The anti-aging gene klotho plays an important role in Ca 2ϩ and phosphate homeostasis. Membrane-bound klotho is an essential coreceptor for fibroblast growth factor-23 and can be cleaved by proteases, including a disintegrin and metalloproteinase (ADAM)10 and ADAM17. Cleavage of klotho occurs at a site directly above the plasma membrane (␣-cut) or between the KL1 and KL2 domain (␤-cut), resulting in soluble full-length klotho or KL1 and KL2 fragments, respectively. The aim of the present study was to gain insights into the mechanisms behind klotho cleavage processes in the kidney. Klotho shedding was demonstrated using a Madin-Darby canine kidney cell line stably expressing klotho and human embryonic kidney-293 cells transiently transfected with klotho. Here, we report klotho expression on both the basolateral and apical membrane, with a higher abundance of klotho at the apical membrane and in the apical media. mRNA expression of ADAM17 and klotho were enriched in mouse distal convoluted and connecting tubules. In vitro ADAM/matrix metalloproteinase inhibition by TNF484 resulted in a concentration-dependent inhibition of the ␣-cut, with a less specific effect on ␤-cut shedding. In vivo TNF484 treatment in wild-type mice did not change urinary klotho levels. However, ADAM/matrix metalloproteinase inhibition did increase renal and duodenal mRNA expression of phosphate transporters, whereas serum phosphate levels were significantly decreased. In conclusion, our data show that renal cells preferentially secrete klotho to the apical side and suggest that ADAMs are responsible for ␣-cut cleavage.klotho; a disintegrin and metalloproteinase 10; a disintegrin and metalloproteinase 17; klotho shedding; a disintegrin and metalloproteinase/matrix metalloproteinase inhibitor CA 2ϩ AND PHOSPHATE HOMEOSTASIS is a tightly regulated process involving several hormones. Klotho is emerging as a key factor in this process (5,18,51). Initially, the klotho gene was discovered as an anti-aging gene, since lack of klotho in mice resulted in early aging and a premature death (23, 26), whereas overexpression of klotho led to an increased lifespan (28).Klotho is a single-pass type 1 transmembrane protein consisting of two extracellular domains, which are the KL1 and KL2 subunits. Full-length klotho has potential enzymatic activity (30, 49), and in the body, it exists in two forms, namely, membrane-bound and soluble klotho. Membrane-bound klotho is predominantly present in the renal distal convolution, consisting of the distal convoluted tubule (DCT) and connecting tubule (CNT), parathyroid glands, and choroid plexus in the brain (26,31). A key function of membrane-bound klotho is to act as an obligate cofactor for the fibroblast growth factor (FGF) receptor, thereby enabling binding and downstream signaling of FGF23 via the ERK1/2 MAPK pathway (27, 51). In the kidney, binding of FGF23 to the receptor complex results in enhanced phosphate excretion via reduced expression of the Na ϩ -phosphate transporters NaPi-IIa and NaPiIIc (12). Moreov...

In conclusion, impaired peripheral endothelium-dependent vasodilatation in 5/6Nx mice is mediated by FGF23 and can be prevented by blocking FGF23. These data corroborate FGF23 as an important target to combat cardiovascular disease in CKD.

Calcium (Ca2+) is vital for multiple processes in the body, and maintenance of the electrolyte concentration is required for everyday physiological function. In the kidney, and more specifically, in the late distal convoluted tubule and connecting tubule, the fine-tuning of Ca2+ reabsorption from the pro-urine takes place. Here, Ca2+ enters the epithelial cell via the transient receptor potential vanilloid receptor type 5 (TRPV5) channel, diffuses to the basolateral side bound to calbindin-D28k and is extruded to the blood compartment via the Na+/Ca2+ exchanger 1 (NCX1) and the plasma membrane Ca2+ ATPase (PMCA). Traditionally, PMCA1 was considered to be the primary Ca2+ pump in this process. However, in recent studies TRPV5-expressing tubules were shown to highly express PMCA4. Therefore, PMCA4 may have a predominant role in renal Ca2+ handling. This study aimed to elucidate the role of PMCA4 in Ca2+ homeostasis by characterizing the Ca2+ balance, and renal and duodenal Ca2+-related gene expression in PMCA4 knockout mice. The daily water intake of PMCA4 knockout mice was significantly lower compared to wild type littermates. There was no significant difference in serum Ca2+ level or urinary Ca2+ excretion between groups. In addition, renal and duodenal mRNA expression levels of Ca2+-related genes, including TRPV5, TRPV6, calbindin-D28k, calbindin-D9k, NCX1 and PMCA1 were similar in wild type and knockout mice. Serum FGF23 levels were significantly increased in PMCA4 knockout mice. In conclusion, PMCA4 has no discernible role in normal renal Ca2+ handling as no urinary Ca2+ wasting was observed. Further investigation of the exact role of PMCA4 in the distal convoluted tubule and connecting tubule is required.

Background/Aims: Fine-tuning of renal calcium (Ca²⁺) reabsorption takes place in the late distal convoluted and connecting tubules (DCT2/CNT) of the kidney via transcellular Ca²⁺ transport. Here, Ca²⁺ enters the cell at the apical side via the epithelial Ca²⁺ channel transient receptor potential vanilloid 5 and is subsequently extruded at the basolateral side by the concerted actions of the plasma membrane Ca²⁺ ATPases and the Na⁺/Ca²⁺ exchanger 1 (NCX1). NCX1 is responsible for ∼70% of basolateral Ca²⁺ extrusion. The aim of this study was to determine the predominant NCX1 variant in the kidney and its role in Ca²⁺ transport. Methods: DCT2/CNT specific tubules were used to show the abundance of NCX1 specific isoforms. Renal NCX1 variants were cloned from mouse kidney tissue. Human Embryonic Kidney 293(T) cells were transiently transfected with NCX1.3, and Fura-2 measurements and ⁴⁵Ca²⁺ uptake assays were performed to determine several characteristics of NCX1.3 in the reverse mode. Results: NCX1.3 was demonstrated to be the predominant NCX1 variant in the DCT2/CNT, next to NCX1.2 and NCX1.7. NCX1.3 could be inhibited by SN-6, an NCX-specific inhibitor, whereas stimulation of the cAMP/PKA or PKC-mediated pathway did not affect Ca²⁺ influx as measured in the reverse mode. Lowering intracellular Ca²⁺ concentrations resulted in a decreased Ca²⁺ uptake. Conclusion: NCX1.3 is the predominant NCX variant in the DCT2/CNT tubules. Its function is dependent on intracellular Ca²⁺ concentrations.