Renal expression of FGF23 and peripheral resistance to elevated FGF23 in rodent models of polycystic kidney disease
2014; Elsevier BV; Volume: 85; Issue: 6 Linguagem: Inglês
10.1038/ki.2013.526
ISSN1523-1755
AutoresDaniela Egli-Spichtig, Hongbo Zhang, Nilufar Mohebbi, Ivana Pavik, Katja Petzold, Gerti Stange, Lanja Saleh, Ilka Edenhofer, Stephan Segerer, Jürg Biber, Philippe Jaeger, Andreas L. Serra, Carsten A. Wagner,
Tópico(s)Biomedical Research and Pathophysiology
ResumoFibroblast growth factor 23 (FGF23) regulates phosphate homeostasis and is linked to cardiovascular disease and all-cause mortality in chronic kidney disease. FGF23 rises in patients with CKD stages 2–3, but in patients with autosomal dominant polycystic kidney disease, the increase of FGF23 precedes the first measurable decline in renal function. The mechanisms governing FGF23 production and effects in kidney disease are largely unknown. Here we studied the relation between FGF23 and mineral homeostasis in two animal models of PKD. Plasma FGF23 levels were increased 10-fold in 4-week-old cy/+ Han:SPRD rats, whereas plasma urea and creatinine concentrations were similar to controls. Plasma calcium and phosphate levels as well as TmP/GFR were similar in PKD and control rats at all time points examined. Expression and activity of renal phosphate transporters, the vitamin D3–metabolizing enzymes, and the FGF23 co-ligand Klotho in the kidney were similar in PKD and control rats through 8 weeks of age, indicating resistance to FGF23, although phosphorylation of the FGF receptor substrate 2α protein was enhanced. In the kidneys of rats with PKD, FGF23 mRNA was highly expressed and FGF23 protein was detected in cells lining renal cysts. FGF23 expression in bone and spleen was similar in control rats and rats with PKD. Similarly, in an inducible Pkd1 knockout mouse model, plasma FGF23 levels were elevated, FGF23 was expressed in kidneys, but renal phosphate excretion was normal. Thus, the polycystic kidney produces FGF23 but is resistant to its action. Fibroblast growth factor 23 (FGF23) regulates phosphate homeostasis and is linked to cardiovascular disease and all-cause mortality in chronic kidney disease. FGF23 rises in patients with CKD stages 2–3, but in patients with autosomal dominant polycystic kidney disease, the increase of FGF23 precedes the first measurable decline in renal function. The mechanisms governing FGF23 production and effects in kidney disease are largely unknown. Here we studied the relation between FGF23 and mineral homeostasis in two animal models of PKD. Plasma FGF23 levels were increased 10-fold in 4-week-old cy/+ Han:SPRD rats, whereas plasma urea and creatinine concentrations were similar to controls. Plasma calcium and phosphate levels as well as TmP/GFR were similar in PKD and control rats at all time points examined. Expression and activity of renal phosphate transporters, the vitamin D3–metabolizing enzymes, and the FGF23 co-ligand Klotho in the kidney were similar in PKD and control rats through 8 weeks of age, indicating resistance to FGF23, although phosphorylation of the FGF receptor substrate 2α protein was enhanced. In the kidneys of rats with PKD, FGF23 mRNA was highly expressed and FGF23 protein was detected in cells lining renal cysts. FGF23 expression in bone and spleen was similar in control rats and rats with PKD. Similarly, in an inducible Pkd1 knockout mouse model, plasma FGF23 levels were elevated, FGF23 was expressed in kidneys, but renal phosphate excretion was normal. Thus, the polycystic kidney produces FGF23 but is resistant to its action. Systemic phosphate homeostasis is regulated by a variety of factors including dietary intake, intestinal absorption, skeletal turnover, renal excretion, and systemic acid–base status as well as by many hormones. Among them, parathyroid hormone (PTH), vitamin D3, and fibroblast growth factor 23 (FGF23) act in concert at various levels of systemic phosphate homeostasis.1.Bacic D. Capuano P. 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Urakawa I. et al.Direct evidence for a causative role of FGF23 in the abnormal renal phosphate handling and vitamin D metabolism in rats with early-stage chronic kidney disease.Kidney Int. 2010; 78: 975-980Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar Autosomal dominant polycystic kidney disease (ADPKD) is a slowly progressive disease that is manifested by the replacement of functional renal tissue with growing cysts.25.Torres V.E. Harris P.C. Pirson Y. Autosomal dominant polycystic kidney disease.Lancet. 2007; 369: 1287-1301Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar The responsible mutations lie in the PKD1 and PKD2 genes, encoding the two transmembrane proteins polycystin-1 and polycystin-2, respectively.26.Reeders S.T. Breuning M.H. Davies K.E. et al.A highly polymorphic DNA marker linked to adult polycystic kidney disease on chromosome 16.Nature. 1985; 317: 542-544Crossref PubMed Scopus (555) Google Scholar, 27.Peters D.J. Spruit L. 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Kistler A.D. et al.Patients with autosomal dominant polycystic kidney disease have elevated fibroblast growth factor 23 levels and a renal leak of phosphate.Kidney Int. 2011; 79: 234-240Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar Among these patients, only those with normal soluble α-Klotho showed a mild decrease in plasma phosphate values.31.Pavik I. Jaeger P. Kistler A.D. et al.Patients with autosomal dominant polycystic kidney disease have elevated fibroblast growth factor 23 levels and a renal leak of phosphate.Kidney Int. 2011; 79: 234-240Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar,32.Pavik I. Jaeger P. Ebner L. et al.Soluble klotho and autosomal dominant polycystic kidney disease.Clin J Am Soc Nephrol. 2012; 7: 248-257Crossref PubMed Scopus (58) Google Scholar The Han:SPRD rat is a well-established PKD animal model with a mutation in the Pkdr1 gene that encodes the SamCystin protein.33.Bihoreau M.T. Ceccherini I. 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Le Hir M. et al.Inhibition of mTOR with sirolimus slows disease progression in Han:SPRD rats with autosomal dominant polycystic kidney disease (ADPKD).Nephrol Dial Transplant. 2006; 21: 598-604Crossref PubMed Scopus (237) Google Scholar, 40.Wu M. Arcaro A. Varga Z. et al.Pulse mTOR inhibitor treatment effectively controls cyst growth but leads to severe parenchymal and glomerular hypertrophy in rat polycystic kidney disease.Am J Physiol Renal Physiol. 2009; 297: F1597-F1605Crossref PubMed Scopus (35) Google Scholar We investigated the regulation of renal phosphate handling, FGF23 expression, and production in two PKD animal models. We found that the polycystic kidney produces FGF23 but is resistant to it. FGF23 plasma concentrations were increased in cy/+ Han:SPRD rats compared with wild-type (+/+) animals at all time points studied (Figure 1a). At week 2, the difference in FGF23 concentration between cy/+ (55±27pg/ml) and +/+ animals (35±11pg/ml) was 20pg/ml (95% confidence interval (CI) 0.5–39pg/ml). FGF23 plasma levels further increased in cy/+ animals to 549pg/ml after 4 weeks (mean difference 497pg/ml, 95% CI 444–549pg/ml). FGF23 concentration in cy/+ animals remained ∼10-fold higher at 6 and 8 weeks compared with controls. PTH plasma concentrations were similar in cy/+ and +/+ animals at 2 weeks (Figure 1b), whereas after 4 weeks, PTH levels of cy/+ animals were twofold elevated (141±16pg/ml) compared with +/+ animals (63±6pg/ml; mean difference 78pg/ml, 95% CI 64–92pg/ml). PTH concentration in cy/+ animals remained twofold higher at 6 and 8 weeks. The renal function parameters plasma creatinine and urea were similar for cy/+ and +/+ animals from birth to week 4; thereafter, the values were higher in cy/+ compared with +/+ animals (Figure 1c and d). The estimated change of plasma creatinine per week in cy/+ animals was 2μmol/l (95% CI 1–3μmol/l) and 0.8μmol/l (95% CI 0.7–1μmol/l) in +/+ animals. For urea, the estimated change per week was 0.6mmol/l (95% CI 0.4–0.9mmol/l) in cy/+ animals and 0.1mmol/l (95% CI 0.1–0.2mmol/l) in +/+ animals. At week 4, we measured plasma phosphate and calcium as well as the ratio of the maximum rate of tubular phosphate reabsorption to the glomerular filtration rate (TmP/GFR) and creatinine clearance of cy/+ and control Han:SPRD rats after an overnight fast (Supplementary Figure S1 online). All parameters were similar in both groups. Download .ppt (1.76 MB) Help with ppt files Supplementary Figures In order to identify the source(s) of elevated FGF23 in cy/+ Han:SPRD rats, we extracted mRNA from bone, kidney, spleen, heart, and liver, and assessed FGF23 mRNA expression by semiquantitative real-time PCR. In bone, FGF23 expression increased over time in cy/+ and healthy animals, but there were no differences between the groups at all time points studied (Figure 2a). In addition, we detected stable FGF23 mRNA expression in spleen, whereas no FGF23 mRNA expression was found in liver and heart tissue in both animal groups (results not shown). In polycystic kidneys, increasing levels of FGF23 mRNA were detectable after 4 weeks. However, in healthy kidneys, no FGF23 mRNA expression was detected (Figure 2a). FGF23 protein expression was detected by immunohistochemistry in cells lining the cysts in kidneys from cy/+ Han:SPRD rats but not in control kidneys (Figure 2b–f). Dmp1 (dentin matrix protein 1) and Fam20c (family with sequence similarity 20, member C) modulate FGF23 expression in bone. We detected mRNA of both molecules in rat kidney at high levels. Fam20c expression was similar in cy/+ and control rats but Dmp1 expression was increased in kidneys from 8-week-old cy/+ rats (Supplementary Figure S2 online). Plasma 1,25(OH)2 vitamin D3 levels were similar among cy/+ and control animals at 2, 4, and 8 weeks (Figure 3a). In both groups, 1,25(OH)2 vitamin D3 levels decreased similarly over time. The relative mRNA expression of vitamin D receptor is similar in both groups, whereas there is a reduction of vitamin D receptor from weeks 4 to 8 in cy/+ animals (Figure 3b). In addition, the relative mRNA expression of Cyp27b1 and Cyp24a1, the major enzymes responsible for vitamin D3 metabolism, was measured (Figure 3c and d). The mRNA expression of the catabolic enzyme Cyp24a1 was higher at 2 weeks in cy/+ animals. However, the expression of Cyp24a1 was similar in cy/+ and +/+ animals after 4 and 8 weeks. For the anabolic enzyme Cyp27b1, we detected similar mRNA expression levels in the cy/+ and +/+ groups at all time points studied. To evaluate the biological effect of high FGF23 plasma levels, we measured phosphate and calcium concentrations in blood and urine. The plasma phosphate and calcium levels as well as the renal excretion of calcium and phosphate were similar among cy/+ and +/+ animals at all time points studied (Figure 4). The renal phosphate cotransporters NaPi-IIa and NaPi-IIc are downregulated by high levels of FGF23. In our study, the mRNA and protein expression levels of NaPi-IIa and NaPi-IIc were similar in both groups at 2, 4, and 8 weeks (Figures 5 and 6). Immunolocalization of NaPi-IIa protein in kidney sections of 4- and 8-week-old cy/+ and +/+ animals demonstrated expression of NaPi-IIa protein in the brush border membrane of proximal tubules and smaller cysts as evident from colocalization with actin (Supplementary Figure S3 online). At these time points, basolateral NaPi-IIa staining was absent, whereas a prior report demonstrated aberrant NaPi-IIa localization in 32-week-old Han:SPRD rats.41.Vogel M. Kränzlin B. Biber J. et al.Altered expression of type II sodium/phosphate cotransporter in polycystic kidney disease.J Am Soc Nephrol. 2000; 11: 1926-1932PubMed Google ScholarFigure 6Phosphate transporter protein expression. Relative protein expression of NaPi-IIa and NaPi-IIc to β-actin after (a) 2, (b) 4, and (c) 8 weeks in cy/+ (black bars) and +/+ (wild type; white bars) Han:SPRD rats (mean ±s.d.); number of animals (median): cy/+=6 and +/+=6.View Large Image Figure ViewerDownload (PPT) We tested the sodium-dependent transport rate of 32-labeled phosphate into brush border membrane vesicles in the absence and presence of phosphonoformic acid (6mmol/l), an inhibitor of NaPi-IIa and NaPi-IIc, and found similar transport rates among cy/+ and control animals at 2 and 4 weeks (Figure 7). Na+-dependent phosphate uptake in the absence of phosphonoformic acid was significantly increased in the kidneys of 8-week-old cy/+ rats. 3H-glucose uptake was similar in both groups at all time points studied. High FGF23 plasma levels are associated with left ventricular hypertrophy.42.Faul C. Amaral A.P. Oskouei B. et al.FGF23 induces left ventricular hypertrophy.J Clin Invest. 2011; 121: 4393-4408Crossref PubMed Scopus (1476) Google Scholar We analyzed whether high FGF23 levels in 4- and 8-week-old rats were associated with left ventricular hypertrophy, but no difference was found for heart wall or septum thickness, heart to body weight, or heart weight to tibia length (Supplementary Figure S4 online). As cy/+ animals appear to be resistant to the expected normal biological actions of FGF23, we examined whether Klotho availability or FGF23 signaling via the FGFR-1 receptor may be altered. We observed similar relative Klotho mRNA expression in cy/+ and +/+ animals at 2 and 4 weeks, whereas at 8 weeks, Klotho mRNA expression was reduced in cy/+ animals (Figure 8a). Klotho protein abundance was similar in 8-week-old cy/+ and +/+ animals (Figure 8b). Relative mRNA expression levels of FGFR-1 in kidney and bone were similar among cy/+ and +/+ animals (Supplementary Figure S5 online). The downstream signaling of FGF23, Klotho, and FGFR-1 complex leads to the phosphorylation of the FGF receptor substrate 2α (FRS2a). Total FRS2a was similar in kidneys from 8-week-old cy/+ and +/+ rats, whereas phosphorylated FRS2a protein was increased (Figure 8c). We employed a second model of PKD, the conditional Pkd1 knockout (KO) mice where deletion of Pkd1 gene can be induced. We induced Pkd1 deletion at days 15–19 after birth that results in a slow progression of cystic kidney disease.43.Piontek K. Menezes L.F. Garcia-Gonzalez M.A. et al.A critical developmental switch defines the kinetics of kidney cyst formation after loss of Pkd1.Nat Med. 2007; 13: 1490-1495Crossref PubMed Scopus (310) Google Scholar Pkd1 mRNA was strongly reduced in kidneys and bone from 10-week-old Pkd1fl/fl, cre+ mice as compared with Pkd1fl/fl, cre- mice (Supplementary Figure S6 online). Conditional Pkd1 KO elevated intact FGF23 plasma levels in 10-week-old Pkd1fl/fl, cre+ (277±130pg/ml) compared with Pkd1fl/fl, cre- mice (199±57pg/ml), whereas TmP/GFR and creatinine clearance was similar in KO and healthy animals (Figure 9). Intact PTH did not differ between Pkd1fl/fl, cre+ (176±71pg/ml) and Pkd1fl/fl, cre- (253±126pg/ml) mice (Figure 9). Similar to the rat model, Pkd1fl/fl, cre+ mice expressed similar FGF23 mRNA levels in bone as the control mice. Kidneys from Pkd1fl/fl, cre+ mice expressed FGF23 mRNA and protein but not kidneys from Pkd1fl/fl, cre- mice (Figure 10 and Supplementary Figure S7 online).Figure 10Fibroblast growth factor 23 (FGF23) and Klotho expression in bone and kidneys of Pkd1fl/fl mice. Relative mRNA expression of FGF23 in (a) kidney and (b) bone as well as (c) Klotho in kidney to 18S rRNA after 10 weeks in Pkd1fl/fl, cre+ (black bars) and Pkd1fl/fl, cre- (white bars) mice (mean ±s.d.); number of animals (median): Pkd1fl/fl, cre+=8 and Pkd1fl/fl, cre-=15. **P<0.001.View Large Image Figure ViewerDownload (PPT) Our study provides four major findings: (1) the Han:SPRD rat model accurately reproduces the observed increase of FGF23 levels in the early disease course of human ADPKD with normal kidney function, (2) Han:SPRD rats show resistance to highly elevated FGF23 levels as evident from normal phosphatemia, unchanged renal phosphate excretion, preserved expression of renal phosphate cotransporters, increased PTH, normal 1,25(OH)2 vitamin D3 levels, and normal expression of Cyp27b1, (3) expression of FGF23 in polycystic kidneys, and (4) reproduction of the major findings in a second model of ADPKD, the inducible Pkd1 KO mouse. ADPKD patients with an estimated GFR of ≥60ml/min per 1.73m2 display strongly elevated FGF23 levels, but only a small fraction of these patients present a reduction in renal phosphate reabsorption—although small—and a very mild hypophosphatemia.31.Pavik I. Jaeger P. Kistler A.D. et al.Patients with autosomal dominant polycystic kidney disease have elevated fibroblast growth factor 23 levels and a renal leak of phosphate.Kidney Int. 2011; 79: 234-240Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar In ADPKD patients with normal renal phosphate handling despite high FGF23 levels, reduced soluble Klotho levels, were found whereas ADPKD patients who appeared to retain sensitivity to high FGF23 levels had preserved soluble Klotho levels.32.Pavik I. Jaeger P. Ebner L. et al.Soluble klotho and autosomal dominant polycystic kidney disease.Clin J Am Soc Nephrol. 2012; 7: 248-257Crossref PubMed Scopus (58) Google Scholar However, the temporal sequence of rising FGF23 in PKD, the mechanisms underlying resistance to the biological actions of FGF23, and the source and cause of the elevated FGF23 levels remained unclear. Herein, we demonstrate that intact FGF23 concentration in the cy/+ Han:SPRD rats increases early after birth before renal function decreases. The high FGF23 levels were accompanied by a twofold increase in PTH and normal 1,25(OH)2 vitamin D3 levels. Plasma creatinine and urea indicated a decrease in renal function in cy/+ animals at 6
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