engleski

Distribution of metallothionein along the rat nephron and its source in the urine

Metallothionein (MT) is a small (MW 6-7 kDa), cysteine-rich protein expressed in a variety of mammalian organs, where it participates in homeostasis of essential metals (Zn, Cu). Treatment of rats with toxic heavy metals, such as Cd, Hg or Pt, induces liver and kidney damage and overexpression of MT in these organs, indicating its possible role in detoxification. MT is excreted in the urine, where it is assumed to originate from the blood plasma by glomerular filtration. An increased excretion of urinary MT can be used as a marker of heavy metal toxicity. Although overexpression of the renal MT in heavy metal toxicity is well known, a detailed localization of MT in various cell types along the nephron in intact rats has not been performed. In this work we studied localization and intracellular distribution of MT along the rat nephron by using a commercial anti-MT antibody and performing Western blots (WB), immunofluorescence cytochemistry (IFC), and electron microscopy (EM). By WB, the specific MT band was detected in tissue homogenates from all the kidney zones ; a band density (relative units) in the cortex, outer stripe, inner stripe (IS), and inner medulla (IM) was 80:10:6:17, respectively. By IFC, the glomerulus and the proximal tubule (PT) S1 segment were unstained, whereas the S2 segment exhibited a heterogeneous cytoplasmic and nuclear staining. The cytoplasm and the nuclei of some cells in the S3 segment were weakly positive. No significant staining was observed in the cells of the IS. However, a relatively strong cytoplasmic and/or nuclear staining was observed in the cells of the thin ascending limb of the proximal IM. In addition to its intracellular localization in the specific parts of the nephron, distinct vesicles rich in MT and carboanhydrase II (typical cytoplasmic proteins) were observed in the lumen of some PT S2 segments. Their limiting membrane was positive for aquaporin 1, sodium hydrogen exchanger type 3, megalin, and carboanhydrase IV (brush-border membrane markers), indicating their formation from the PT cell apical domain by exocytosis. The presence of these vesicles in the PT lumen were confirmed by EM. We conclude that in intact adult rats, MT is localized to distinct nephron segments in a heterogeneous abundance in the cell cytoplasm and/or nucleus. Glomerular filtration may not be the only source of urinary MT ; the MT-rich vesicles in the PT may contribute to the excretion of this and other proteins in the mammalian urine.

heavy metals; immunocytochemistry; kidney; nephrotoxicity

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