Cyclosporine A-induced hypercalciuria in calbindin-D28k knockout and wild-type mice.
BACKGROUND
It is known that cyclosporine A (CsA) treatment induces high bone-turnover osteopenia and hypercalciuria. It has been proposed that down-regulation of renal calbindin-D28k by CsA results in renal calcium wasting. We investigated the role of the kidney and bone in CsA-induced hypercalciuria in calbindin-D28k knockout (KO) and wild-type (WT) mice.


METHODS
Two sets of experiments were performed. In experiment 1, KO and WT mice were treated with CsA 20 mg/kg/day intraperitoneally (IP) for 7 days. In experiment 2, to eliminate the CsA effect on bone resorption, pamidronate (APD) 2.5 mg/kg IP was given every 4 days with the first dose given 4 days prior to the 7-day course of CsA. Serum levels of creatinine, calcium, and osteocalcin, as well as renal calcium excretion were measured to assess CsA's effects on calcium homeostasis. Effects of CsA on the expression of calbindin-D28k, and two calcium channels in the apical membrane of the distal tubule, epithelial calcium channel (ECaC) and alpha1G-subunit of a voltage-dependent Ca channel (alpha1G), in the kidney were examined by semiquantitative reverse transcription polymerase chain reaction (RT-PCR).


RESULTS
KO mice had a threefold increase in renal calcium excretion when compared with WT mice at the baseline. This difference disappeared when calcium load was reduced by overnight fasting. After the CsA treatment, both WT and KO mice had a significant increase of renal calcium excretion (urine Ca/Cr ratio in WT, 0.11 +/- 0.01 to 1.29 +/- 0.17; in KO, 0.39 +/- 0.04 to 1.18 +/- 0.13; both P < 0.01). CsA treatment decreased renal calbindin-D28k mRNA by 61%, but did not affect the expression of ECaC and alpha1G. Baseline serum osteocalcin level of KO mice was significantly lower than that of WT mice. After CsA treatment, both groups had a 50% increase in the serum osteocalcin level, indicating increased bone turnover. When mice were treated with both CsA and APD, the increase in serum osteocalcin level was prevented, and renal calcium excretion was significantly lower than that in mice treated with CsA alone. However, there was still a significant increase in the urine Ca/Cr ratio in WT and KO mice compared with pretreatment levels (urine Ca/Cr in WT, 0.11 +/- 0.01 to 0.76 +/- 0.05, P < 0.01; in KO, 0.39 +/- 0.05 to 0.79 +/- 0.06; P < 0.01).


CONCLUSIONS
Calbindin-D28k KO mice have diet-dependent hypercalciuria and a lower bone turnover rate. CsA treatment suppresses the expression of calbindin-D28k in mice, but has no effects on ECaC and alpha1G gene expression at the mRNA level. The pathogenesis of CsA-induced hypercalciuria involves both down-regulation of calbindin-D28k with subsequent impaired renal calcium reabsorption and CsA-induced high turnover bone disease. Additionally, our results suggest that mechanism(s) independent of calbindin-D28k within the kidney also may contribute to the CsA-induced calcium leak.
Lee
Chien-Te
CT
Department of Medicine, University of Arizona Health Sciences Centers, Tucson, Arizona 85724, USA.
Huynh
Viet M
VM
Lai
Li-Wen
LW
Lien
Yeong-Hau H
YH
D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D001851:Bone Diseases, Metabolic / Q000139:chemically induced* / Q000503:physiopathology / Q000652:urine; D064092:Calbindin 1; D064026:Calbindins; D002118:Calcium / Q000652:urine*; D015220:Calcium Channels / Q000235:genetics; D016572:Cyclosporine / Q000633:toxicity*; D004164:Diphosphonates / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D007166:Immunosuppressive Agents / Q000633:toxicity*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D000077268:Pamidronate; D064030:S100 Calcium Binding Protein G / Q000235:genetics*
D000893:Anti-Inflammatory Agents; C576499:Calb1 protein, mouse; D064092:Calbindin 1; D064026:Calbindins; D015220:Calcium Channels; D004164:Diphosphonates; D007166:Immunosuppressive Agents; D064030:S100 Calcium Binding Protein G; D016572:Cyclosporine; D000077268:Pamidronate; D002118:Calcium