Susan C. Schiavi

Regulation of Fibroblast Growth Factor-23 Signaling by Klotho

The aging suppressor gene Klotho encodes a single-pass transmembrane protein. Klotho-deficient mice exhibit a variety of aging-like phenotypes, many of which are similar to those observed in fibroblast growth factor-23 (FGF23)-deficient mice. To test the possibility that Klotho and FGF23 may function in a common signal transduction pathway(s), we investigated whether Klotho is involved in FGF signaling. Here we show that Klotho protein directly binds to multiple FGF receptors (FGFRs). The Klotho-FGFR complex binds to FGF23 with higher affinity than FGFR or Klotho alone. In addition, Klotho significantly enhanced the ability of FGF23 to induce phosphorylation of FGF receptor substrate and ERK in various types of cells. Thus, Klotho functions as a cofactor essential for activation of FGF signaling by FGF23.

Klotho Inhibits Transforming Growth Factor-β1 (TGF-β1) Signaling and Suppresses Renal Fibrosis and Cancer Metastasis in Mice

Fibrosis is a pathological process characterized by infiltration and proliferation of mesenchymal cells in interstitial space. A substantial portion of these cells is derived from residing non-epithelial and/or epithelial cells that have acquired the ability to migrate and proliferate. The mesenchymal transition is also observed in cancer cells to confer the ability to metastasize. Here, we show that renal fibrosis induced by unilateral ureteral obstruction and metastasis of human cancer xenografts are suppressed by administration of secreted Klotho protein to mice. Klotho is a single-pass transmembrane protein expressed in renal tubular epithelial cells. The extracellular domain of Klotho is secreted by ectodomain shedding. Secreted Klotho protein directly binds to the type-II TGF-β receptor and inhibits TGF-β1 binding to cell surface receptors, thereby inhibiting TGF-β1 signaling. Klotho suppresses TGF-β1-induced epithelial-to-mesenchymal transition (EMT) responses in cultured cells, including decreased epithelial marker expression, increased mesenchymal marker expression, and/or increased cell migration. In addition to TGF-β1 signaling, secreted Klotho has been shown to inhibit Wnt and IGF-1 signaling that can promote EMT. These results have raised the possibility that secreted Klotho may function as an endogenous anti-EMT factor by inhibiting multiple growth factor signaling pathways simultaneously.

Secreted frizzled-related protein 4 is a potent tumor-derived phosphaturic agent

Tumors associated with osteomalacia elaborate the novel factor(s), phosphatonin(s), which causes phosphaturia and hypophosphatemia by cAMP-independent pathways. We show that secreted frizzled-related protein-4 (sFRP-4), a protein highly expressed in such tumors, is a circulating phosphaturic factor that antagonizes renal Wnt-signaling. In cultured opossum renal epithelial cells, sFRP-4 specifically inhibited sodium-dependent phosphate transport. Infusions of sFRP-4 in normal rats over 2 hours specifically increased renal fractional excretion of inorganic phosphate (FEPi) from 14% +/- 2% to 34% +/- 5% (mean +/- SEM, P < 0.01). Urinary cAMP and calcium excretion were unchanged. In thyro-parathyroidectomized rats, sFRP-4 increased FEPi from 0.7% +/- 0.2% to 3.8% +/- 1.2% (P < 0.05), demonstrating that sFRP-4 inhibits renal inorganic phosphate reabsorption by PTH-independent mechanisms. Administration of sFRP-4 to intact rats over 8 hours increased FEPi, decreased serum phosphate (1.95 +/- 0.1 to 1.53 +/- 0.09 mmol/l, P < 0.05) but did not alter serum 1alpha, 25-dihydroxyvitamin D, renal 25-hydroxyvitamin D 1alpha-hydroxylase cytochrome P450, and sodium-phosphate cotransporter mRNA concentrations. Infusion of sFRP-4 antagonizes Wnt action as demonstrated by reduced renal beta-catenin and increased phosphorylated beta-catenin concentrations. The sFRP-4 is detectable in normal human serum and in the serum of a patient with tumor-induced osteomalacia. Thus, sFRP-4 displays phosphatonin-like properties, because it is a circulating protein that promotes phosphaturia and hypophosphatemia and blunts compensatory increases in 1alpha, 25-dihydroxyvitamin D.

Induction of TRPC6 Channel in Acquired Forms of Proteinuric Kidney Disease

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.

Tumors associated with oncogenic osteomalacia express genes important in bone and mineral metabolism

Oncogenic osteomalacia (OOM) is associated with primitive mesenchymal tumors that secrete phosphaturic factors resulting in low serum concentrations of phosphate and calcitriol, phosphaturia, and defective bone mineralization. To identify overexpressed genes in these tumors, we compared gene expression profiles of tumors resected from patients with OOM and histologically similar control tumors using serial analysis of gene expression (SAGE). Three hundred and sixty‐four genes were expressed at least twofold greater in OOM tumors compared with control tumors. A subset of 67 highly expressed genes underwent validation with an extended set of OOM and control tumors using array analysis or reverse‐transcription polymerase chain reaction (RT‐PCR). Ten of these validated genes were consistently overexpressed in all OOM tumors relative to control tumors. Strikingly, genes with roles in bone matrix formation, mineral ion transport, and bone mineralization were highly expressed in the OOM tumors.

Intestinal Npt2b Plays a Major Role in Phosphate Absorption and Homeostasis

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(-/-) (Npt2b(fl/fl):Cre(+/-)). Npt2b(-/-) animals had increased fecal phosphate excretion and hypophosphaturia, but serum phosphate remained unchanged. Decreased urinary phosphate excretion correlated with reduced serum levels of the phosphaturic hormone FGF23 and increased protein expression of the renal phosphate transporter Npt2a. These results demonstrate that the absence of Npt2b triggers compensatory renal mechanisms to maintain phosphate homeostasis. In animals fed a low phosphate diet followed by acute administration of a phosphate bolus, Npt2b(-/-) animals absorbed approximately 50% less phosphate than wild-type animals, confirming a major role of this transporter in phosphate regulation. In vitro analysis of active phosphate transport in ileum segments isolated from wild-type or Npt2b(-/-) mice demonstrated that Npt2b contributes to >90% of total active phosphate absorption. In summary, Npt2b is largely responsible for intestinal phosphate absorption and contributes to the maintenance of systemic phosphate homeostasis.

Wnt-Dependent β-Catenin Signaling Is Activated after Unilateral Ureteral Obstruction, and Recombinant Secreted Frizzled-Related Protein 4 Alters the Progression of Renal Fibrosis

beta-Catenin functions as a transducer of Wnt signals to the nucleus, where it interacts with the T cell factor (TCF) family of DNA binding proteins to regulate gene expression. On the basis of the genes regulated by beta-catenin and TCF in various biologic settings, two predicted functions of beta-catenin/TCF-dependent transcription are to mediate the loss of epithelial polarity and to promote fibroblast activities, such as the increased synthesis of fibronectin during chronic renal disease. These predictions were tested by determination of the expression and function of an inhibitor of Wnt signaling, secreted frizzled-related protein 4 (sFRP4), during renal tubular epithelial injury initiated by unilateral ureteral obstruction (UUO). Despite increased sFRP4 gene expression in perivascular regions of injured kidneys, total sFRP4 protein levels decreased after injury. The decreased sFRP4 protein levels after UUO accompanied increased Wnt-dependent beta-catenin signaling in tubular epithelial and interstitial cells, along with increased expression of markers of fibrosis. Administration of recombinant sFRP4 protein caused a reduction in tubular epithelial beta-catenin signaling and suppressed the progression of renal fibrosis, as evidenced by a partial maintenance of E-cadherin mRNA expression and a reduction in the amount of fibronectin and alpha-smooth muscle actin proteins. Furthermore, recombinant sFRP4 reduced the number of myofibroblasts, a central mediator of fibrosis. It is concluded that beta-catenin signaling is activated in tubular epithelial and interstitial cells after renal injury, and recombinant sFRP4 can interfere with epithelial de-differentiation and with fibroblast differentiation and function during progression of renal fibrosis.

Repression of osteocyte Wnt/β-catenin signaling is an early event in the progression of renal osteodystrophy

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.

Thyroid-stimulating hormone restores bone volume, microarchitecture, and strength in aged ovariectomized rats.

We show the systemic administration of low levels of TSH increases bone volume and improves bone microarchitecture and strength in aged OVX rats. TSHs actions are mediated by its inhibitory effects on RANKL‐induced osteoclast formation and bone resorption coupled with stimulatory effects on osteoblast differentiation and bone formation, suggesting TSH directly affects bone remodeling in vivo.

FGF-23 and sFRP-4 in Chronic Kidney Disease and Post Renal Transplantation

Background: The phosphatonins fibroblast growth factor-23 (FGF-23) and FRP-4 are inhibitors of tubular phosphate reabsorption that may play a role in the hyperphosphatemia associated with chronic kidney disease (CKD) or in the hypophosphatemia associated with renal transplants. Methods: Plasma FGF-23, FRP-4, phosphorus and parathyroid hormone were measured in patients at all stages of CKD. Phosphate regulation of FGF-23 and secreted frizzled related protein-4 (sFRP-4) was examined in end-stage renal disease patients in the presence and absence of therapeutic phosphate binder usage. In renal transplant patients, plasma FGF-23, sFRP-4 and phosphorus concentrations were determined before and 4–5 days after transplantation. Results: Plasma FGF-23 correlated with creatinine clearance (r2 = –0.584, p < 0.0001) and plasma phosphorus (r2 = 0.347, p < 0.001) in CKD patients and with plasma phosphorus (r2 = 0.448, p < 0.001) in end-stage renal disease patients. Phosphate binder withdrawal increased FGF-23 levels. In kidney transplant patients, dramatic decreases in FGF-23 (–88.8 ± 5.4%) and phosphorus (–64 ± 10.2%) were observed by 4–5 days post-transplantation. In patients with post-transplant hypophosphatemia, FGF-23 levels correlated inversely with plasma phosphorus (r2 = 0.661, p < 0.05). sFRP-4 levels did not change with creatinine clearance or hyperphosphatemia in CKD or end-stage renal disease patients, and no relation was noted between post-transplant sFRP-4 levels and hypophosphatemia. Conclusions: In CKD, FGF-23 levels rose with decreasing creatinine clearance rates and increasing plasma phosphorus levels, and rapidly decreased post-transplantation suggesting FGF-23 is cleared by the kidney. Residual FGF-23 may contribute to the hypophosphatemia in post-transplant patients.