Klotho prevents renal calcium loss

Abstract

Disturbed calcium (Ca(2+)) homeostasis, which is implicit to the aging phenotype of klotho-deficient mice, has been attributed to altered vitamin D metabolism, but alternative possibilities exist. We hypothesized that failed tubular Ca(2+) absorption is primary, which causes increased urinary Ca(2+) excretion, leading to elevated 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and its sequelae. Here, we assessed intestinal Ca(2+) absorption, bone densitometry, renal Ca(2+) excretion, and renal morphology via energy-dispersive x-ray microanalysis in wild-type and klotho(-/-) mice. We observed elevated serum Ca(2+) and fractional excretion of Ca(2+) (FE(Ca)) in klotho(-/-) mice. Klotho(-/-) mice also showed intestinal Ca(2+) hyperabsorption, osteopenia, and renal precipitation of calcium-phosphate. Duodenal mRNA levels of transient receptor potential vanilloid 6 (TRPV6) and calbindin-D(9K) increased. In the kidney, klotho(-/-) mice exhibited increased expression of TRPV5 and decreased expression of the sodium/calcium exchanger (NCX1) and calbindin-D(28K), implying a failure to absorb Ca(2+) through the distal convoluted tubule/connecting tubule (DCT/CNT) via TRPV5. Gene and protein expression of the vitamin D receptor (VDR), 25-hydroxyvitamin D-1-alpha-hydroxylase (1alphaOHase), and calbindin-D(9K) excluded renal vitamin D resistance. By modulating the diet, we showed that the renal Ca(2+) wasting was not secondary to hypercalcemia and/or hypervitaminosis D. In summary, these findings illustrate a primary defect in tubular Ca(2+) handling that contributes to the precipitation of calcium-phosphate in DCT/CNT. This highlights the importance of klotho to the prevention of renal Ca(2+) loss, secondary hypervitaminosis D, osteopenia, and nephrocalcinosis.

Figure 1.

Klotho^−/− mice characteristics. Plots of serum Ca²⁺ concentration (mM) (A), fractional excretion (FE) of Ca²⁺ (B), urine pH (C), and urine osmolarity (D) from wild-type and klotho^−/− (KO) mice, n = 10 for both groups. *P < 0.05 in comparison to wild-type.

Figure 2.

Characterization of intestinal Ca²⁺ handling. qPCR analysis of TRPV6 (A), calbindin-D_9K (B), and PMCA1b (C) expression in duodenum. The results are expressed as a percentage of wild-type and are normalized to the expression of HPRT, n = 8 per group. A representative immunoblot (D) and quantification (E) of calbindin-D_9K protein expression from wild-type and klotho^−/− duodenum, n = 7 per group; note β-actin has been blotted (bottom panel) as a loading control. (F) ⁴⁵Ca²⁺ absorption into serum of wild-type (●) and klotho^−/− (♢) mice after gastric gavage, n = 8 per group. *P < 0.05 in comparison to wild-type.

Figure 3.

Characterization of the molecular mediators of renal transepithelial Ca²⁺ reabsorption. qPCR analysis of TRPV5 (A), NCX1 (B), calbindin-D_28K (C), and PMCA1b (D) mRNA expression in kidney. The results are expressed as a percentage of wild-type; both are normalized to the expression of HPRT, n = 8 per group. Representative immunoblot and quantification of calbindin-D_28K (E and F) and aquaporin-2 (I and J) protein expression from wild-type and klotho^−/− (KO) whole kidney lysate, n = 7 per group. Note β-actin has been blotted (bottom panel) as a loading control. Quantification of TRPV5 protein expression in kidney of wild-type and klotho^−/− (KO) mice (G), n = 6 per group. Representative images from the quantification are shown in H. *P < 0.05 in comparison to wild-type.

Figure 4.

Klotho knockout mice show Ca²⁺-phosphate precipitates. Low-power images of von Kossa–stained renal sections of wild-type (A) and klotho^−/− (B) mice. Toluidine blue–stained renal section of a peri-glomerular, stone-containing region of a klotho^−/− renal section (C); note arrows point to calcium precipitations. Transmission electron micrograph of renal cortex from a klotho^−/− renal section (D); note arrows point to calcium precipitations. A representative energy map (E) and x-ray spectra (F) from EDX measurements performed on the Ca²⁺-containing deposits. A high-magnification image of DCT/CNT (G–I) stained first with the von Kossa method to visualize Ca²⁺ and then immunostained with anti-calbindin-D_28K to localize the DCT/CNT.

Figure 5.

Characteristics of wild-type and klotho^−/− mice on control diet and LVD. Serum levels of 1,25(OH)₂D₃ (A) and Ca²⁺ (B) from wild-type (WT) and klotho^−/− (KO) mice on control diet (CD) or LVD, n = 6 per group. The fractional excretion (FE) of Ca²⁺ from wild-type and klotho^−/− mice on either CD or LVD, n = 6 per group. Renal TRPV5 (D), calbindin-D_28K (E), or duodenal calbindin-D_9K (F) protein expression from wild-type and klotho^−/− mice on either CD or LVD, n = 6 per group. (E and F) β-actin has been blotted as a loading control (bottom panel). (G) Representative three-dimensional reconstructions of femurs from wild-type and klotho^−/− mice on either CD or LVD, n = 4 per group. Note the thinner cortices (arrows) and reduced trabecular bone volume (arrowhead) in the CD-KO mice. The black boxes indicate the scan areas for the analyses of (1) trabecular and (2) cortical bone, respectively. *P < 0.05 compared with CD-WT.