Lee Chien-Te, Huynh Viet M, Lai Li-Wen, Lien Yeong-Hau H
Department of Medicine, University of Arizona Health Sciences Centers, Tucson, Arizona 85724, USA.
Kidney Int. 2002 Dec;62(6):2055-61. doi: 10.1046/j.1523-1755.2002.00670.x.
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.
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).
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).
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.
已知环孢素A（CsA）治疗可导致高骨转换型骨质减少和高钙尿症。有人提出，CsA使肾钙结合蛋白-D28k下调导致肾性钙流失。我们研究了肾脏和骨骼在CsA诱导的钙结合蛋白-D28k基因敲除（KO）和野生型（WT）小鼠高钙尿症中的作用。
进行了两组实验。在实验1中，KO和WT小鼠腹腔内注射（IP）20mg/kg/天的CsA，持续7天。在实验2中，为消除CsA对骨吸收的影响，每4天给予2.5mg/kg IP的帕米膦酸（APD），首剂在7天CsA疗程前4天给予。测量血清肌酐、钙和骨钙素水平以及肾钙排泄，以评估CsA对钙稳态的影响。通过半定量逆转录聚合酶链反应（RT-PCR）检测CsA对肾钙结合蛋白-D28k以及远端小管顶端膜中两个钙通道，即上皮钙通道（ECaC）和电压依赖性钙通道α1G亚基（α1G）表达的影响。
与WT小鼠相比，KO小鼠在基线时肾钙排泄增加了两倍。禁食过夜降低钙负荷后，这种差异消失。CsA治疗后，WT和KO小鼠的肾钙排泄均显著增加（WT小鼠尿钙/肌酐比值从0.11±0.01增至1.29±0.17；KO小鼠从0.39±0.04增至1.18±0.13；均P<0.01）。CsA治疗使肾钙结合蛋白-D28k mRNA降低61%，但不影响ECaC和α-1G的表达。KO小鼠的基线血清骨钙素水平显著低于WT小鼠。CsA治疗后，两组血清骨钙素水平均增加50%，表明骨转换增加。当小鼠同时接受CsA和APD治疗时，血清骨钙素水平的增加得到预防，且肾钙排泄显著低于单独接受CsA治疗的小鼠。然而，与治疗前水平相比，WT和KO小鼠的尿钙/肌酐比值仍显著增加（WT小鼠尿钙/肌酐比值从0.11±0.01增至0.76±0.05，P<0.01；KO小鼠从0.39±0.05增至0.79±0.06；P<0.01）。
钙结合蛋白-D28k基因敲除小鼠有饮食依赖性高钙尿症且骨转换率较低。CsA治疗可抑制小鼠钙结合蛋白-D28k的表达，但在mRNA水平上对ECaC和α1G基因表达无影响。CsA诱导的高钙尿症的发病机制涉及钙结合蛋白-D28k下调及随后的肾钙重吸收受损，以及CsA诱导的高转换型骨病。此外，我们的结果表明，肾脏内独立于钙结合蛋白-D28k的机制也可能导致CsA诱导的钙泄漏。
