What is the function of the ANKH protein in the kidney?

Abstract

Pyrophosphate (PPi) has been known since the 1960s as an inhibitor of calcium renal stone formation. Naturally occurring mutations in a putative PPi transporter, ANK, causes renal calcification in mice. We hypothesised that the human homologue, ANKH, plays a role in PPi transport in the human kidney. Immunocytochemical localisation of ANKH in human kidney showed greater abundance in the cortical collecting duct than elsewhere in the nephron. The transport function of ANKH was investigated by heterologous expression of ANKH in Xenopus oocytes. Despite confirmation of ANKH expression at the oocyte plasma membrane, neither ANKH-mediated PPi efflux (the physiological mode of operation) nor influx was detectable compared to water-injected oocytes. Pyrophosphatase activity was detected at the surface of oocytes, suggesting hydrolysis of PPi to inorganic phosphate. Screening using a yeast two-hybrid method of the N-terminal of ANKH against a mouse renal library identified a possible protein-protein interaction with the fatty acid transporter SLC27A2, whose acyl-CoA synthetase activity yields PPi as an end product. This suggests that ANKH and protein partners such as SLC27A2 may form a biochemical couple whereby PPi is sequestered by transmembrane transport rather than by hydrolysis. Since there is no pyrophosphatase activity in peroxisomes, we suggest that the ANKH/SLC27A2 complex is a candidate protein for the peroxisomal membrane PPi transporter. AVP mediates increased expression and localisation of ANK to the apical membrane of a collecting duct model (mpkCCDcl4) in vitro, suggesting physiologically appropriate regulation, analogous to that of aquaporin-2. These findings offer insights into the cellular homeostasis of PPi. Instead of cytosolic hydrolysis, coupling of PPi generation and ANKH-mediated transport as part of a protein complex may allow PPi to be compartmentalised, preserving it for use within vesicular structures elsewhere in the cell or allowing export to the extracellular medium to assist in the regulation of apatite deposition.