include proteinuria, phosphaturia, glucosuria, aminoaciduria, and hyperosmolar polyuria (Adams et al., 1969, Kim et al., 1988, Kim et al., 1990, Lee et al., 1991, Ahn and Park, 1995, Herak-Kramberger et al., 1996), indicate that Cd preferentially targets various functional proteins in the brush-border membrane (BBM) of proximal tubule (PT) cells.

Several BBM transporters, such as the Na+-phosphate (NaPi-2) (Ahn and Park, 1995, Herak-Kramberger et al., 1996, Ahn et al., 1999), Na+-glucose (Lee et al., 1991), and Na+-amino acid (Kim et al., 1990) cotransporters, were inhibited by Cd in vitro, in isolated BBM vesicles, and in vivo, in experimental animals treated with CdCl2 for 2–3 weeks. Whereas the inhibition in vitro may reflect a direct interaction of the transporter with Cd or/and the loss of BBM vesicle integrity (Herak-Kramberger and Sabolic, 2001), the nature of Cd action in PT cells in vivo is less clear. Tubular disfunctions may result from the loss of functional BBM capacity due to (a) shortening and/or loss of microvilli (Condron et al., 1994, Herak-Kramberger et al., 1998), (b) loss of specific transporters from the BBM (Herak-Kramberger et al., 1996, Herak-Kramberger et al., 1998), (c) direct inhibition of the BBM transporter activity by Cd (Kim et al., 1990, Lee et al., 1991, Ahn and Park, 1995, Kinne et al., 1995, Sato et al., 1995, Park et al., 1997, Ahn et al., 1999), (d) indirect inhibition of the transporter activity through a change in membrane fluidity due to oxidative stress and lipid peroxidation (Shaikh et al., 1999), and (e) combination of these phenomena.

The detailed intracellular mechanisms that lead to PT disfunctions in Cd-nephrotoxicity are poorly known. Studies by Dorian et al. (1995) showed that an acute exposure to a high dose of CdCl2 in rats was hepatotoxic but not nephrotoxic. Nephrotoxicity developed only following repeated small daily doses of CdCl2 that in the liver stimulated the synthesis of metallothionein, a 6–7 kDa cystein-rich metal-binding protein. The nephrotoxic action of Cd may be mediated by Cd-metallothionenin complex released from the damaged liver cells, filtered in the glomeruli, reabsorbed by endocytosis in PT cells, and degraded in lysosomes (Dorian et al., 1992). The released Cd may then stimulate production of metallothionein in PT cells (Sendelbach and Klaassen, 1988), diminish expression of mRNA for some specific BBM transporters (Herak-Kramberger et al., 1996), directly damage the integrity of microvilli and intracellular vesicles (Herak-Kramberger and Sabolic, 2001), decrease membrane fluidity and increase lipid peroxidation by binding to phospholipids (Shaikh et al., 1999), and target various intracellular proteins and membrane transporters at their cytoplasmic side by binding to their reactive SH- or COO- groups (Vallee and Ulmer, 1972).

We have demonstrated that the in vivo intoxication of rats with CdCl2 for 2 weeks caused a strongly diminished expression of NaPi-2 and vacuolar H+-ATPase (V-ATPase) in the renal cortical BBM (Herak-Kramberger et al., 1996, Herak-Kramberger et al., 1998). Cd also directly inhibited the activity of V-ATPase in vitro, in BBM and endosomal vesicles isolated from the rat kidney cortex, and acidification in isolated endosomes (Herak-Kramberger et al., 1998). The V-ATPase-mediated acidification in the cell vacuolar system is pivotally important for continuing intracellular vesicle trafficking and recycling of plasma membrane proteins (Mellman et al., 1986). By inhibiting V-ATPase activity and deranging formation of transmembrane pH gradients in various organelles of the vacuolar system, Cd may impair endocytosis of the filtered proteins and recycling of BBM transporters in PT cells (Herak-Kramberger et al., 1998).

A variety of recent data have indicated cell cytoskeleton in addition to the V-ATPase as an important player in intracellular vesicle trafficking and recycling of the cell membrane proteins (Brown and Stow, 1996). Thus depolymerization of microtubules in PT cells by colchicine inhibited intracellular vesicle trafficking and recycling of some BBM transporters, and caused their redistribution in intracellular vesicles (Brown and Stow, 1996, Baus et al., 2000). Furthermore, the abundance and the arrangement of microtubules (Abbate et al., 1994), actin filaments (Molitoris et al., 1992, Brown et al., 1997) and villin (Brown et al., 1997) in PT cells were heavily compromised in reperfusion following renal ischemia, with a consecutive loss of polarity for some plasma membrane transporters. In in vitro studies in various cultured cells, Cd was found to depolymerize microtubules (Miura et al., 1984, Gan et al., 1986, Perrino and Chou, 1986, Li et al., 1993) and block polymerization of actin (Mills and Ferm, 1989, Prozialeck and Niewenhuis, 1991, Wang and Templeton, 1996). A possible effect of Cd on cytoskeletal elements in PT cells in vivo, a phenomenon that could affect intracellular vesicle trafficking and recycling of BBM transporters, and lead to PT disfunction in Cd-nephrotoxicity, has not been investigated to our knowledge, and is the subject of this study.

Section snippets

Animals and treatment

Three months old male Wistar rats from the breeding colony at the Institute in Zagreb were used. Animals were bred and maintained according to the Guide for Care and Use of Laboratory Animals (NIH, 1996). Before and during experiments, animals had free access to standard laboratory food and tap water. The studies were approved by the Institutional Ethic Committee.

Cd nephrotoxicity was induced by injecting CdCl2 (2 mg Cd/kg b.m., s.c., daily for up to 16 days). Control animals received an

Induction of Cd-nephrotoxicity: Cd and metallothionein in renal cortex

A day-to-day treatment of rats with CdCl2 (2 mg Cd/kg b.m., s.c.) resulted in a time-dependent accumulation of Cd in the liver and kidney cortex tissue (Fig. 1). At each time point the accumulation in the liver was higher than in the kidney cortex. After 14 days of treatment, Cd in the renal cortical tissue reached the concentration of 200 μg/g wet mass, e.g. it was about the level proposed to be ‘critical’ for the onset of nephrotoxicity in man and experimental animals (Friberg et al., 1974).

Discussion

By immunocytochemistry and immunoblotting we studied the labeling pattern of cytoskeletal proteins: actin, villin, and tubulin, in kidney cortex from control and Cd-intoxicated rats. In tissues from rats treated with CdCl2 for 2 weeks we observed: (a) reduced staining of actin in the apical domain of PT cells, without a visible change in its intracellular distribution, (b) reduced staining of villin in the PT cell apical domain, with its possible redistribution intracellularly, and an increased

Acknowledgements

The authors thank Mrs. Eva Hersak, Djurdja Breski, and Margaret McLoughlin for technical assistance. This work is supported by the grants No. 00220101 from the Croatian Ministry of Science and Technology and by the Fogarty International Research Collaborative Award (National Institute of Health, USA) No. 1-R03-TW01057-01 (I.S. and D.B.).

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