Identification of the vitamin D receptor in various cells of the mouse kidney
Authors: Yongji Wang, Megan L. Borchert, Hector F. DeLuca
Journal: Kidney International (2012)

The kidney is the major, if not sole, site for the production of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), the biologically active form of vitamin D that can stimulate calcium reabsorption in the kidney and may provide renoprotective benefits. The biological effects of 1,25(OH)2D3 are mediated through a nuclear hormone receptor, known as the vitamin D receptor (VDR). It is well accepted that the VDR is present in the distal renal convoluted tubule cells; however, whether VDR is present in other kidney cell types is uncertain. Using a highly specific and sensitive anti-VDR antibody, we determined its distribution in the mouse kidney by immunohistochemistry. Our results show that the VDR is not only present in the distal but is also found in the proximal tubules, but at 24-fold lower levels. The VDR was also found in the macula densa of the juxtaglomerular apparatus, glomerular parietal epithelial cells, and podocytes. In contrast, the VDR is either very low or absent in interstitial fibroblasts, glomerular mesangial cells, and juxtaglomerular cells. Thus, identification of VDR in the proximal tubule, macula densa, and podocytes suggests that 1,25(OH)2D3 plays a direct role in these cells under normal conditions. The kidney is the major, if not sole, site for the production of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), the biologically active form of vitamin D that can stimulate calcium reabsorption in the kidney and may provide renoprotective benefits. The biological effects of 1,25(OH)2D3 are mediated through a nuclear hormone receptor, known as the vitamin D receptor (VDR). It is well accepted that the VDR is present in the distal renal convoluted tubule cells; however, whether VDR is present in other kidney cell types is uncertain. Using a highly specific and sensitive anti-VDR antibody, we determined its distribution in the mouse kidney by immunohistochemistry. Our results show that the VDR is not only present in the distal but is also found in the proximal tubules, but at 24-fold lower levels. The VDR was also found in the macula densa of the juxtaglomerular apparatus, glomerular parietal epithelial cells, and podocytes. In contrast, the VDR is either very low or absent in interstitial fibroblasts, glomerular mesangial cells, and juxtaglomerular cells. Thus, identification of VDR in the proximal tubule, macula densa, and podocytes suggests that 1,25(OH)2D3 plays a direct role in these cells under normal conditions. The primary functions of the kidney include waste removal, maintenance of homeostatic functions, such as calcium and phosphate and electrolyte regulation and acid–base balance.1Reilly R.F. Ellison D.H. Mammalian distal tubule: physiology, pathophysiology, and molecular anatomy.Physiol Rev. 2000; 80: 277-313Crossref PubMed Scopus (301) Google Scholar Renal functions are performed within nephrons, the basic structural and functional units of the kidney. Each nephron is composed of a glomerulus and a number of structurally and functionally different renal tubules, including the proximal tubule, loop of Henle, and the distal tubule. Collecting ducts are not part of the nephron but connect with the distal tubule through connecting tubule, a part of the distal tubule. 1α, 25-Dihydroxyvitamin D3 regulates calcium reabsorption in the distal tubules and collecting ducts of the kidney.2.Yamamoto M. Kawanobe Y. Takahashi H. et al.Vitamin D deficiency and renal calcium transport in the rat.J Clin Invest. 1984; 74: 507-513Crossref PubMed Scopus (103) Google Scholar, 3.DeLuca H.F. 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Verlinden L. et al.Vitamin D and human health: lessons from vitamin D receptor null mice.Endocr Rev. 2008; 29: 726-776Crossref PubMed Scopus (1308) Google Scholar The kidney is also a major site for vitamin D metabolism. 25-Hydroxyvitamin D3 (25(OH)D3) is converted into the biologically active 1,25(OH)2D3, by 25-(OH)-vitamin D3 1α-hydroxylase. 1α-Hydroxylase is a cytochrome P450 enzyme localized in both convoluted and straight proximal tubules.5.Brunette M.G. Chan M. Ferriere C. et al.Site of 1,25(OH)2 vitamin D3 synthesis in the kidney.Nature. 1978; 276: 287-289Crossref PubMed Scopus (134) Google Scholar, 6.Kawashima H. Kurokawa K. Metabolism and sites of action of vitamin D in the kidney.Kidney Int. 1986; 29: 98-107Abstract Full Text PDF PubMed Scopus (45) Google Scholar, 7.Kawashima H. Torikai S. Kurokawa K. 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Wei M. et al.1,25-Dihydroxyvitamin D(3) is a negative endocrine regulator of the renin-angiotensin system.J Clin Invest. 2002; 110: 229-238Crossref PubMed Scopus (1750) Google Scholar Renin is a circulating proteinase produced by juxtaglomerular cells of the juxtaglomerular apparatus (JGA) in adult animals and human. This hormone involves the renin–angiotensin system and regulates blood pressure. Using renin gene promoter, which is restricted to the juxtaglomerular cells of the JGA in adult animals and human, Dr Li's group has showed that selective overexpression of human vitamin D receptor (VDR) in the juxtaglomerular cells either in the wild-type or the receptor knockout mice can significantly reduce the production of renin.12.Kong J. Qiao G. 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Reid F.A. et al.Target cells for 1,25-dihydroxyvitamin D3 in intestinal tract, stomach, kidney, skin, pituitary, and parathyroid.Science. 1979; 206: 1188-1190Crossref PubMed Scopus (593) Google Scholar In addition, several studies using animal models have shown that 1,25(OH)2D3 exerts a renoprotective function in several chronic kidney diseases. The hormone seems to protect the podocytes and prevent proteinuria.17.Migliori M. Giovannini L. Panichi V. et al.Treatment with 1,25-dihydroxyvitamin D3 preserves glomerular slit diaphragm-associated protein expression in experimental glomerulonephritis.Int J Immunopathol Pharmacol. 2005; 18: 779-790Crossref PubMed Scopus (40) Google Scholar, 18Xiao H. Shi W. Liu S. et al.1,25-Dihydroxyvitamin D(3) prevents puromycin aminonucleoside-induced apoptosis of glomerular podocytes by activating the phosphatidylinositol 3-kinase/Akt-signaling pathway.Am J Nephrol. 2009; 30: 34-43Crossref PubMed Scopus (54) Google Scholar, 19.Xiao H.Q. Shi W. 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Role of calcium-phosphorous disorders in the progression of renal failure.Kidney Int Suppl. 2005; 68: S66-S70Abstract Full Text Full Text PDF Scopus (26) Google Scholar The cellular and molecular mechanisms of 1,25(OH)2D3-mediated renoprotective actions remain largely unknown. The actions of 1,25(OH)2D3 are mediated through binding with its nuclear receptor, VDR. The receptor is a ligand-activated transcription factor that interacts with coregulators and transcriptional complexes to alter the rate of target gene transcription.3.DeLuca H.F. Overview of general physiologic features and functions of vitamin D.Am J Clin Nutr. 2004; 80: 1689S-1696SCrossref PubMed Google Scholar,23.Dusso A.S. Brown A.J. Slatopolsky E. Vitamin D.Am J Physiol Renal Physiol. 2005; 289: F8-F28Crossref PubMed Scopus (1069) Google Scholar Ligand activation of the VDR promotes adequate calcium reabsorption in the distal renal tubules and collecting ducts by regulating the expression of several calcium transport proteins in the kidney.3.DeLuca H.F. Overview of general physiologic features and functions of vitamin D.Am J Clin Nutr. 2004; 80: 1689S-1696SCrossref PubMed Google Scholar,24.Bouillon R. Carmeliet G. Verlinden L. et al.Vitamin D and human health: lessons from vitamin D receptor null mice.Endocr Rev. 2008; 29: 726-776Crossref PubMed Scopus (876) Google Scholar The upregulation of calcium-binding proteins, such as calbindin D28k (CaBP-D28k) and D9k (CaBP-D9k),25.Darwish H.M. DeLuca H.F. Identification of a 1,25-dihydroxyvitamin D3-response element in the 5′-flanking region of the rat calbindin D-9k gene.Proc Natl Acad Sci USA. 1992; 89: 603-607Crossref PubMed Scopus (188) Google Scholar and calcium transporters, such as TRVP5 and TRVP6,26.van de Graaf S.F. Boullart I. Hoenderop J.G. et al.Regulation of the epithelial Ca2+ channels TRPV5 and TRPV6 by 1alpha,25-dihydroxy vitamin D3 and dietary Ca2+.J Steroid Biochem Mol Biol. 2004; 89–90: 303-308Crossref PubMed Scopus (49) Google Scholar,27.Hsu Y.J. Hoenderop J.G. Bindels R.J. TRP channels in kidney disease.Biochim Biophys Acta. 2007; 1772: 928-936Crossref PubMed Scopus (55) Google Scholar are believed to have a role in calcium reabsorption in the distal renal tubules. Additionally, the genes involved in vitamin D metabolism in the kidney are known to respond to 1,25(OH)2D3.3.DeLuca H.F. Overview of general physiologic features and functions of vitamin D.Am J Clin Nutr. 2004; 80: 1689S-1696SCrossref PubMed Google Scholar For example, the 24-hydroxylase gene transcription is activated by 1,25(OH)2D3, while 1α-hydroxylase gene transcription is suppressed by 1,25(OH)2D3.10.Plum L.A. DeLuca H.F. Vitamin D, disease and therapeutic opportunities.Nat Rev Drug Discov. 2010; 9: 941-955Crossref PubMed Scopus (349) Google Scholar Consequently, identification of VDR within renal structures is important for understanding the mechanism of 1,25(OH)2D3 in these functions. Currently, the segment-specific distribution of VDR is inconclusive. During the last three decades, multiple assays including autoradiography, polymerase chain reaction/quantitative polymerase chain reaction, in situ hybridization, and/or immunohistochemistry have been used to determine the distribution of VDR in the kidney. These studies demonstrate that VDR is present in distal renal tubules.9.Iida K. Taniguchi S. 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McClelland R.A. et al.Immunocytochemical detection of 1,25-dihydroxyvitamin D receptors in normal human tissues.J Clin Endocrinol Metab. 1988; 67: 607-613Crossref PubMed Scopus (194) Google Scholar, 29.Liu L. Ng M. Iacopino A.M. et al.Vitamin D receptor gene expression in mammalian kidney.J Am Soc Nephrol. 1994; 5: 1251-1258PubMed Google Scholar, 30.Liu L. Khastgir A. McCauley J.M. et al.RT-PCR microlocalization of mRNAs for calbindin D28k and vitamin D receptor in the murine nephron.Am J Physiol. 1996; 270: F677-F681PubMed Google Scholar, 34.Kawashima H. Kurokawa K. Localization of receptors for 1,25-dihydroxyvitamin D3 along the rat nephron. Direct evidence for presence of the receptors in both proximal and distal nephron.J Biol Chem. 1982; 257: 13428-13432PubMed Google Scholar as well as the absence of VDR expression.16.Stumpf W.E. Sar M. Reid F.A. et al.Target cells for 1,25-dihydroxyvitamin D3 in intestinal tract, stomach, kidney, skin, pituitary, and parathyroid.Science. 1979; 206: 1188-1190Crossref PubMed Scopus (593) Google Scholar, 28.Liu W. Tretiakova M. Kong J. et al.Expression of vitamin D3 receptor in kidney tumors.Hum Pathol. 2006; 37: 1268-1278Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 32.Narbaitz R. Stumpf W.E. Sar M. et al.The distal nephron in the chick embryo as a target tissue for 1-alpha-25-dihydroxycholecalciferol.Acta Anat (Basel). 1982; 112: 208-216Crossref PubMed Scopus (15) Google Scholar, 33.Stumpf W.E. Sar M. Narbaitz R. et al.Cellular and subcellular localization of 1,25-(OH)2-vitamin D3 in rat kidney: comparison with localization of parathyroid hormone and estradiol.Proc Natl Acad Sci USA. 1980; 77: 1149-1153Crossref PubMed Scopus (100) Google Scholar More recently, immunohistochemical studies using a rabbit polyclonal antibody, C-20, showed the absence of VDR in proximal tubules of normal human kidneys.28.Liu W. Tretiakova M. Kong J. et al.Expression of vitamin D3 receptor in kidney tumors.Hum Pathol. 2006; 37: 1268-1278Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar In addition to the uncertainty regarding VDR expression within the proximal tubules, the presence of VDR in the JGA and glomerulus requires confirmative study, using a highly specific and sensitive method. The antibody to VDR is essential for determination of the receptor in a target. We have previously characterized a set of VDR antibodies (nine antibodies) from different sources, including the widely used antibodies such as rat monoclonal 9A7 and rabbit polyclonal antibody C-20,35.Wang Y. Becklund B.R. DeLuca H.F. Identification of a highly specific and versatile vitamin D receptor antibody.Arch Biochem Biophys. 2010; 494: 166-177Crossref PubMed Scopus (0) Google Scholar and successfully applied the selected antibody to address if the VDR is present in the muscle.36.Wang Y. Deluca H.F. Is the vitamin D receptor found in muscle?.Endocrinology. 2011; 152: 354-363Crossref PubMed Scopus (193) Google Scholar The sensitivity and specificity of these antibodies were carefully characterized by multiple immunological assays, such as immunoblotting, immunocytochemistry, and immunohistochemistry. Most importantly, we included samples from Demay VDR knockout mice, which have no native or truncated VDR. Using this negative control, we were able to learn the specificity of each antibody. In addition, comparing the intensity of the VDR band, we were able to learn the sensitivity of each antibody to the receptor. We concluded that the D-6 VDR antibody is highly specific and sensitive. Even more relevant to the questions regarding the expression of the receptor in certain segments of the kidney, the D-6 antibody selectively reacts with the VDR in whole-kidney lysates of wild-type mice. This antibody does not react with whole-renal lysate from the Demay VDR knockout mice. Thus, the D-6 antibody shows no nonspecific binding in any renal tissue. In contrast, other antibodies, such as C-20 and 9A7, bind to proteins in the lysates from the Demay mice. Using the highly specific and sensitive antibody to VDR identified previously,35.Wang Y. Becklund B.R. DeLuca H.F. Identification of a highly specific and versatile vitamin D receptor antibody.Arch Biochem Biophys. 2010; 494: 166-177Crossref PubMed Scopus (0) Google Scholar the presence of VDR in the proximal renal tubule has been clearly confirmed. In addition, the VDR is clearly found in the glomerular parietal epithelial cells, podocytes, and the macula densa of the JGA. In contrast, the interstitial fibroblasts, intraglomerular mesangial cells, and juxtaglomerular cells either do not contain VDR or have undetectable low levels. To address if the VDR is present in the proximal renal tubules, glomeruli, and JGA, we used immunohistochemistry employing a well-characterized D-6 VDR antibody.35.Wang Y. Becklund B.R. DeLuca H.F. Identification of a highly specific and versatile vitamin D receptor antibody.Arch Biochem Biophys. 2010; 494: 166-177Crossref PubMed Scopus (0) Google Scholar We first differentiated the proximal tubules from the distal tubules, and cortical and medullary collecting ducts. The kidney sections were stained with molecular markers, including E-cadherin and CaBP-D28k, for identifying the distal tubules and collecting ducts. E-cadherin is highly expressed in the distal tubules and (cortical) collecting ducts, and is absent or expressed at a low level in the proximal tubules.37.Prozialeck W.C. Lamar P.C. Appelt D.M. Differential expression of E-cadherin, N-cadherin and beta-catenin in proximal and distal segments of the rat nephron.BMC Physiol. 2004; 4: 10Crossref PubMed Google Scholar Therefore, the CaBP-D28k and E-cadherin negative tubules denote the proximal tubules. As shown in Figure 1, the renal tubule with high levels of E-cadherin and CaBP-D28k is the distal tubule, while the tubule with a high level of E-cadherin and with a low level of CaBP-D28k is the cortical collecting duct or the medullary collecting duct. The tubules lacking both E-cadherin and CaBP-D28k in the cortical region are the proximal tubules. The spatial relationship between VDR and CaBP-D28k can be used to identify the distal tubules and collecting ducts. The VDR and CaBP-D28k costaining showed that VDR is specifically expressed in distal tubules in the renal cortex and medulla (Figure 2). The expression of VDR was very low in collecting ducts in the medulla. Analysis of the staining intensities of VDR and CaBP-D28k indicated that their expression levels were highly correlated and colocalized (Figure 2). Both VDR and E-cadherin were highly expressed and colocalized within the distal tubules (Figure 3). The cortical collecting ducts (strong E-cadherin-positive staining in the cortex) and medullary collecting ducts highly expressed E-cadherin but expressed VDR at lower levels. In contrast, the proximal tubules were negative or extremely weak for the E-cadherin staining. Interestingly, these tubules were positive for VDR but its levels were low (Figure 3, middle and lower panels). We found that double staining could alter the results of VDR antibody staining, such as losing weak immunosignals from the proximal tubules and collecting ducts. To avoid potential interference, we stained kidney sections using only VDR antibody to accurately assess VDR expression in kidney. We also used the kidney sections from Demay VDR knockout mice as proper negative controls to insure specific staining because these mice express neither native VDR nor truncated VDR protein.38.Wang Y. Becklund B.R. DeLuca H.F. Identification of a highly specific and versatile vitamin D receptor antibody.Arch Biochem Biophys. 2010; 494: 166-177Crossref PubMed Scopus (70) Google Scholar The staining showed that VDR was present in the entire renal tubules located in the cortex and medulla of the wild-type mouse kidney (Figures 4a–g). The distal tubules expressed VDR at higher levels and were mainly located within the cortex of the kidney. Levels of VDR in the distal tubules varied from tubule to tubule. The collecting ducts in the medulla were weakly stained. Besides these tubules, the majority of the renal tubules in the cortex were weakly stained. These tubules were likely the proximal tubules and cortical collecting ducts. In contrast to the renal tubular epithelial cells, VDR staining was not detected in the renal interstitial fibroblasts, located between renal tubules (Figures 4a, b, f, and g). There were also no significant signals in sections of the Demay VDR knockout kidneys (Figures 4h and i), and in the sections from wild-type mice stained only with the secondary antibody and mouse isotype IgG (Figure 3, upper panel). Together, these results demonstrate that the VDR immunosignals in wild-type mouse kidney sections were highly specific. The relative contents of VDR in these segments were measured in a semiquantitative manner using Image J software to quantitate the intensity of immunostaining in the nuclei of the renal tubular epithelial cells as described in the Materials and Methods section. Our results showed that VDR expression in the distal tubules was 24-fold higher than the proximal tubules. Differences between distal and proximal tubules ranged between 4- and 69-fold (Table 1). These results demonstrated that the VDR expression was low in proximal tubules and further indicated that the D-6 antibody was extremely sensitive and highly specific.Table 1Relative levels of VDR expression in mouse renal distal tubule and proximal tubuleType of tubulesDistal tubuleProximal tubuleAverage foldRangeVDR intensity7745±5994320±121244–69Abbreviation: VDR, vitamin D receptor.N=5 mice. Open table in a new tab Abbreviation: VDR, vitamin D receptor. N=5 mice. The VDR is highly expressed in distal tubules of the human kidney and is present in the proximal tubules and collecting ducts (Figure 5a and b). Parietal epithelial cells of glomerulus were also positive for VDR staining (Figures 5c and d). Noticeably, the VDR signal was not found in nuclei of the VDR-positive cells. In contrast, the hnRNP, a nuclear protein, which is normally localized in the nucleus, was found in the nuclei of human renal cells (Figure 5e), suggesting the nucleus–cytoplasm relocation of VDR in the human kidney. The reasons for the relocation were not clear. However, a number of factors, such as vitamin D status and tissue processing method, could alter the subcellular location of the receptor.38.Wang Y. Becklund B.R. DeLuca H.F. Identification of a highly specific and versatile vitamin D receptor antibody.Arch Biochem Biophys. 2010; 494: 166-177Crossref PubMed Scopus (70) Google Scholar,39Barsony J. Pike J.W. DeLuca H.F. et al.Immunocytology with microwave-fixed fibroblasts shows 1 alpha, 25-dihydroxyvitamin D3-dependent rapid and estrogen-dependent slow reorganization of vitamin D receptors.J Cell Biol. 1990; 111: 2385-2395Crossref PubMed Scopus (81) Google Scholar The glomerulus contains several cell types including glomerular capsule (parietal epithelial cells), parietal and visceral podocytes, mesangial cells, and endothelial cells. We showed that the VDR immunosignals were present within the glomerulus from the wild-type mice (Figures 6a–g), but not from the kidney samples from the Demay VDR knockout mice (Figure 6c). The expression of VDR seemed to be lower in podocytes than that in the proximal tubular cells. The VDR-positive cells located in the capsule and visceral areas of the glomerulus were likely parietal epithelial cells and podocytes. The parietal epithelial cells and podocytes have the same embryonic origin, but Wilms' tumor-1 (WT-1) is only present in podocytes.40.Bariety J. Mandet C. Hill G.S. et al.Parietal podocytes in normal human glomeruli.J Am Soc Nephrol. 2006; 17: 2770-2780Crossref PubMed Scopus (129) Google Scholar Costaining of kidney sections with the antibodies to VDR and WT-1 showed that VDR colocalized with WT-1, thereby demonstrating that these were podocytes (Figures 6f and g). Our results also suggest that VDR is very low or not found in the other cell types such as intraglomerular mesangial and endothelial cells. JGA is located at the vascular pole of each glomerulus.41.Peti-Peterdi J. Komlosi P. Fuson A.L. et al.Luminal NaCl delivery regulates basolateral PGE2 release from macula densa cells.J Clin Invest. 2003; 112: 76-82Crossref PubMed Scopus (138) Google Scholar We addressed if VDR was present in the juxtaglomerular cells and macula densa cells of JGA. Our results showed that the VDR was highly expressed in the macula densa (Figure 6a, b, d, and e). In contrast, the juxtaglomerular cells were not stained by the VDR antibody (Figures 6d and e). To specify the juxtaglomerular cells (smooth muscle origin), we first costained the glomeruli using antibodies to WT-1 and α-smooth muscle actin (αSMA), which is a marker for juxtaglomerular cells (Figures 6h and i). The juxtaglomerular cells strongly expressed αSMA, while the intraglomerular mesangial cells and some interstitial fibroblasts expressed αSMA at lower levels. Importantly, the αSMA staining was not colocalized with WT-1 staining within the JGA. Therefore, the αSMA-positive intraglomerular mesangial and ju