Increased bone resorption by osteoclast-specific deletion of the sodium/calcium exchanger isoform 1 (NCX1).

Calcium is a key component of the bone mineral hydroxyapatite. During osteoclast-mediated bone resorption, hydroxyapatite is dissolved and significant quantities of calcium are released. Several calcium transport systems have previously been identified in osteoclasts, including members of the sodium/calcium exchanger (NCX) family. Expression pattern and physiological role of NCX isoforms in osteoclasts, however, remain largely unknown at the moment. Our data indicate that all three NCX isoforms (NCX1, NCX2, and NCX3) are present in murine osteoclasts. RANKL-induced differentiation of murine osteoclast precursors into mature osteoclasts significantly attenuated the expression of NCX1, while NCX2 and NCX3 expressions were largely unaffected. To study the role of NCX1 during osteoclast differentiation and bone resorption, we crossed mice with exon 11 of the NCX1 gene flanked by loxP sites with cathepsin K-Cre transgenic mice. Mature osteoclasts derived from transgenic mice exhibited an 80-90% reduction of NCX1 protein. In vitro studies indicate that NCX1 is dispensable for osteoclast differentiation, but NCX1-deficient osteoclasts exhibited increased resorptive activity. In line with these in vitro findings, mice with an osteoclast-targeted deletion of the NCX1 gene locus displayed an age-dependent loss of bone mass. Thus, in summary, our data reveal NCX1 as a regulator of osteoclast-mediated bone resorption.

Insulin receptor substrate-2 is expressed in kidney epithelium and up-regulated in diabetic nephropathy.

Diabetic nephropathy (DN) is a progressive fibrotic condition that may lead to end-stage renal disease and kidney failure. Transforming growth factor-ß1 and bone morphogenetic protein-7 (BMP7) have been shown to induce DN-like changes in the kidney and protect the kidney from such changes, respectively. Recent data identified insulin action at the level of the nephron as a crucial factor in the development and progression of DN. Insulin requires a family of insulin receptor substrate (IRS) proteins for its physiological effects, and many reports have highlighted the role of insulin and IRS proteins in kidney physiology and disease. Here, we observed IRS2 expression predominantly in the developing and adult kidney epithelium in mouse and human. BMP7 treatment of human kidney proximal tubule epithelial cells (HK-2 cells) increases IRS2 transcription. In addition, BMP7 treatment of HK-2 cells induces an electrophoretic shift in IRS2 migration on SDS/PAGE, and increased association with phosphatidylinositol-3-kinase, probably due to increased tyrosine/serine phosphorylation. In a cohort of DN patients with a range of chronic kidney disease severity, IRS2 mRNA levels were elevated approximately ninefold, with the majority of IRS2 staining evident in the kidney tubules in DN patients. These data show that IRS2 is expressed in the kidney epithelium and may play a role in the downstream protective events triggered by BMP7 in the kidney. The specific up-regulation of IRS2 in the kidney tubules of DN patients also indicates a novel role for IRS2 as a marker and/or mediator of human DN progression.

Role of SPAK and OSR1 signalling in the regulation of NaCl cotransporters.

PURPOSE OF REVIEW: Sodium and chloride transport play a fundamental role in many physiological processes. In the kidney, sodium secretion and reabsorption are essential to maintain the extracellular volume and, thus, blood pressure (BP). In vascular smooth muscle, it is important for contractility and in the nervous system for the functioning of GABAergic neurons. Hence, the emergence of a WNK/SPAK/OSR1 kinase cascade that activates NaCl cotransporters has widespread physiological implications. This review gives an overview of the actions of SPAK and OSR1 kinases on NaCl cotransporters and highlights their possible therapeutic potential. RECENT FINDINGS: Evidence has emerged from in-vitro phosphorylation assays that WNK kinases can activate SPAK and OSR1 kinases by phosphorylation of a key Thr residue in their catalytic domains. Once activated, SPAK and OSR1 in turn activate members of the SCL12A family of solute carriers by phosphorylation of conserved Ser/Thr residues in the N-terminal domain of these carrier proteins. The importance of this pathway has recently emerged from studies on mice that lack a catalytically active SPAK enzyme. These models are strikingly hypotensive with marked reduction in the phosphorylation of Na⁺/Cl⁻ cotransporter (NCC) in the kidney, and reduced Na⁺/K⁺/2Cl⁻ cotransporter (NKCC1) phosphorylation in the vessel wall. SUMMARY: SPAK and OSR1 kinases regulate SCL12A transporters with important physiological effects for sodium homeostasis by the kidney, aortic contractility and neuronal excitability. In vivo, SPAK plays a major role in the regulation of blood pressure and represents a potential target for the development of novel diuretics.

The activity of the thiazide-sensitive Na(+)-Cl(-) cotransporter is regulated by protein phosphatase PP4.

Cation transport in the distal mammalian nephron relies on the SLC12 family of membrane cotransporters that include the thiazide-sensitive Na(+)-Cl⁻ cotransporter (NCC). NCC is regulated through a scaffold of interacting proteins, including the WNK kinases, WNK 1 and WNK 4, which are mutated in the hypertensive Gordon's syndrome. Dynamic regulation of NCC function by kinases must involve dephosphorylation by phosphatases, as illustrated by the role of PP1 and PP2B in the regulation of KCC members of the SLC12 family. There are 2 phosphorylation-controlled regulatory pathways for NCC: type 1, mediated by WNK4 and affecting trafficking to the surface membrane, and type 2, affecting intrinsic transporter kinetics by phosphorylation of conserved N-terminal S/T amino acids. Using the Xenopus oocyte expression system, we show that PP4 inhibits NCC activity - but not trafficking to the surface membrane - by a mechanism that requires phosphatase activity and a conserved N-terminal amino acid of NCC, threonine 58. This action is distinct from WNK4 regulation of membrane trafficking. In the mouse kidney, PP4 is selectively expressed in the distal nephron, including cells of the distal convoluted tubule cells, suggesting that PP4 may have a physiological role in regulating NCC and hence NaCl reabsorption in vivo.