The Klotho gene encodes a 130-kDa single-pass transmembrane protein with a short cytoplasmic domain (10 amino acids) and is expressed predominantly in the kidney. Mice carrying a loss-of-function mutation in the Klotho gene develop a syndrome resembling human aging, including shortened life span, skin atrophy, muscle atrophy, osteoporosis, arteriosclerosis, and pulmonary emphysema (1). Conversely, overexpression of the Klotho gene extends the life span and increases resistance to oxidative stress in mice (2-4). These observations suggest that the Klotho gene functions as an aging suppressor gene. The extracellular domain of Klotho protein is shed and secreted in the blood (2, 5), potentially functioning as a humoral factor that signals suppression of intracellular insulin/IGF1 signaling, which partly contributes to its anti-aging properties (2). However, a signaling pathway(s) directly activated by Klotho protein, including the identity of the Klotho receptor, has not been determined. The function of the transmembrane form of Klotho protein also remains to be determined.Fibroblast growth factor-23 (FGF23) 2 was originally identified as a gene mutated in patients with autosomal dominant hypophosphatemic rickets (6), where mutations in the FGF23 gene conferred resistance to inactivation by protease cleavage, resulting in elevated serum levels of FGF23 (7-12). FGF23 inhibits phosphate transport in renal proximal tubular cells and in proximal tubules perfused in vitro (13). Consistent with these findings, mice defective in FGF23 expression show increased renal phosphate reabsorption and hyperphosphatemia (14). Although FGF23 binds to multiple FGF receptors (FGFRs) (15), it has modest receptor affinity (K D ϭ 200 -700 nM) and often requires cofactors such as heparin or glycosaminoglycan (15, 16) to activate FGF signaling in cultured cells and to inhibit phosphate transport in proximal tubules perfused in vitro (13).Klotho-deficient mice (Klotho Ϫ/Ϫ mice) and FGF23 deficient mice (Fgf23 Ϫ/Ϫ mice) develop many common phenotypes, including shortened life span, growth retardation, infertility, muscle atrophy, hypoglycemia, and vascular calcification in the kidneys. Notably, they both have increased serum levels of phosphate (14, 17). These observations have led us to the hypothesis that Klotho and FGF23 may function via a common signal transduction pathway. In this report we show that Klotho binds to multiple FGFRs and functions as a cofactor necessary for FGF signaling activation by FGF23. MATERIALS AND METHODSExpression Vectors-Complementary DNA containing the mouse FGFRs coding region (IMAGE Clone, Invitrogen, supplemental Fig. 1) were cloned into pcDNA3.1(ϩ) expression vector (Invitrogen). Before subcloning, a V5-epitope tag was added to the C terminus and appropriate restriction enzyme sites to the both ends using synthetic oligonucleotides and polymerase chain reaction. Expression vectors for the mouse FGF23 resistant to proteolytic inactivation (R179Q) (18), the transmembrane form of mouse Klotho, and the extracel...

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Intestinal phosphate absorption occurs through both a paracellular mechanism involving tight junctions and an active transcellular mechanism involving the type II sodium-dependent phosphate cotransporter NPT2b (SLC34a2). To define the contribution of NPT2b to total intestinal phosphate absorption, we generated an inducible conditional knockout mouse, Npt2b Inorganic phosphate is an essential mineral critical for cellular processes and bone mineralization. Severe disruptions in serum phosphate have pathologic consequences. 1,2 Hypophosphatemic disorders are associated with rickets, osteomalacia, and a host of secondary dysfunctions. 3 In contrast, hyperphosphatemia associated with chronic kidney disease (CKD) is linked tightly to increased risk of cardiovascular morbidity and mortality. 4 -6 Recent studies show that elevated phosphate concentrations within the high normal range in individuals with functional kidneys also are correlated with increased cardiovascular risk and mortality. 7,8 Thus, an elevated serum phosphate level is an emerging health risk. Despite the importance of maintaining a relatively narrow serum phosphate range, nearly 70% of dietary phosphate is absorbed, resulting in transient postprandial increases in serum phosphate concentrations. 9 Normalization of serum phosphate appears to be managed primarily within the renal proximal tubule by the type II sodium-dependent phosphate cotransporters NPT2a (SLC34a1) and NPT2c (SLC34a3). Genetic knockout mouse models demonstrate that 80% and 20% of total urinary phosphorus are managed by the Npt2a and Npt2c transporters, respectively. 10,11 Chronic and acute regulation of these renal transporters is modulated by changes in dietary and serum phosphate

Injury to podocytes and their slit diaphragms typically leads to marked proteinuria. Mutations in the TRPC6 gene that codes for a slit diaphragm-associated, cation-permeable ion channel have been shown recently to co-segregate with hereditary forms of progressive kidney failure. Herein is shown that induced expression of wild-type TRPC6 is a common feature of human proteinuric kidney diseases, with highest induction observed in membranous nephropathy. Cultured podocytes that are exposed to complement upregulate TRPC6 protein. Stimulation of receptor-operated channels in puromycin aminonucleoside-treated podocytes leads to increased calcium influx in a time-and dosage-dependent manner. Mechanistically, it is shown that TRPC6 is functionally connected to the podocyte actin cytoskeleton, which is rearranged upon overexpression of TRPC6. Transient in vivo gene delivery of TRPC6 into mice leads to expression of TRPC6 protein at the slit diaphragm and causes proteinuria. These studies suggest the involvement of TRPC6 in the pathology of nongenetic forms of proteinuric disease.

Chronic kidney disease–mineral bone disorder (CKD‐MBD) is defined by abnormalities in mineral and hormone metabolism, bone histomorphometric changes, and/or the presence of soft‐tissue calcification. Emerging evidence suggests that features of CKD‐MBD may occur early in disease progression and are associated with changes in osteocyte function. To identify early changes in bone, we utilized the jck mouse, a genetic model of polycystic kidney disease that exhibits progressive renal disease. At 6 weeks of age, jck mice have normal renal function and no evidence of bone disease but exhibit continual decline in renal function and death by 20 weeks of age, when approximately 40% to 60% of them have vascular calcification. Temporal changes in serum parameters were identified in jck relative to wild‐type mice from 6 through 18 weeks of age and were subsequently shown to largely mirror serum changes commonly associated with clinical CKD‐MBD. Bone histomorphometry revealed progressive changes associated with increased osteoclast activity and elevated bone formation relative to wild‐type mice. To capture the early molecular and cellular events in the progression of CKD‐MBD we examined cell‐specific pathways associated with bone remodeling at the protein and/or gene expression level. Importantly, a steady increase in the number of cells expressing phosphor‐Ser33/37‐β‐catenin was observed both in mouse and human bones. Overall repression of Wnt/β‐catenin signaling within osteocytes occurred in conjunction with increased expression of Wnt antagonists (SOST and sFRP4) and genes associated with osteoclast activity, including receptor activator of NF‐κB ligand (RANKL). The resulting increase in the RANKL/osteoprotegerin (OPG) ratio correlated with increased osteoclast activity. In late‐stage disease, an apparent repression of genes associated with osteoblast function was observed. These data confirm that jck mice develop progressive biochemical changes in CKD‐MBD and suggest that repression of the Wnt/β‐catenin pathway is involved in the pathogenesis of renal osteodystrophy. © 2012 American Society for Bone and Mineral Research.

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BackgroundIn vivo models of uremia are important tools to study numerous aspects of acute and chronic kidney disease. Mouse models are pivotal because most genetically engineered animal models are mice, which allow dissecting the impact of selected target genes in renal failure. Adenine-based protocols to induce renal failure are available in rats, but have not been adapted in mice due to their reluctance to consume adenine. In the current paper we developed a novel method for induction of renal failure through dietary delivery of adenine mixed in a casein-based diet.ResultsAfter an induction phase, a stable model of renal impairment was obtained (target urea range 80–100 mg/dL), mimicking several aspects of chronic kidney disease - mineral and bone disorder including secondary hyperparathyroidism, bone abnormalities and pathological elevation of FGF23. No deaths occurred and the level of uremia was adaptable through adjustments of the adenine content, providing significant advantages compared to existing models. In an 8-week proof-of-concept study, renal histology showed mainly a tubulointerstitial damage with infiltrating leukocytes, interstitial edema and widening of the Bownman's space. Fibrosis was present in most animals as defined by histology and gene expression changes of fibrosis markers. Parathyroid cell proliferation was markedly increased but without signs of glandular hypertrophy. Skeletal histology showed increased trabecular bone and bone marrow adiposity whereas bone biomarkers (CTX and PINP) suggested higher bone formation, but surprisingly, lower bone resorption and perturbations in mineral metabolism.ConclusionsWe present a novel, non-surgical method for induction of renal failure in mice. This is an important complement to existing uremic models for pathophysiological studies in acute and chronic kidney disease, especially in terms of tubulointerstitial lesions.