engleski

Immunolocalization of metallothionein along the rat nephron

Metallothionein (MT) is a small (MW 7 kDa), cysteine-rich protein with an unresolved function. Its reactive SH groups exhibit a high binding affinity to some nontoxic and toxic metals. Being expressed in a variety of mammalian organs, with highest concentrations found in liver, it is assumed to participate in homeostatic control of essential metals, such as Zn and Cu. The treatment of experimental animals with Cd, Hg or Pt, induces liver and/or kidney damage, and increases expression of MT in these and other organs, indicating its possible role in diminishing toxicity of these metals. Although the upregulation of MT expression in heavy metal nephrotoxicity has been described, a detailed localization of MT in various cell types along the intact and heavy metal-affected nephrons has been poorly studied. Also, a phenomenon that Cd nephrotoxicity in female (F) rats is less severe that in male (M) rats has not been correlated with the level of MT in their kidneys. To study localization and intracellular distribution of MT in cells along the rat nephron, we performed Western blots (WB) with tissue homogenates from the kidney cortex (C), outer stripe (OS), outer medulla (OM), and inner medulla (IM), and immunofluorescence cytochemistry (IF) in cryostat sections of the same tissues in adult M and F Wistar rats by using a commercially available monoclonal anti-MT antibody. By WB, a 6-7 kDa MT band was found in tissue homogenates from all the zones of the M kidney, with relative densities (arbitrary units): C:OS:IS:IM = 80:10:6:17. By IF, the glomerulus and the proximal tubule S1 segment were unstained, whereas the S2 segment exhibited a heterogeneous cytoplasmic and nuclear staining, which in various tubule profiles was either strong or weak, or negative. When positive, the cytoplasmic staining was either diffuse or concentrated more subapically and/or basolaterally. However, WB of isolated renal cortical brush-border, basolateral and endosomal vesicles, revealed an absence of a specific MT band in these organelles, indicating that the observed polar distribution of MT was a fixation artefact. The cytoplasm and the nuclei of some cells in the S3 segment were weakly positive. No significant staining was observed in cells of the IS. However, a relatively strong cytoplasmic and/or nuclear staining was observed in some cells of the proximal IM. Double staining of MT and aquaporin 1 (a marker for descending thin limbs and nonfenestrated capillaries) or aquaporin 2 (a marker for collecting duct principal cells), or thrombomodulin (a marker for endothelial cells), showed that MT was absent in the respective structures. Thus, the positive MT staining in the proximal papilla could be attributed to the thin ascending limb. WB of tissue homogenates and IF studies in tissue cryosections in F revealed no significant differences in the abundance or localization of MT in F and M kidneys. We conclude that in intact adult rats, MT can be localized to the distinct nephron segments in a heterogeneous abundance in the cytoplasm and/or nucleus of the respective cells, with similar level of expression in M and F kidneys.

heavy metal nephrotoxicity; kidney; immunocytochemistry; gender differences

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