FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2412505484> ?p ?o ?g. }

• W2412505484 endingPage "362" @default.
• W2412505484 startingPage "348" @default.
• W2412505484 abstract "Klotho is a transmembrane protein expressed in the renal tubules where it acts as a permissive coreceptor for fibroblast growth factor 23 (FGF23). FGF23 signaling reduces the abundance of CYP27b1 and phosphate cotransporters NPT2a and NPT2c, leading to a decrease in 1,25(OH)2D3 synthesis and a rise in urinary phosphate excretion, respectively. Systemic or whole-nephron deletion of Klotho in mice results in renal FGF23 resistance characterized by high 1,25(OH)2D3 and phosphate levels and premature aging. Expression of Klotho is highest in the distal tubules, whereas 25OH vitamin D 1α hydroxylation and phosphate reabsorption predominantly occur in the proximal tubules. Currently, the segment-specific roles of Klotho in renal tubules are not fully understood. Here we have generated mice with Klotho specifically ablated from the proximal tubules using 3 different Cre mouse strains. All 3 models displayed impaired urinary phosphate excretion and increased abundance of NPT2a in the brush border membrane. Notably, hyperphosphatemia in knockout mice was mild or nonexistent under basal conditions but occurred upon high phosphate loading, indicating the presence of compensatory mechanisms. Effects on 1,25(OH)2D3 varied between mouse strains but were modest overall. Thus, Klotho expressed in the proximal tubules has a defined but limited role in renal phosphate handling in vivo. Klotho is a transmembrane protein expressed in the renal tubules where it acts as a permissive coreceptor for fibroblast growth factor 23 (FGF23). FGF23 signaling reduces the abundance of CYP27b1 and phosphate cotransporters NPT2a and NPT2c, leading to a decrease in 1,25(OH)2D3 synthesis and a rise in urinary phosphate excretion, respectively. Systemic or whole-nephron deletion of Klotho in mice results in renal FGF23 resistance characterized by high 1,25(OH)2D3 and phosphate levels and premature aging. Expression of Klotho is highest in the distal tubules, whereas 25OH vitamin D 1α hydroxylation and phosphate reabsorption predominantly occur in the proximal tubules. Currently, the segment-specific roles of Klotho in renal tubules are not fully understood. Here we have generated mice with Klotho specifically ablated from the proximal tubules using 3 different Cre mouse strains. All 3 models displayed impaired urinary phosphate excretion and increased abundance of NPT2a in the brush border membrane. Notably, hyperphosphatemia in knockout mice was mild or nonexistent under basal conditions but occurred upon high phosphate loading, indicating the presence of compensatory mechanisms. Effects on 1,25(OH)2D3 varied between mouse strains but were modest overall. Thus, Klotho expressed in the proximal tubules has a defined but limited role in renal phosphate handling in vivo. Type I membrane-bound alpha-Klotho (Klotho) is predominantly expressed in the renal tubules,1Kuro-o M. Matsumura Y. Aizawa H. et al.Mutation of the mouse klotho gene leads to a syndrome resembling ageing.Nature. 1997; 390: 45-51Crossref PubMed Scopus (2797) Google Scholar where it acts as an obligate coreceptor for the phosphaturic hormone fibroblast growth factor 23 (FGF23).2Kuro-o M. Klotho as a regulator of fibroblast growth factor signaling and phosphate/calcium metabolism.Curr Opin Nephrol Hypertens. 2006; 15: 437-441Crossref PubMed Scopus (199) Google Scholar, 3Kurosu H. Ogawa Y. Miyoshi M. et al.Regulation of fibroblast growth factor-23 signaling by klotho.J Biol Chem. 2006; 281: 6120-6123Crossref PubMed Scopus (1067) Google Scholar, 4Urakawa I. Yamazaki Y. Shimada T. et al.Klotho converts canonical FGF receptor into a specific receptor for FGF23.Nature. 2006; 444: 770-774Crossref PubMed Scopus (1455) Google Scholar FGF23 binds to a Klotho-FGF-receptor complex4Urakawa I. Yamazaki Y. Shimada T. et al.Klotho converts canonical FGF receptor into a specific receptor for FGF23.Nature. 2006; 444: 770-774Crossref PubMed Scopus (1455) Google Scholar and inhibits renal phosphate reabsorption by internalizing the sodium-dependent phosphate cotransporters NPT2a and NPT2c.5Larsson T. Marsell R. Schipani E. et al.Transgenic mice expressing fibroblast growth factor 23 under the control of the alpha1(I) collagen promoter exhibit growth retardation, osteomalacia, and disturbed phosphate homeostasis.Endocrinology. 2004; 145: 3087-3094Crossref PubMed Scopus (438) Google Scholar, 6Bai X. Miao D. Li J. et al.Transgenic mice overexpressing human fibroblast growth factor 23 (R176Q) delineate a putative role for parathyroid hormone in renal phosphate wasting disorders.Endocrinology. 2004; 145: 5269-5279Crossref PubMed Scopus (301) Google Scholar, 7Baum M. Schiavi S. Dwarakanath V. Quigley R. Effect of fibroblast growth factor-23 on phosphate transport in proximal tubules.Kidney Int. 2005; 68: 1148-1153Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar FGF23 signaling also suppresses 1,25 dihydroxyvitamin D (1,25(OH)2D3) synthesis by decreasing the activating enzyme CYP27b1, and by increasing the catabolic enzyme CYP24a1.8Shimada T. Hasegawa H. Yamazaki Y. et al.FGF-23 is a potent regulator of vitamin D metabolism and phosphate homeostasis.J Bone Miner Res. 2004; 19: 429-435Crossref PubMed Scopus (1418) Google Scholar, 9Perwad F. Zhang M.Y. Tenenhouse H.S. et al.Fibroblast growth factor 23 impairs phosphorus and vitamin D metabolism in vivo and suppresses 25-hydroxyvitamin D-1alpha-hydroxylase expression in vitro.Am J Physiol Renal Physiol. 2007; 293: F1577-F1583Crossref PubMed Scopus (253) Google Scholar Systemic deletion of Klotho causes severe disturbances in mineral metabolism and an accelerated aging phenotype, involving growth retardation, organ dysfunction, vascular and soft tissue calcification, and premature death.1Kuro-o M. Matsumura Y. Aizawa H. et al.Mutation of the mouse klotho gene leads to a syndrome resembling ageing.Nature. 1997; 390: 45-51Crossref PubMed Scopus (2797) Google Scholar An almost identical phenotype is observed in mice with whole-nephron Klotho deletion, underlining the functional significance of renal Klotho.10Lindberg K. Amin R. Moe O.W. et al.The kidney is the principal organ mediating klotho effects.J Am Soc Nephrol. 2014; 25: 2169-2175Crossref PubMed Scopus (203) Google Scholar Because genetic or dietary correction of the hyperphosphatemia and high 1,25(OH)2D3 ameliorates the phenotype in Klotho-deficient mice,11Tsujikawa H. Kurotaki Y. Fujimori T. et al.Klotho, a gene related to a syndrome resembling human premature aging, functions in a negative regulatory circuit of vitamin D endocrine system.Mol Endocrinol. 2003; 17: 2393-2403Crossref PubMed Scopus (429) Google Scholar, 12Woudenberg-Vrenken T.E. van der Eerden B.C. van der Kemp A.W. et al.Characterization of vitamin D-deficient klotho(-/-) mice: do increased levels of serum 1,25(OH)2D3 cause disturbed calcium and phosphate homeostasis in klotho(-/-) mice?.Nephrol Dial Transplant. 2012; 27: 4061-4068Crossref PubMed Scopus (17) Google Scholar, 13Ohnishi M. Nakatani T. Lanske B. Razzaque M.S. In vivo genetic evidence for suppressing vascular and soft-tissue calcification through the reduction of serum phosphate levels, even in the presence of high serum calcium and 1,25-dihydroxyvitamin D levels.Circ Cardiovasc Genet. 2009; 2: 583-590Crossref PubMed Scopus (95) Google Scholar, 14Ohnishi M. Nakatani T. Lanske B. Razzaque M.S. Reversal of mineral ion homeostasis and soft-tissue calcification of klotho knockout mice by deletion of vitamin D 1alpha-hydroxylase.Kidney Int. 2009; 75: 1166-1172Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 15Kuro-o M. Phosphate and Klotho.Kidney Int Suppl. 2011; : S20-S23Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar the phenotype is at least in part related to the role of Klotho in renal phosphate and vitamin D handling. In the kidney, Klotho is expressed primarily in the distal tubules, and to a much lower extent in the proximal tubules.16Hu M.C. Shi M. Zhang J. et al.Klotho: a novel phosphaturic substance acting as an autocrine enzyme in the renal proximal tubule.FASEB J. 2010; 24: 3438-3450Crossref PubMed Scopus (469) Google Scholar However, renal phosphate is reabsorbed mainly in the proximal tubules17Blaine J. Chonchol M. Levi M. Renal control of calcium, phosphate, and magnesium homeostasis.Clin J Am Soc Nephrol. 2015; 10: 1257-1272Crossref PubMed Scopus (431) Google Scholar, 18Biber J. Hernando N. Forster I. Phosphate transporters and their function.Annu Rev Physiol. 2013; 75: 535-550Crossref PubMed Scopus (129) Google Scholar, 19Prasad N. Bhadauria D. Renal phosphate handling: physiology.Indian J Endocrinol Metab. 2013; 17: 620-627Crossref PubMed Google Scholar and activation of vitamin D occurs primarily in the proximal tubules.20Nykjaer A. Dragun D. Walther D. et al.An endocytic pathway essential for renal uptake and activation of the steroid 25-(OH) vitamin D3.Cell. 1999; 96: 507-515Abstract Full Text Full Text PDF PubMed Scopus (847) Google Scholar It is currently unknown whether Klotho expressed in the proximal tubule is sufficient to mediate the phosphaturic and anti–vitamin D actions of FGF23, or whether cross talk between the distal and proximal tubules is needed for such regulation. Whereas one study21Farrow E.G. Davis S.I. Summers L.J. White K.E. Initial FGF23-mediated signaling occurs in the distal convoluted tubule.J Am Soc Nephrol. 2009; 20: 955-960Crossref PubMed Scopus (191) Google Scholar reported activation of the mitogen-activated protein kinase/extracellular signal–regulated kinase (MAPK/ERK) pathway exclusively in the distal tubules upon administration of FGF23, a subsequent study reported direct effects of FGF23 in microdissected proximal tubules.22Andrukhova O. Zeitz U. Goetz R. et al.FGF23 acts directly on renal proximal tubules to induce phosphaturia through activation of the ERK1/2-SGK1 signaling pathway.Bone. 2012; 51: 621-628Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar We have previously shown that ablation of Klotho specifically from the distal tubules results in a hyperphosphatemic phenotype, although not as pronounced as in the systemic or whole-nephron knockouts.23Olauson H. Lindberg K. Amin R. et al.Targeted deletion of Klotho in kidney distal tubule disrupts mineral metabolism.J Am Soc Nephrol. 2012; 23: 1641-1651Crossref PubMed Scopus (124) Google Scholar Also, mice with a distal tubule-specific deletion of Klotho have unchanged serum levels of 1,25(OH)2D3. This proposes a functional role for proximal tubular Klotho in regulating phosphate reabsorption and controlling 1,25(OH)2D3 synthesis. To explore the functional role of Klotho in proximal tubules, we generated mice with Klotho specifically ablated from the proximal tubules using Cre-Lox recombination. Because no single Cre strain provides efficient recombination in all proximal tubular segments, 3 different models were established. Floxed Klotho mice (Klothofl/fl) were crossed with mice expressing Cre recombinase under the kidney androgen-regulated protein (KapCre), the phosphoenolpyruvate carboxykinase (PEPCKCre), and the type II sodium phosphate cotransporter, member 1 (Slc34a1Cre) promoters, respectively. Using this approach, we demonstrate a key role for proximal tubular Klotho in renal phosphate handling and vitamin D metabolism. Importantly, while global and whole-nephron deletion of Klotho result in a severe phenotype, deletion in either the distal or the proximal renal tubules alone results only in minor changes in mineral homeostasis and a normal gross phenotype. These results are the first to suggest an integrated regulatory function of Klotho in the distal and proximal tubules on mineral metabolism. First, the renal expression pattern of Klotho was examined using immunohistochemistry. Klotho staining was detected in the renal cortex but not in the medulla of wild-type mice (Figure 1a, left panel). In the cortex, distal tubules had a strong positive staining, whereas proximal tubules stained substantially weaker (Figure 1a, right panel). Next, we explored the expression of Klotho mRNA using RNAScope, a novel method for in situ hybridization.24Wang F. Flanagan J. Su N. et al.RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.J Mol Diagn. 2012; 14: 22-29Abstract Full Text Full Text PDF PubMed Scopus (1463) Google Scholar In concordance with the immunohistochemical findings, Klotho RNA was predominately localized to distal tubules, with weaker but positive expression in proximal tubules (Figure 1b and c). The expression of Klotho in distal tubules was too high to allow for exact quantification and thus it was not possible to calculate a ratio of expression in proximal-distal tubules. KapCre;Klothofl/fl (Kap-KL) and Slc34a1Cre;Klothofl/fl (Slc34a1-KL) were generated and analyzed at Harvard School of Dental Medicine (Boston, MA, USA) and PEPCKCre;Klothofl/fl (PEPCK-KL) was generated and analyzed at Karolinska Institutet (Stockholm, Sweden). Testosterone pellets were implanted at 6 weeks of age in order to increase Cre expression in Kap-KL mice. Tamoxifen was administered at 6 weeks of age to induce Cre expression in Slc34a1-KL mice. Mice were sacrificed and analyzed at 14, 12, and 9 weeks, respectively. The phenotypes of the 3 strains are summarized and compared in Table 1.Table 1Phenotype comparisonKAP-KLPEPCK-KLSLC34a1-KLSerum Calcium−−↑ Phosphate−/↑ *−↑ Fgf23−↑↑− PTH−−− 1,25(OH)2D3−−−Urine Ca−−− Pi−/↓ *↓↓↓BBM NaPi2a↑↑↑↑↑↑Each strain is compared with its appropriate wild-type control. −, unchanged and/or normal; *, on high phosphate drinking water.BBM, brush border membrane; Ca, calcium; Fgf23, fibroblast growth factor 23; Pi, phosphate; PTH, parathyroid hormone. Open table in a new tab Each strain is compared with its appropriate wild-type control. −, unchanged and/or normal; *, on high phosphate drinking water. BBM, brush border membrane; Ca, calcium; Fgf23, fibroblast growth factor 23; Pi, phosphate; PTH, parathyroid hormone. First, we used mice expressing Cre recombinase under the Kap promoter, as this is the best characterized proximal tubular-specific Cre strain available through public, open mice repositories. Cre expression in these mice has previously been shown to be restricted to the proximal tubules.25Li H. Zhou X. Davis D.R. et al.An androgen-inducible proximal tubule-specific Cre recombinase transgenic model.Am J Physiol Renal Physiol. 2008; 294: F1481-F1486Crossref PubMed Scopus (19) Google Scholar, 26Ding Y. Sigmund C.D. Androgen-dependent regulation of human angiotensinogen expression in KAP-hAGT transgenic mice.Am J Physiol Renal Physiol. 2001; 280: F54-F60PubMed Google Scholar To analyze the recombination specificity and efficiency of this line, mice were crossed with a tdTomato reporter strain. For our experiments we used KapCre:Klothofl/fl;tdTomatofl/+ (Kap-KL) and KapCre;tdTomatofl/+ (Kap-WT) mice. Kap-KL mice had a normal gross phenotype (Figure 2a) and survived up to 12 months with none of the mice dying spontaneously. First, we identified the cells that express Cre recombinase in sections of kidney that were double-stained with Lotus tetragonolobus lectin (LTL) that specifically labels differentiated proximal tubules and an anti–red fluorescent protein (RFP) antibody to detect tdTomato protein. The recombination efficiency, as determined by the tdTomato/LTL ratio, was found to be 66% in Kap-KL mice at 14 weeks of age (Figure 2b). Deletion of Klotho from proximal tubules was confirmed using immunohistochemistry (Figure 2c). In isolated kidney proximal tubular cell homogenates, Klotho mRNA expression was reduced by 66% (P < 0.05, n = 6) (Figure 2d) and Klotho protein expression was reduced by 53% (P < 0.01, n = 6) (Figure 2e). Analyses of biochemical parameters demonstrated no significant changes in urine or serum levels of calcium and phosphate in Kap-KL mice when compared with control mice (Figure 2f). Next, we measured the regulatory factors that control serum calcium and phosphate levels. Mineral metabolism-regulating hormones, such as intact Fgf23, parathyroid hormone (PTH), and 1,25(OH)2D3 were not different compared with wild-type control mice (Figure 2f). Renal gene expression was examined by quantitative polymerase chain reaction (qPCR). As expected, transcript levels of Npt2a were significantly increased in Kap-KL mice, whereas Npt2c expression levels were unchanged. In addition, both Cyp27b1 and Cyp24a1 were significantly increased in Kap-KL mice (Figure 2g). Western blot analysis revealed significantly increased Npt2a expression at brush border membrane (BBM) in Kap-KL mice compared with Kap-WT mice (Figure 2h, Supplementary Figure S1A).Figure 2Characterization of Kap-KL mice at 14 weeks of age. (a) Kap-WT (left) mice and Kap-KL (right) mice had a normal gross appearance. Bar = 1 cm. (b) Kidney sections were double stained with Lotus tetragonolobus lectin (LTL) and an antired fluorescent protein (RFP) antibody. Bar = 500 μm. (c) Immunohistochemical staining of Klotho revealed deletion of Klotho in Kap-KL kidney by reduced brown 3,3′-diaminobenzidine staining in PT. Bar = 50 μm. Arrows point to stronger and remaining Klotho staining in DT. Klotho mRNA and protein levels were measured by (d) quantitative polymerase chain reaction and (e) Western blot analysis from isolated kidney proximal tubules of Kap-KL and Kap-WT mice (n = 6). (f) Measurements of serum and urine Ca and Pi levels and regulatory factors in Kap-KL and Kap-WT mice (n = 4–6). (g) Relative mRNA levels of renal transcripts (Npt2a, Npt2c, Cyp27b1, and Cyp24a1) were measured by quantitative polymerase chain reaction in Kap-KL mice and Kap-WT mice (n = 4–6). (h) Npt2a expression was detected by Western blot analysis. Brush border membrane was isolated from the kidney of Kap-KL and Kap-WT mice. β-Actin was used as an internal control (P < 0.01, n = 3). Data represent the means ± SEM. *P< 0.05; **P< 0.01. AU, arbitrary unit; DAPI, 4′,6-diamidino-2-phenylindole; DT, distal tubules; Fgf23, fibroblast growth factor 23; G, glomerular; KL, Klotho; PT, proximal tubules; PTH, parathyroid hormone; S-Ca, serum calcium; S-Pi, serum phosphate; U-Ca, urinary calcium excretion per urinary creatinine; U-Cr, urinary creatinine excretion; U-Pi, urinary phosphate excretion per urinary creatinine; WT, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Next, we examined mice expressing Cre recombinase under the rat PEPCK promoter. In the kidney, PEPCK-Cre is expressed in most S1 and S2 and all S3 segments of the proximal tubule.27Rankin E.B. Tomaszewski J.E. Haase V.H. Renal cyst development in mice with conditional inactivation of the von Hippel-Lindau tumor suppressor.Cancer Res. 2006; 66: 2576-2583Crossref PubMed Scopus (287) Google Scholar Knockout mice (PEPCKCre;Klothofl/fl–PEPCK-KL) were generated and compared with floxed Klotho mice (Klothofl/fl–PEPCK-WT) at 12 weeks of age. Because PEPCK-Cre is X-linked, only male mice were analyzed. Efficient Klotho deletion in the proximal tubules was confirmed by in situ hybridization, immunohistochemistry, and qPCR (Figure 3a and b). Klotho mRNA expression levels were reduced by 36% in the renal cortex of PEPCK-KL mice (P < 0.01, n = 5) (Figure 3b). Serum calcium and phosphate levels were unchanged in PEPCK-KL mice, but urinary phosphate excretion was significantly lower than in control littermates (Figure 3c). Serum intact Fgf23 levels were significantly increased in PEPCK-KL mice, whereas 1,25(OH)2D3 and PTH levels were comparable to those of PEPCK-WT mice (Figure 3c). Gene expression analysis revealed that Npt2a and Npt2c expression levels were unchanged, Cyp27b1 was slightly but not statistically increased, whereas Cyp24a1 was significantly up-regulated in the renal cortex of PEPCK-KL mice (Supplementary Figure S2A). Western blot analysis showed significantly increased expression of Npt2a at BBM in PEPCK-KL mice compared with PEPCK-WT (P < 0.001, n = 4) (Figure 3d, Supplementary Figure S1B). As a third model for proximal tubular Klotho deletion, we used mice expressing Cre under the Slc34a1 promoter. These mice have previously been shown to have efficient recombination in all cortical segments of the proximal tubules upon Tamoxifen administration, but most efficiently in the S1 and S2 segments.28Kusaba T. Lalli M. Kramann R. et al.Differentiated kidney epithelial cells repair injured proximal tubule.Proc Natl Acad Sci U S A. 2014; 111: 1527-1532Crossref PubMed Scopus (309) Google Scholar In our analysis, we compared conditional Klotho knockout mice carrying a tomato reporter gene (Slc34a1cre;Tomatofl/+;Klothofl/fl–Slc34a1-KL) with mice only expressing the reporter gene (Slc34a1cre;Tomatofl/+–Slc34a1-WT). Slc34a1-KL mice displayed no gross abnormalities (Figure 4a). Kidney sections were double-stained with LTL and RFP antibody to detect tdTomato protein. The recombination efficiency, as determined by the tdTomato/LTL overlap, was found to be 95% at 9 weeks of age (Figure 4b). Deletion of Klotho was confirmed in Slc34a1-KL using immunohistochemistry (Figure 4c). Klotho deletion was also examined by qPCR, Western blot analysis, and flow cytometry (Figure 4d–f). Klotho mRNA expression was reduced by 68% in isolated kidney proximal tubular cells of Slc34a1-KL mice as determined by qPCR (P < 0.05, n = 6) (Figure 4d), and Klotho protein expression was reduced by 62% and 64% as determined by Western blot analysis (P < 0.01, n = 6) and flow cytometry (P < 0.0001, n = 3), respectively (Figure 4e and f). Importantly, at 9 weeks of age, Slc34a1-KL mice had significantly increased serum calcium and phosphate levels, and markedly decreased urinary phosphate excretion (Figure 4g). In contrast, hormones regulating mineral metabolism (intact Fgf23, PTH, and 1,25(OH)2D3) were not different compared with those of control mice (Figure 4g). Gene expression analysis revealed that Npt2a expression levels were significantly increased, whereas Npt2c and Cyp24a1 were unchanged in Slc34a1-KL mice. Cyp27b1 mRNA was slightly decreased but without reaching statistical significance (Supplementary Figure S2B). Western blot analysis of Npt2a in BBM showed significantly higher expression in Slc34a1-KL than in Slc34a1-WT mice (Figure 4h, Supplementary Figure S1C).Figure 4Characterization of Slc34a1-KL mice at 9 weeks of age. (a) Slc34a1-WT (left) and Slc34a1-KL mice (right) had a normal gross appearance. Bar = 1 cm. (b) Kidney sections were double stained with Lotus tetragonolobus lectin (LTL) and an antired fluorescent protein (RFP) antibody (left, bar = 2 mm; right, bar = 500 μm). (c) Immunohistochemical staining of Klotho revealed deletion of proximal tubular Klotho in Slc34a1-KL (bar = 100 μm). Klotho mRNA and protein levels were measured by (d) quantitative polymerase chain reaction and (e) Western blot analysis from isolated kidney proximal tubules of Slc34a1-KL and Slc34a1-WT mice (P < 0.01, n = 6). (f) Quantification of Klotho and tdTomato double positive cells were determined by flow cytometry (P < 0.0001, n = 3). (g) Serum and urinary biochemistry, and regulatory factors (intact Fgf23, 1,25(OH)2D3, and PTH) were measured in Slc34a1-KL mice and Slc34a1-WT mice (n = 4–6). (h) Western blot analysis of Npt2a expression in brush border membrane from Slc34a1-KL mice and Slc34a1-WT mice. β-Actin was used as an internal control (P < 0.05, n = 4). The data represent the means ± SEM. *P < 0.05; **P < 0.01; ****P < 0.0001. AU, arbitrary unit; DAPI, 4′,6-diamidino-2-phenylindole; Fgf23, fibroblast growth factor 23; KL, Klotho; PTH, parathyroid hormone; S-Ca, serum calcium; S-Pi, serum phosphate; U-Ca, urinary calcium excretion per urinary creatinine; U-Cr, urinary creatinine excretion; U-Pi, urinary phosphate excretion per urinary creatinine; WT, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To put further stress on renal phosphate handling, Kap-KL and Kap-WT mice were challenged with high phosphate (HP) in the drinking water for 8 weeks. Serum phosphate levels were markedly increased in Kap-KL on HP, whereas they remained unchanged on normal phosphate (NP) (Figure 5a). We evaluated the effect of Klotho deletion and HP using a 2-way analysis of variance (ANOVA). Klotho deletion had a significant main effect on serum phosphate (F(1,19) = 24.62, P < 0.001), whereas HP did not have this effect (F(1,19) = 4.297, P = NS). No interaction effect of Klotho deletion and HP was observed (F(1,19) = 3.478, P = NS). These data indicate that mice with conditional deletion of Klotho in proximal tubules had significantly increased serum phosphate levels when compared with control mice (Supplementary Table S1). Serum calcium and urinary calcium excretion did not differ between the groups on NP and HP (Supplementary Figure S3A). Importantly, Kap-WT mice substantially increased their urinary phosphate excretion after HP loading, whereas urinary phosphate excretion in Kap-KL mice on HP did not change (Figure 5a). This effect was not due to decreased renal function as serum creatinine and blood urea nitrogen were similar between groups (Supplementary Figure S3B). Serum intact Fgf23 was significantly increased in both Kap-KL and Kap-WT mice on HP, whereas 1,25(OH)2D3 levels were significantly increased only in Kap-KL mice on HP (Figure 5b). PTH levels increased significantly in Kap-WT mice on HP when compared with unchallenged mice, whereas no changes were noted in Kap-KL mice (Figure 5b). Gene expression analysis revealed that Cyp27b1 expression levels were significantly higher in Kap-KL mice on HP compared with Kap-WT control mice, whereas expression levels of Cyp24a1 significantly increased in Kap-WT mice and remained high in Kap-KL mice (Figure 5c). All results of a 2-way ANOVA for Klotho deletion and HP in Kap-KL and Kap-WT mice are shown in Supplementary Table S1. Slc34a1-KL and Slc34a1-WT mice were challenged with HP in the drinking water starting at 6 weeks of age for a period of 8 weeks. Serum phosphate levels in Slc34a1-WT did not significantly increase upon HP, and Slc34a1-KL did not further increase their already higher basal serum Pi concentrations (Supplementary Figure S3C). Urinary phosphate excretion levels were significantly higher in Slc34a1-WT mice compared with Slc34a1-KL mice after HP exposure (Supplementary Figure S3C). To prevent potential systemic compensatory effects, 9-week-old Slc34a1-KL and Slc34a1-WT mice were provided with high serum phosphate drinking water for 24 hours. Serum phosphate levels were significantly increased in Slc34a1-KL mice, whereas they remained normal in Slc34a1-WT mice (Figure 5d). The rate of urinary phosphate excretion increased in Slc34a1-WT mice as compensatory effect to the additional phosphate uptake but remained unchanged in Slc34a1-KL mice (Figure 5d), leading to hyperphosphatemia. Renal function as measured by serum creatinine and blood urea nitrogen did not change in neither Slc34a1-WT nor Slc34a1-KL mice on 24-hour HP (Supplementary Figure S3D). All results of a 2-way ANOVA for Klotho deletion and HP in Slc34a1-KL mice and Slc34a1-WT mice are shown in Supplementary Table S1. Serum soluble Klotho has been suggested to derive mainly from renal distal tubules10Lindberg K. Amin R. Moe O.W. et al.The kidney is the principal organ mediating klotho effects.J Am Soc Nephrol. 2014; 25: 2169-2175Crossref PubMed Scopus (203) Google Scholar and to exhibit phosphaturic effects, thereby providing compensatory mechanisms to mask the loss of Klotho in renal proximal tubules. We, therefore, measured serum levels of soluble Klotho in 2 of our mouse lines. No significant differences in serum soluble Klotho levels could be detected in adult Kap-KL, Kap-WT, Slc34a1-KL, and Slc34a1-WT mice (Figure 6). To examine whether deletion of Klotho from renal proximal tubules and potential uncovered associated changes in phosphate metabolism could affect bone metabolism, we measured serum CTX (C-terminal telopeptides of type I collagen) levels, a well-known bone resorption marker, in our 3 mouse lines. No significant difference could be detected between control and conditional mutant mice for any of the lines (Supplementary Figure S4A). Next, we performed routine histological analysis of the long bones of Slc34a1-KL mice, as this line had the most efficient deletion of Klotho and also showed slight hyperphosphatemia. Hematoxylin and eosin stained paraffin sections of the Slc34a1-KL tibia showed a marked increase in trabecular bone volume and also thicker cortical bone (Supplementary Figure S4B and C). Moreover, the size of the growth plate was narrower when compared with that of Slc34a1-WT control mice (Supplementary Figure S4D). The phenotype of the 3 proximal tubule-specific Klotho knockout mouse lines is compared and summarized in Table 1. In addition, all 3 models displayed normal gross appearance, normal growth, and the absence of premature aging features. No effect on survival was found in any of the 3 mouse lines with observations of Kap-KL and Slc34a1-KL mice for up to 12 months and PEPCK-KL mice up to 9 months. All models exhibited hypophosphaturia (unchallenged or on HP) and increased renal Npt2a expression. Other factors varied among the models. A schematic outline of the effects of Klotho deletion from different tubular segments is illustrated in Figure 7. In the current study, we provide the first in vivo evidence for a functional role of proximal tubular Klotho in renal phosphate handling and vitamin D metabolis" @default.
• W2412505484 created "2016-06-24" @default.
• W2412505484 creator A5004405514 @default.
• W2412505484 creator A5007789093 @default.
• W2412505484 creator A5051341882 @default.
• W2412505484 creator A5055588517 @default.
• W2412505484 creator A5056680996 @default.
• W2412505484 creator A5062845949 @default.
• W2412505484 creator A5071947921 @default.
• W2412505484 creator A5088544652 @default.
• W2412505484 date "2016-08-01" @default.
• W2412505484 modified "2023-10-13" @default.
• W2412505484 title "In vivo evidence for a limited role of proximal tubular Klotho in renal phosphate handling" @default.
• W2412505484 cites W1902909046 @default.
• W2412505484 cites W1960895270 @default.
• W2412505484 cites W1966074338 @default.
• W2412505484 cites W1996966304 @default.
• W2412505484 cites W2005107495 @default.
• W2412505484 cites W2007546762 @default.
• W2412505484 cites W2012675371 @default.
• W2412505484 cites W2015668150 @default.
• W2412505484 cites W2019253822 @default.
• W2412505484 cites W2019620862 @default.
• W2412505484 cites W2021852822 @default.
• W2412505484 cites W2028480953 @default.
• W2412505484 cites W2028927099 @default.
• W2412505484 cites W2045131606 @default.
• W2412505484 cites W2045867494 @default.
• W2412505484 cites W2047329464 @default.
• W2412505484 cites W2052887604 @default.
• W2412505484 cites W2055071554 @default.
• W2412505484 cites W2056109978 @default.
• W2412505484 cites W2059629870 @default.
• W2412505484 cites W2065463926 @default.
• W2412505484 cites W2066802616 @default.
• W2412505484 cites W2076129076 @default.
• W2412505484 cites W2091948921 @default.
• W2412505484 cites W2092830611 @default.
• W2412505484 cites W2097034311 @default.
• W2412505484 cites W2103313018 @default.
• W2412505484 cites W2105755732 @default.
• W2412505484 cites W2106223721 @default.
• W2412505484 cites W2106711282 @default.
• W2412505484 cites W2111387279 @default.
• W2412505484 cites W2111410841 @default.
• W2412505484 cites W2114360920 @default.
• W2412505484 cites W2114372930 @default.
• W2412505484 cites W2114669693 @default.
• W2412505484 cites W2123072873 @default.
• W2412505484 cites W2127396817 @default.
• W2412505484 cites W2128059069 @default.
• W2412505484 cites W2132961875 @default.
• W2412505484 cites W2133492406 @default.
• W2412505484 cites W2142139309 @default.
• W2412505484 cites W2144414222 @default.
• W2412505484 cites W2146666133 @default.
• W2412505484 cites W2148382170 @default.
• W2412505484 cites W2149900560 @default.
• W2412505484 cites W2154287224 @default.
• W2412505484 cites W2161470994 @default.
• W2412505484 cites W2164533721 @default.
• W2412505484 cites W2169367958 @default.
• W2412505484 cites W2169669025 @default.
• W2412505484 cites W2270636614 @default.
• W2412505484 cites W2334275353 @default.
• W2412505484 cites W4245958271 @default.
• W2412505484 doi "https://doi.org/10.1016/j.kint.2016.04.009" @default.
• W2412505484 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/27292223" @default.
• W2412505484 hasPublicationYear "2016" @default.
• W2412505484 type Work @default.
• W2412505484 sameAs 2412505484 @default.
• W2412505484 citedByCount "58" @default.
• W2412505484 countsByYear W24125054842016 @default.
• W2412505484 countsByYear W24125054842017 @default.
• W2412505484 countsByYear W24125054842018 @default.
• W2412505484 countsByYear W24125054842019 @default.
• W2412505484 countsByYear W24125054842020 @default.
• W2412505484 countsByYear W24125054842021 @default.
• W2412505484 countsByYear W24125054842022 @default.
• W2412505484 countsByYear W24125054842023 @default.
• W2412505484 crossrefType "journal-article" @default.
• W2412505484 hasAuthorship W2412505484A5004405514 @default.
• W2412505484 hasAuthorship W2412505484A5007789093 @default.
• W2412505484 hasAuthorship W2412505484A5051341882 @default.
• W2412505484 hasAuthorship W2412505484A5055588517 @default.
• W2412505484 hasAuthorship W2412505484A5056680996 @default.
• W2412505484 hasAuthorship W2412505484A5062845949 @default.
• W2412505484 hasAuthorship W2412505484A5071947921 @default.
• W2412505484 hasAuthorship W2412505484A5088544652 @default.
• W2412505484 hasBestOaLocation W24125054841 @default.
• W2412505484 hasConcept C126322002 @default.
• W2412505484 hasConcept C126894567 @default.
• W2412505484 hasConcept C134018914 @default.
• W2412505484 hasConcept C150903083 @default.
• W2412505484 hasConcept C185592680 @default.
• W2412505484 hasConcept C207001950 @default.
• W2412505484 hasConcept C2777132085 @default.
• W2412505484 hasConcept C2780091579 @default.

• next