Carlton M. Bates

FGF23 decreases renal NaPi-2a and NaPi-2c expression and induces hypophosphatemia in vivo predominantly via FGF receptor 1

Fibroblast growth factor-23 (FGF23) is a phosphaturic hormone that contributes to several hypophosphatemic disorders by reducing the expression of the type II sodium-phosphate cotransporters (NaPi-2a and NaPi-2c) in the kidney proximal tubule and by reducing serum 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] levels. The FGF receptor(s) mediating the hypophosphatemic action of FGF23 in vivo have remained elusive. In this study, we show that proximal tubules express FGFR1, -3, and -4 but not FGFR2 mRNA. To determine which of these three FGFRs mediates FGF23s hypophosphatemic actions, we characterized phosphate homeostasis in FGFR3(-/-) and FGFR4(-/-) null mice, and in conditional FGFR1(-/-) mice, with targeted deletion of FGFR1 expression in the metanephric mesenchyme. Basal serum phosphorus levels and renal cortical brush-border membrane (BBM) NaPi-2a and NaPi-2c expression were comparable between FGFR1(-/-), FGFR3(-/-), and FGFR4(-/-) mice and their wild-type counterparts. Administration of FGF23 to FGFR3(-/-) mice induced hypophosphatemia in these mice (8.0 +/- 0.4 vs. 5.4 +/- 0.3 mg/dl; p < or = 0.001) and a decrease in renal BBM NaPi-2a and NaPi-2c protein expression. Similarly, in FGFR4(-/-) mice, administration of FGF23 caused a small but significant decrease in serum phosphorus levels (8.7 +/- 0.3 vs. 7.6 +/- 0.4 mg/dl; p < or = 0.001) and in renal BBM NaPi-2a and NaPi-2c protein abundance. In contrast, injection of FGF23 into FGFR1(-/-) mice had no effects on serum phosphorus levels (5.6 +/- 0.3 vs. 5.2 +/- 0.5 mg/dl) or BBM NaPi-2a and NaPi-2c expression. These data show that FGFR1 is the predominant receptor for the hypophosphatemic action of FGF23 in vivo, with FGFR4 likely playing a minor role.

Management and etiology of the unilateral multicystic dysplastic kidney: a review

In children, unilateral multicystic dysplastic kidney (MCDK) is one of the most frequently identified urinary tract abnormalities. A variety of proposed etiologies has been associated with the underlying pathogenesis of MCDK. These include genetic disturbances, teratogens, in utero infections, and urinary outflow tract obstruction. From 5–43% of the time, MCDK has associated genito-urinary anomalies, both structural and functional in nature. A review of the literature reveals that involution rates are reported to be 19–73%, compensatory hypertrophy of the contralateral kidney occurs from 24–81% of the time, and estimated glomerular filtration rates (GFRs) (by the Schwartz formula) range from 86–122xa0ml/min per 1.73xa0m2 body surface area. Most authors suggest serial ultrasonography to monitor contralateral growth, routine blood pressure monitoring, and a serum creatinine monitoring algorithm. The risk of hypertension in those with MCDKs does not appear to be greater than that of the general population, and the rates of malignant transformation of MCDK are small, if at all increased, in comparison with those in the general population. If the patient develops a urinary tract infection or has abnormalities of the contralateral kidney, shown on ultrasound, a voiding cystourethrogram is recommended. Finally, the body of literature does not support the routine surgical removal of MCDKs.

Role of fibroblast growth factor receptor signaling in kidney development

Fibroblast growth factor receptors (Fgfrs) are expressed throughout the developing kidney. Several early studies have shown that exogenous fibroblast growth factors (Fgfs) affect growth and maturation of the metanephric mesenchyme (MM) and ureteric bud (UB). Transgenic mice that over-express a dominant negative receptor isoform develop renal aplasia/severe dysplasia, confirming the importance of Fgfrs in renal development. Furthermore, global deletion of Fgf7, Fgf10, and Fgfr2IIIb (isoform that binds Fgf7 and Fgf10) in mice leads to small kidneys with fewer collecting ducts and nephrons. Deletion of Fgfrl1, a receptor lacking intracellular signaling domains, causes severe renal dysgenesis. Conditional targeting of Fgf8 from the MM interrupts nephron formation. Deletion of Fgfr2 from the UB results in severe ureteric branching and stromal mesenchymal defects, although loss of Frs2α (major signaling adapter for Fgfrs) in the UB causes only mild renal hypoplasia. Deletion of both Fgfr1 and Fgfr2 in the MM results in renal aplasia with defects in MM formation and initial UB elongation and branching. Loss of Fgfr2 in the MM leads to many renal and urinary tract anomalies as well as vesicoureteral reflux. Thus, Fgfr signaling is critical for patterning of virtually all renal lineages at early and later stages of development.

Coordinate integrin and c-Met signaling regulate Wnt gene expression during epithelial morphogenesis

Integrin receptors for the extracellular matrix and receptor tyrosine kinase growth factor receptors represent two of the major families of receptors that transduce into cells information about the surrounding environment. Wnt proteins are a major family of signaling molecules that regulate morphogenetic events. There is presently little understanding of how the expression of Wnt genes themselves is regulated. In this study, we demonstrate that α3β1 integrin, a major laminin receptor involved in the development of the kidney, and c-Met, the receptor for hepatocyte growth factor, signal coordinately to regulate the expression of Wnt7b in the mouse. Wnt signals in turn appear to regulate epithelial cell survival in the papilla of the developing kidney, allowing for the elongation of epithelial tubules to form a mature papilla. Together, these results demonstrate how signals from integrins and growth factor receptors can be integrated to regulate the expression of an important family of signaling molecules so as to regulate morphogenetic events.

Extracellular calcium is a mediator of astroglial injury during combined glucose-oxygen deprivation

We tested the hypothesis that extracellular calcium is a mediator of astroglial injury during combined glucose-oxygen deprivation. Both differentiated and undifferentiated astroglial cultures were exposed to combined glucose-oxygen deprivation in the presence and absence of extracellular calcium. Lactate dehydrogenase efflux was used as an index of cellular injury. Both types of cultures exhibited significantly less cellular injury when exposed to combined glucose-oxygen deprivation in the absence of extracellular calcium (e.g. lactate dehydrogenase efflux in undifferentiated cultures after 12 h of exposure: presence of calcium, 65.2 +/- 2.5% vs. absence of calcium, 21.4 +/- 1.3%). To further elucidate the mechanism by which extracellular calcium produces injury, we studied the effect of nimodipine, an L-type calcium channel blocker, on astroglial injury resulting from combined glucose-oxygen deprivation. Nimodipine decreased cellular injury in both types of cultures (e.g. lactate dehydrogenase efflux in undifferentiated cultures after 12 h of exposure: untreated, 65.4 +/- 2.2% vs. 10 nM nimodipine, 44.6 +/- 4.2%). Extracellular calcium appears to be a mediator of astroglial injury during combined glucose-oxygen deprivation. These results suggest that influx of extracellular calcium via L-type voltage-gated calcium channels may contribute to astroglial injury during cerebral ischemia.

Endothelial Progenitors Exist within the Kidney and Lung Mesenchyme

The renal endothelium has been debated as arising from resident hemangioblast precursors that transdifferentiate from the nephrogenic mesenchyme (vasculogenesis) and/or from invading vessels (angiogenesis). While the Foxd1-positive renal cortical stroma has been shown to differentiate into cells that support the vasculature in the kidney (including vascular smooth muscle and pericytes) it has not been considered as a source of endothelial cell progenitors. In addition, it is unclear if Foxd1-positive mesenchymal cells in other organs such as the lung have the potential to form endothelium. This study examines the potential for Foxd1-positive cells of the kidney and lung to give rise to endothelial progenitors. We utilized immunofluorescence (IF) and fluorescence-activated cell sorting (FACS) to co-label Foxd1-expressing cells (including permanently lineage-tagged cells) with endothelial markers in embryonic and postnatal mice. We also cultured FACsorted Foxd1-positive cells, performed in vitro endothelial cell tubulogenesis assays and examined for endocytosis of acetylated low-density lipoprotein (Ac-LDL), a functional assay for endothelial cells. Immunofluorescence and FACS revealed that a subset of Foxd1-positive cells from kidney and lung co-expressed endothelial cell markers throughout embryogenesis. In vitro, cultured embryonic Foxd1-positive cells were able to differentiate into tubular networks that expressed endothelial cell markers and were able to endocytose Ac-LDL. IF and FACS in both the kidney and lung revealed that lineage-tagged Foxd1-positive cells gave rise to a significant portion of the endothelium in postnatal mice. In the kidney, the stromal-derived cells gave rise to a portion of the peritubular capillary endothelium, but not of the glomerular or large vessel endothelium. These findings reveal the heterogeneity of endothelial cell lineages; moreover, Foxd1-positive mesenchymal cells of the developing kidney and lung are a source of endothelial progenitors that are likely critical to patterning the vasculature.

Regulation of renal phosphate transport by FGF23 is mediated by FGFR1 and FGFR4

Fibroblast growth factor 23 (FGF23) is a bone-derived hormone that acts on the proximal tubule to decrease phosphate reabsorption and serum levels of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂ Vitamin D₃]. Abnormal FGF23 metabolism has been implicated in several debilitating hypophosphatemic and hyperphosphatemic disorders. The renal receptors responsible for the phosphaturic actions of FGF23 have not been elucidated. There are four fibroblast growth factor receptors (FGFR); 1-4 with b and c isoforms for receptors 1, 2, and 3. FGFR1, 3, and 4 are expressed in the mouse proximal tubule, and deletion of any one receptor did not affect serum phosphate levels, suggesting that more than one receptor is involved in mediating the phosphaturic actions of FGF23. To determine the receptors responsible for the phosphaturic actions of FGF23, we studied Fgfr1 (kidney conditional) and Fgfr4 (global) double mutant mice (Fgfr1⁻/⁻/Fgfr4⁻/⁻). Fgfr1⁻/⁻/Fgfr4⁻/⁻ mice have higher FGF23 levels than their wild-type counterparts (108.1 ± 7.3 vs. 4,953.6 ± 675.0 pg/ml; P < 0.001). Despite the elevated FGF23 levels, Fgfr1⁻/⁻/Fgfr4⁻/⁻ mice have elevated serum phosphorus levels, increased brush-border membrane vesicle (BBMV) phosphate transport, and increased Na-P(i) cotransporter 2c (NaPi-2c) protein expression compared with wild-type mice. These data are consistent with FGFR1 and FGFR4 being the critical receptors for the phosphaturic actions of FGF23.

Lipid alterations following impact spinal cord injury in the rat

A computer-controlled impactor was used to produce a severe spinal cord injury in the rat thoracic spinal cord. Cords were rapidly frozen in situ at 5, 15, 30, and 60 min and 6, 12, and 24 h postinjury. Control cords were noninjured cords from animals having undergone a laminectomy and allowed to recover for 90 min postlaminectomy. The cords were assayed for alterations in lipid metabolism. Specifically, there were rapid increases in prostaglandin F2 alpha and thromboxane, with a peak increase in thromboxane levels at 30 min. Prostaglandin F2 alpha levels peaked at 15 min with levels remaining nearly constant for 12 h. There were no detectable changes in phospholipid levels, although diacylglycerol levels and free fatty acid levels were increased. Total free fatty acids were increased at 12 and 24 h postinjury by 2.3- and 3.2-fold over control levels, respectively. Arachidonic acid levels were not significantly elevated at early time points, however, these early time points correspond to elevated eicosanoid synthesis and this may account for the lack of early detectable increases in arachidonic acid. After 6 h postinjury, arachidonic acid levels were 20-fold greater than control levels and remained elevated at 24 h. There were minimal decreases in cholesterol and no decrease in either choline or ethanolamine plasmalogen levels. These results suggest a rapid turnover of arachidonic acid following spinal cord injury with a concomitant increase in vasoconstrictive eicosanoid synthesis. The lack of changes in major membrane constituents suggests the mechanisms may not involve general membrane degradation, but an over-stimulation of phospholipase A2-linked membrane receptors.

Role of fibroblast growth factor receptor 2 in kidney mesenchyme.

Conditional deletion of murine fibroblast growth factor receptors (Fgfrs) 1 and 2 in metanephric mesenchyme leads to renal agenesis with unbranched ureteric buds; however, there are occasionally two buds per nephric duct. Our goal was to determine whether conditional deletion of Fgfr1 or Fgfr2 alone resulted in multiple ureteric bud induction sites. Although deletion of Fgfr1 alone results in no abnormalities, loss of Fgfr2 often leads to multiple ureteric buds and anomalies including renal aplasia, misshaped kidneys, partially duplicated kidneys, duplicated ureters, and obstructed hydroureter. Deletion of Fgfr2 did not change expression domains of glial cell line-derived neurotrophic factor (GDNF), Robo2, bone morphogenetic protein 4, or Sprouty1, all of which regulate ureteric bud induction. Cultured Fgfr2 mutant nephric ducts were also not more sensitive to exogenous GDNF than controls. Whole mount in situ hybridization revealed that in mutant embryos, Fgfr2 was deleted from stromal cells around the nephric duct and ureteric bud base, which correlates well with the ureteric bud induction abnormalities. Thus, Fgfr2 is critical in ensuring that there is a single ureteric bud from the nephric duct. The plethora of later stage defects in Fgfr2 conditional knockouts is reminiscent of many human cases of genetic urogenital anomalies.

High Incidence of Vesicoureteral Reflux in Mice With Fgfr2 Deletion in Kidney Mesenchyma

PURPOSEnMice with Fgfr2 conditional deletion in metanephric mesenchyma (Fgfr2(Mes-/-)) have ureteral bud induction abnormalities. We determined whether Fgfr2(Mes-/-) mutants developed abnormally positioned ureters predisposing to vesicoureteral reflux.nnnMATERIALS AND METHODSnWe measured common nephric duct length and assayed for apoptosis in embryonic day 11.5 mice. We performed 3-dimensional reconstruction of, and real-time polymerase chain reaction and whole mount in situ hybridization for Fgfr2 in urinary tracts in embryonic day 15.5 embryos. We also performed cystograms followed by 3-dimensional reconstruction in postnatal animals.nnnRESULTSnCompared with controls Fgfr2(Mes-/-) embryos had increased common nephric duct length with no difference in apoptosis, indicating cranially displaced ureteral buds. Three-dimensional reconstruction at embryonic day 15.5 showed low ureteral insertion into the bladder near the bladder neck in Fgfr2(Mes-/-) mice. Postnatal Fgfr2(Mes-/-) mutants had a high rate of vesicoureteral reflux compared with controls (47.4% vs 4.0%, p = 0.00006). In postnatal mutants with unilateral reflux the refluxing ureters inserted closer to the bladder neck than nonrefluxing ureters. External ureteral insertional angles at the outer bladder wall formed by the ureteral insertion points and the bladder neck were greater in mutant refluxing ureters than in contralateral nonrefluxing ureters or control ureters. At embryonic day 15.5 Fgfr2 was decreased in Fgfr2(Mes-/-) kidneys compared with that in controls but not statistically different in ureters or bladders.nnnCONCLUSIONSnFgfr2(Mes-/-) mice have ureteral induction abnormalities associated with abnormal ureteral insertion in the bladder and subsequent vesicoureteral reflux, consistent with the Mackie and Stephens hypothesis.