Figure 1.
Dwarfism and organ atrophy in Klotho−/− mice is rescued by ablation of VDR function.
(A) X-ray image of 4-week-old WT, Kl−/−, VDRΔ/Δ, and Kl−/−/VDRΔ/Δ mice on rescue diet shows dwarfism in Kl−/− mice. VDRΔ/Δ and compound mutants display normal body size. (B–F) Relative to WT controls, Kl−/− mice show reduced body weight (BW), reduced absolute and relative organ weight of the pancreas, and reduced absolute but not relative organ weight of the spleen. Body weight and organ weights are indistinguishable between VDRΔ/Δ and compound mutants. All animals were fed the rescue diet. Each data point is the mean ± SEM of 4–8 animals per genotype. * denotes P<0.05 vs. WT mice, 1-way ANOVA followed by Student-Newman-Keuls test.
Figure 2.
Ablation of VDR signaling rescues skin atrophy and pulmonary emphysema in Klotho−/− mice.
Upper panel shows representative von Kossa-stained sections counterstained with nuclear fast red of thoracic aortae from 4-week-old WT, Kl−/−, VDRΔ/Δ, and Kl−/−/VDRΔ/Δ mice. No ectopic calcifications are present in Kl−/− mice but a thinning of the aortic wall is evident. Middle and lower panels show HE stained sections. Pulmonary emphysema and severe skin atrophy with almost complete lack of subcutaneous fat tissue is clearly evident in Kl−/− mice, but absent in Kl−/−/VDRΔ/Δ compound mutants. All animals were fed the rescue diet. Five-µm-thick paraffin sections. Bar = 200 µm.
Figure 3.
Defects in mineral homeostasis observed in Klotho−/− mice are normalized in Klotho−/−/VDRΔ/Δ double mutant mice.
(A–D) Analysis of mineral homeostasis in serum and urine shows hypercalcemia, hyperphosphatemia, and hyperphosphaturia in Kl−/− mice compared with WT controls, whereas compound mutants are normocalcemic and normophosphatemic. (E) Kl−/− mice display about 2- to 3-fold higher serum urea values than WT mice. (F) Serum intact parathyroid hormone (PTH) tends to be higher in VDR and compound mutants, relative to WT mice (n = 8–10 each). (G) mRNA abundance assessed in isolated parathyroid glands by qRT-PCR reveals higher PTH mRNA abundance in VDR and compound mutants relative to WT mice. Relative expression of Fgf receptor 1c (FGFR) and PTH is not different between VDRΔ/Δ and compound mutants, but also not different between WT and Kl−/− mice. Calcium-sensing receptor (CaR) mRNA expression was lower in VDRΔ/Δ compared with WT mice. Using primers located in the deleted region of the VDR in gene-targeted VDRΔ/Δ mice, relative VDR mRNA expression was not different between WT and Kl−/− mice, and undetectable in VDRΔ/Δ and compound mutants. (H) Representative Western blotting image of core (75 kD) and complex (95 kD) glycosylated TRPV5 protein expression in renal cortical total membrane fractions of 4-week-old WT, VDRΔ/Δ, Kl−/− and Kl−/−/VDRΔ/Δ mice. Membrane abundance of fully glycosylated TRPV5 is lower in Kl−/− and especially in Kl−/−/VDRΔ/Δ mice, relative to WT and VDRΔ/Δ mice. All animals were fed the rescue diet. Each data point is the mean ± SEM of 4–10 animals per genotype. * denotes P<0.05 vs. WT mice, 1-way ANOVA followed by Student-Newman-Keuls test.
Figure 4.
Bone phenotype of Klotho−/− mice is rescued in Klotho−/−/VDRΔ/Δ compound mutants.
(A–B) Three-µm-thick undecalcified plastic sections of distal femurs of 4-week-old WT, Kl−/−, VDRΔ/Δ, and Kl−/−/VDRΔ/Δ compound mutant mice, stained with von Kossa/McNeal. Low power view (A) shows normal bone architecture in VDR mutants and Kl−/−/VDRΔ/Δ compound mutants, while narrowing and premature ossification of the growth plate area is evident in Kl−/− mice. High power view (B) reveals severe osteomalacia (thickened osteoid seams are indicated by arrows) in cancellous bone of Kl−/− mice, but normal mineralization in the other genotypes. (C) Undecalcified sections of distal femurs histochemically stained for tartrate resistant acid phosphatase (TRACP) activity show normal numbers of osteoclasts in VDRΔ/Δ and Kl−/−/VDRΔ/Δ compound mutants, but profoundly reduced osteoclast numbers in the distal metaphysis of Kl−/− mice. Bar in A–C = 200 µm. (D) Femur length measured by calipers is lower in Kl−/− mice, but remains unchanged in VDR and compound mutants, relative to WT controls. (E) Trabecular volumetric bone mineral density (BMD) of the distal femoral metaphysis measured by peripheral quantitative computed tomography (pQCT). VDR mutants and Kl−/−/VDRΔ/Δ compound mutants show a slightly decreased femoral trabecular BMD, relative to WT mice. (F–G) Measurement of cancellous bone formation rate (F) and osteoclast number (G) in the distal femoral metaphysis by histomorphometry reveals almost undetectable calcein-based bone formation and decreased osteoclast numbers in Kl−/− mice, but normal bone turnover in VDR and compound mutant mice. All animals were fed the rescue diet. Each data point is the mean ± SEM of 6–7 animals per genotype. * denotes P<0.05 vs. WT mice, 1-way ANOVA followed by Student-Newman-Keuls test.
Figure 5.
Normalization of increased insulin sensitivity in Klotho−/− mice by ablation of vitamin D signaling.
(A–D) Glucose tolerance and insulin tolerance tests in 4-week-old WT, Kl−/−, VDRΔ/Δ, and Kl−/−/VDRΔ/Δ mice show hypoglycemia and profoundly improved glucose tolerance (A, C) and enhanced insulin sensitivity (B, D) in Kl−/− mice. Ablation of vitamin D signaling in compound mutants completely normalizes glucose and insulin tolerance. The area under the curve (AUC) for glucose and insulin tolerance tests is shown in C and D. Glucose (1.5 mg/g body weight) was administered subcutaneously at time 0 after two hours of fasting. Insulin (0.6 IE/kg) was injected intraperitoneally at time 0. (E–F) Fasting insulin and insulin serum levels measured 10 min after a subcutaneous glucose challenge (1.5 mg/g) are not significantly different between the groups, but tend to be lower in Kl−/− mice. (G–J) Histomorphometric analysis of anti-insulin-stained pancreatic paraffin sections (G) shows higher number of islets per tissue area in Kl−/− compared with WT mice (H), but unchanged mean islet size (I) and beta cell volume per kg BW (J) in all groups of animals. Bar in G = 200 µm. (K) Serum triglycerides are similar in all groups of animals. All animals were fed the rescue diet. Each data point is the mean ± SEM of 6–8 animals per genotype. * denotes P<0.05 vs. WT mice, 1-way ANOVA followed by Student-Newman-Keuls test.