ARTICLES J Am Soc Nephrol 14: 2731–2740, 2003 Localization and Regulation of the Epithelial Ca2ϩ Channel TRPV6 in the Kidney TOM NIJENHUIS, JOOST G. J. HOENDEROP, ANNEMIETE W. C. M. VAN DER KEMP, and RENE´ J. M. BINDELS Department of Physiology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, the Netherlands. Abstract. The family of epithelial Ca2ϩ channels consists of with the other known Ca2ϩ transport proteins, including two highly homologues members, TRPV5 and TRPV6, which TRPV5 and calbindin-D28K. Together, these data suggest a role 2ϩ 2ϩ 2ϩ constitute the apical Ca entry mechanism in active Ca for TRPV6 in 1,25(OH)2D3-stimulated Ca reabsorption in (re)absorption in kidney and small intestine. In kidney, TRPV5 these segments. Interestingly, distribution of TRPV6 extended expression has been extensively studied, whereas TRPV6 lo- to the CD, where it localized to the apical domain of principal calization and regulation has been largely confined to the small and intercalated cells, which are not generally implicated in intestine. The present study investigated the renal distribution active Ca2ϩ reabsorption. In addition, TRPV6 mRNA levels of TRPV6 and regulation by 1,25-dihydroxyvitamin D3 were quantified in a large set of tissues, and in the order of Ͼ (1,25(OH)2D3). In mouse kidney, TRPV6 was detected by decreasing expression level were detected: prostate stomach, immunohistochemistry at the apical domain of the distal con- brain Ͼ lung Ͼ duodenum, kidney, bone, cecum, heart Ͼ voluted tubules (DCT2), connecting tubules (CNT), and corti- colon Ͼ skeletal muscle Ͼ pancreas. Therefore, additional cal and medullary collecting ducts (CD). Furthermore, several physiologic functions for TRPV6 are feasible. In conclusion, putative vitamin D–responsive elements were detected up- TRPV6 is expressed along the apical domain of DCT2, CNT, stream of the mouse TRPV6 start codon, and 1,25(OH)2D3 and CD, where TRPV6 expression is positively regulated by treatment significantly increased renal TRPV6 mRNA and 1,25(OH)2D3. protein expression. In DCT2 and CNT, TRPV6 co-localizes Active Ca2ϩ absorption plays a key role in Ca2ϩ homeostasis Ca2ϩ-dependent inactivation (7,8). However, significant dif- and takes place in Ca2ϩ-transporting tissues, including kidney ferences exist in the N- and C-termini of TRPV5 and TRPV6, and intestine. In the mammalian genome, a distinct family of which may account for distinct functional and regulatory epithelial Ca2ϩ channels was recently identified, which pro- features. vides the molecular identity of the apical entry mechanism, In the kidney, active transcellular reabsorption of Ca2ϩ takes facilitating the active Ca2ϩ transport process (1). This family place in the distal convoluted tubule (DCT) and the connecting of highly Ca2ϩ-selective channels is restricted to two members, tubule (CNT) and constitutes the fine-tuning mechanism de- TRPV5 (previously ECaC1), which was originally cloned from termining net urinary Ca2ϩ excretion (1,9,10). The renal ex- rabbit kidney, and TRPV6 (previously CaT1/ECaC2), which pression and regulation of TRPV5 has been extensively stud- was identified from rat duodenum (2,3). Genomic analysis ied. Briefly, TRPV5 is localized along the apical membrane of revealed that two genes, juxtaposed on human chromosome DCT2 and CNT and is regulated by vitamin D (1,25(OH)2D3), 7q35 and on mouse chromosome 6, encode these highly ho- dietary Ca2ϩ, and estrogens, substantiating the role of TRPV5 mologous but distinct epithelial Ca2ϩ channels (4–6). In ad- in renal active Ca2ϩ transport (11–13). Data on the localization dition to the conserved pore region, these channels share sev- and regulation of TRPV6 have been largely confined to the eral functional properties, including the permeation profile for small intestine, where the expression and regulation by 2ϩ monovalent and divalent cations, the high Ca selectivity, and 1,25(OH)2D3 strongly supports the involvement of this channel in the absorption of dietary Ca2ϩ (14,15). Therefore, TRPV5 is generally suggested to be the epithelial Ca2ϩ channel primarily Received May 9, 2003. Accepted August 5, 2003. responsible for renal transcellular Ca2ϩ transport, whereas Correspondence to Dr. René J.M. Bindels, 160 Cell Physiology, University TRPV6 would serve as the apical Ca2ϩ entry mechanism in the Medical Center Nijmegen, P.O. Box 9101, NL-6500 HB Nijmegen, the Neth- erlands. Phone: 31-24-3614211; Fax: 31-24-3616413; E-mail: small intestine. [email protected] Interestingly, TRPV6 mRNA has been detected in the kid- 1046-6673/1411-2731 ney, but little is known about the localization and regulation of Journal of the American Society of Nephrology TRPV6 in this organ (16). The elucidation of the precise Copyright © 2003 by the American Society of Nephrology nephron distribution of TRPV6 is crucial in understanding its DOI: 10.1097/01.ASN.0000094081.78893.E8 physiologic role in the kidney in general and particularly in 2732 Journal of the American Society of Nephrology J Am Soc Nephrol 14: 2731–2740, 2003 evaluating the potential contribution of TRPV6 to renal trans- structed. To this end, C57BL6 mice were sacrificed and kidney, bone, cellular Ca2ϩ reabsorption. Importantly, co-localization of prostate, stomach, duodenum, ileum, cecum, colon, pancreas, liver, TRPV5 and TRPV6 in the kidney may have significant func- spleen, brain, lung, heart, and skeletal muscle was sampled. Tissues Ϫ tional relevance, because it was recently shown that TRPV5 were subsequently stored at 80°C. The animal ethics board of the and TRPV6 can form heterotetrameric Ca2ϩ channels with University Medical Center Nijmegen approved all experimental procedures. distinct functionality (17). The aim of the present study was, therefore, to determine the localization and regulation of TRPV6 in the kidney. Immunohis- Immunohistochemistry tochemistry was performed using a new TRPV6 antibody to The renal localization of TRPV6 was assessed by immunohisto- elucidate the tubular and subcellular localization of this epithelial chemistry, including co-localization studies using proteins with estab- Ca2ϩ channel. In animal experiments, the effect of 1,25(OH) D lished distribution patterns as markers for distinct nephron segments. 2 3 Immunohistochemical staining was performed as described previ- on renal TRPV6 mRNA and protein expression was studied using ously (18). In short, either co-immunohistochemical staining or stain- real-time quantitative polymerase chain reaction (PCR) analysis ing of serial sections for TRPV6 with TRPV5, calbindin-D28K, cal- and semiquantitative immunohistochemistry. ϩ Ϫ bindin-D9K, the thiazide-sensitive Na -Cl cotransporter (NCC), aquaporin-2 (AQP2), and peanut lectin-binding intercalated cells was Materials and Methods performed on 7-␮m sections of fixated frozen kidney samples. Generation and Characterization of Affinity-Purified TRPV5 staining involved immersion of the kidney sections in boiled Rabbit TRPV6 Antibody citrate target retrieval buffer (0.01 M sodium citrate and 0.01 M citric Antiserum against TRPV6 was obtained by immunization of rab- acid, pH 6.0), which was then left to cool for 30 min, and subsequent bits with 400 ␮g of a keyhole limpet hemocyanin-coupled synthetic incubation in 0.3% (vol/vol) H2O2 in buffer (0.15 M NaCl, 0.1 M peptide representing the last 15 amino acids of the C-terminal tail of Tris-HCl, pH 7.5) for 30 min. Sections were incubated for 16 h at 4°C with the primary antibodies: affinity-purified rabbit TRPV6 antibody mouse TRPV6 (NH2-INRGLEDGEGWEYQI-COOH). The anti- serum was affinity-purified according to standard procedures. The (1:25), affinity-purified guinea pig TRPV5 antibody (1:1000) (18), specificity of the affinity-purified rabbit TRPV6 antibody was as- guinea pig anti-calbindin-D9K (1:500) (12), mouse anti-calbindin- sessed by immunoblot analysis of Xenopus laevis oocytes heterolo- D28K (Swant, Bellinzona, Switzerland) (1:750), rabbit anti-NCC (1: gously expressing TRPV6 or TRPV5. In addition, the efficacy of the 200) (19), guinea pig anti-AQP2 (1:1000) (20), and FITC-coupled antibody to detect endogenous TRPV6 was assessed by immunoblot peanut lectin (1:100), respectively. In addition, immunohistochemis- analyses of mouse kidney and duodenum lysates. try of kidney sections using pre-immune serum and TRPV6 antiserum pre-absorbed for 1 h with the corresponding peptide was performed as negative controls. For detection of TRPV6, calbindin-D , calbindin- Animal Studies 9K D , NCC, and AQP2 sections were incubated with Alexa-conju- Experiment 1. 28K To study the nephron localization of TRPV6 by gated secondary antibodies. After incubation with biotin-coated goat immunohistochemistry, kidneys were isolated from C57BL6 mice anti–guinea pig secondary antibody, TRPV5 was visualized using a (kindly provided by Dr. J. Loffing, University of Zürich, Switzerland). tyramide signal amplification kit (NEN Life Science Products, The tissue fixation procedure consisted of anesthestizing the animals, Zaventem, Belgium). Images were made using a Nikon Diaphot after which the abdominal aorta was clamped downstream of the renal confocal laser scanning microscope (Tokyo, Japan; MRC-1000; Bio- arteries. Tubing was inserted at the level of the iliac bifurcation into Rad, Richmond, CA) and a Zeiss fluorescence microscope equipped the aorta, pushed up to the aortic clamp, and fixed by a ligature. The with a digital photo camera (Nikon DMX1200). For semiquantitative vena cava was opened, the aortic clamp was removed, and a fixative determination of protein levels, images were analyzed with the Image solution (50 ml) was allowed to flush the mouse vasculature under Pro Plus 4.1 image analysis software (Media Cybernetics, Silver high pressure. The fixative consisted of 3% paraformaldehyde (vol/ Spring, MD), resulting in quantification of the protein levels as the vol) and 0.05% picric acid (vol/vol) dissolved in a 3:2 mixture of 0.1 mean of integrated optical density (IOD). M cacodylate buffer (pH 7.4) and 10% hydroxyethyl starch (vol/vol) in saline (10). After 5-min fixation, the kidneys were rinsed at hy- drostatic pressure by perfusion for 5 min with the cacodylate buffer.