Aminoglycoside antibiotics (AGAs) exhibit nephrotoxicity primarily in the proximal tubule (PT). We have previously shown that the extracellular calcium sensing receptor (CaR), which is stimulated by AGAs including gentamicin and neomycin, is expressed on the apical membrane of rat proximal tubules and in proximal tubular-derived cell lines. To investigate whether altered CaR expression accompanies AGA treatment, we studied the effect of chronic gentamicin administration on the CaR content of a membrane fraction of renal cortex. This fraction is known to be enriched (85-90 %) for PT brush-border vesicles but includes proximal and distal intracellular vesicles (Biber et al. 1981).
Male Sprague-Dawley rats received single, daily, subcutaneous injections of either 50 mg kg-1 gentamicin or 0.9 % saline vehicle for 1, 3 or 6 days according to the provisions of the UK Animal Procedures Act (1986). Animals were humanely killed by cervical dislocation either under anaesthesia using pentobarbitone (50 mg kg-1 I.P.) or following stunning. Kidneys were collected and brush-border vesicle-enriched membranes (BBM) prepared from cortex as described by Biber et al. (1981). Protein equivalent aliquots of BBM from control and gentamicin-treated rats were processed for semi-quantitative immunoblotting using an antibody to CaR. In addition, the expression levels of the divalent metal transporter, DMT1, present in the PT (Ferguson et al. 2001), and that of a distal tubule-marker, the sodium chloride co-transporter NCCT, were also tested. Relative immunoreactivity was quantified by densitometry and expressed as percentage change ± S.E.M., with statistical significance determined by paired t test (N = 3-4 unless otherwise stated).
The CaR content of BBM was not significantly changed 24 h after a single gentamicin injection, but was reduced at day 3 (-23 ± 5 %) with the change tending towards significance (P = 0.07) and was normal again by day 6 (-10 ± 8 %, n.s.).
The expression of DMT1 in BBM was also unchanged on day 1 (-3 ± 10 %, n.s.) but was elevated on day 3 (+48 ± 1 %, P < 0.02), remaining elevated at day 6 (+109 ± 40 %, P < 0.05). This was confirmed by immunofluorescence microscopy, which showed elevated intracellular anti-DMT1 immunoreactivity in proximal tubule of rats treated with gentamicin for 6 days. Expression of NCCT was unchanged at both time points tested (day 1, -3 ± 9 %, n.s.; day 3, +5 ± 12 %, n.s.; n = 2-3).
These data demonstrate altered CaR and DMT1 expression, but not that of NCCT, in renal cortex resulting from chronic gentamicin treatment. Changes in CaR expression could account for the hypercalciuria and hypermagnesiuria, which frequently accompany AGA treatment. In addition, upregulation of DMT1, known to transport iron in the kidney (Ferguson et al. 2001), could alter iron homeostasis and therefore contribute to iron-mediated free radical formation. Finally, it will be necessary to determine whether the apparent CaR downregulation is a direct result of chronic receptor activation or a downstream effect of gentamicin exposure. Work is currently underway to test these hypotheses.
This work was supported by a grant from The Wellcome Trust.