Downregulation of Ca and Mg Transport Proteins in the Kidney Explains Tacrolimus (FK506)-Induced Hypercalciuria and Hypomagnesemia

FK506 (tacrolimus) and dexamethasone are potent immunosuppressants known to induce significant side effects on mineral homeostasis, including hypercalciuria and hypomagnesemia. However, the underlying molecular mechanisms remain unknown. The present study investigated the effects of FK506 and dexamethasone on the expression of proteins involved in active Ca reabsorption: the epithelial Ca channel TRPV5 and the cytosolic Ca -binding protein calbindinD28K. In addition, the renal expression of the putative Mg 2 channel TRPM6, suggested to be involved in transcellular Mg reabsorption, was determined. Administration of FK506 to rats by daily oral gavage during 7 d significantly enhanced the urinary excretion of Ca and Mg and induced a significant hypomagnesemia. FK506 significantly decreased the renal mRNA expression of TRPV5 (62 7% relative to controls), calbindin-D28K (9 1%), and TRPM6 (52 8%), as determined by real-time quantitative PCR analysis. Furthermore, semiquantitative immunohistochemistry showed reduced renal protein abundance of TRPV5 (24 5%) and calbindin-D28K (29 4%), altogether suggesting that downregulation of these transport proteins is responsible for the FK506-induced Ca and Mg wasting. In contrast, dexamethasone significantly enhanced renal TRPV5 (150 15%), calbindin-D28K (177 23%), and TRPM6 (156 20%) mRNA levels along with TRPV5 (211 8%) and calbindinD28K (176 5%) protein abundance in the presence of significantly increased Ca and Mg excretion. This indicated that these proteins are directly or indirectly regulated by dexamethasone. In conclusion, FK506 and dexamethasone induce renal Ca and Mg wasting, albeit by different mechanisms. Downregulation of specific Ca and Mg transport proteins provides a molecular mechanism for FK506-induced hypercalciuria and hypomagnesemia, whereas dexamethasone positively regulates these proteins. Immunosuppressants such as the calcineurin inhibitors cyclosporin A and FK506 (tacrolimus) along with glucocorticoids such as dexamethasone are widely prescribed in numerous disorders and to organ transplant recipients. Although their immunosuppressive actions are achieved by distinct mechanisms, FK506 and dexamethasone both are known to induce significant side effects on mineral homeostasis. These drugs are associated with an increased bone turnover, a negative Ca balance and hypercalciuria, perturbations that can ultimately result in osteoporosis (1–3). Furthermore, hypomagnesemia is a widely known additional side effect of FK506 treatment (4, 5). The kidney is crucial to both Ca and Mg homeostasis by providing the main excretory route for these divalent ions. However, the exact mechanisms by which these immunosuppressants provoke renal cation wasting are unknown. The hypercalciuria during treatment with these drugs has been attributed to increased bone resorption as well as decreased renal Ca reabsorption (2, 6, 7). In the kidney, the bulk of Ca reabsorption is accomplished by Na -driven passive paracellular Ca transport in the proximal tubule and thick ascending limb of Henle (TAL) (8). Fine-tuning of Ca excretion is achieved by active transcellular reabsorption of Ca in the distal convoluted tubule (DCT) and connecting tubule (CNT), which involves apical Ca entry through the epithelial Ca channel TRPV5 (previously ECaC1), intracellular buffering and facilitated diffusion by the Ca -binding protein calbindin-D28K, and basolateral extrusion by the Na / Ca exchanger (NCX1) and the plasma membrane Ca ATPase (PMCA1b) (9, 10). Theoretically, inhibition of active Ca reabsorption could be involved in the immunosuppressant-induced hypercalciuria. FK506 treatment has been associated with an inappropriately high fractional excretion of Mg , suggesting that inhibition of passive or active Mg reabsorption could contribute to the hypomagnesemia (11). Active reabsorption of Mg has been localized to the DCT, but in contrast to Ca reabsorption, little is known about the specific proteins that mediate Received June 13, 2003. Accepted November 27, 2003. Correspondence to René J.M. Bindels, 160 Cell Physiology, University Medical Center Nijmegen, P.O. Box 9101, NL-6500 HB Nijmegen, the Netherlands. Phone: 31-24-3614211; Fax: 31-24-3616413; E-mail: [email protected]