RESUMEN
Klotho plays a critical role in the regulation of ion and fluid homeostasis. A previous study reported that haplo-insufficiency of Klotho in mice results in increased aldosterone synthase (CYP11B2) expression, elevated plasma aldosterone, and high blood pressure. This phenotype was presumed to be the result of diminished Klotho expression in zona glomerulosa (zG) cells of the adrenal cortex; however, systemic effects on adrenal aldosterone production could not be ruled out. To examine whether Klotho expressed in the zG is indeed a critical regulator of aldosterone synthesis, we generated a tamoxifen-inducible, zG-specific mouse model of Klotho deficiency by crossing Klotho-flox mice with Cyp11b2-CreERT mice (zG-Kl-KO). Tamoxifen-treated Cyp11b2-CreERT animals (zG-Cre) served as controls. Rosa26-mTmG reporter mice were used for Cre-dependent lineage-marking. Two weeks after tamoxifen induction, the specificity of the zG-Cre line was verified using immunofluorescence analysis to show that GFP expression was restricted to the zG. RNA in situ hybridization revealed a 65% downregulation of Klotho messenger RNA expression in the zG of zG-Kl-KO female mice at age 12 weeks compared to control mice. Despite this significant decrease, zG-Kl-KO mice exhibited no difference in plasma aldosterone levels. However, adrenal CYP11B2 expression and the CYP11B2 promotor regulatory transcription factors, NGFIB and Nurr1, were enhanced. Together with in vitro experiments, these results suggest that zG-derived Klotho modulates Cyp11b2 but does not evoke a systemic phenotype in young adult mice on a normal diet. Further studies are required to investigate the role of adrenal Klotho on aldosterone synthesis in aged animals.
Asunto(s)
Corteza Suprarrenal , Hiperaldosteronismo , Femenino , Ratones , Animales , Zona Glomerular/metabolismo , Citocromo P-450 CYP11B2/genética , Citocromo P-450 CYP11B2/metabolismo , Aldosterona/metabolismo , Corteza Suprarrenal/metabolismo , Hiperaldosteronismo/genética , Tamoxifeno/farmacologíaRESUMEN
The kidney controls systemic inorganic phosphate (Pi) levels by adapting reabsorption to Pi intake. Renal Pi reabsorption is mostly mediated by sodium-phosphate cotransporters NaPi-IIa (SLC34A1) and NaPi-IIc (SLC34A3) that are tightly controlled by various hormones including parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23). PTH and FGF23 rise in response to Pi intake and decrease NaPi-IIa and NaPi-IIc brush border membrane abundance enhancing phosphaturia. Phosphaturia and transporter regulation occurs even in the absence of PTH and FGF23 signaling. The calcium-sensing receptor (CaSR) regulates PTH and FGF23 secretion, and may also directly affect renal Pi handling. Here, we combined pharmacological and genetic approaches to examine the role of the CaSR in the acute phosphaturic response to Pi loading. Animals pretreated with the calcimimetic cinacalcet were hyperphosphatemic, had blunted PTH levels upon Pi administration, a reduced Pi-induced phosphaturia, and no Pi-induced NaPi-IIa downregulation. The calcilytic NPS-2143 exaggerated the PTH response to Pi loading but did not abolish Pi-induced downregulation of NaPi-IIa. In mice with a dominant inactivating mutation in the Casr (CasrBCH002), baseline NaPi-IIa expression was higher, whereas downregulation of transporter expression was blunted in double CasrBCH002/PTH knockout (KO) transgenic animals. Thus, in response to an acute Pi load, acute modulation of the CaSR affects the endocrine and renal response, whereas chronic genetic inactivation, displays only subtle differences in the downregulation of NaPi-IIa and NaPi-IIc renal expression. We did not find evidence that the CaSR impacts on the acute renal response to oral Pi loading beyond its role in regulating PTH secretion.NEW & NOTEWORTHY Consumption of phosphate-rich diets causes an adaptive response of the body leading to the urinary excretion of phosphate. The underlying mechanisms are still poorly understood. Here, we examined the role of the calcium-sensing receptor (CaSR) that senses both calcium and phosphate. We confirmed that the receptor increases the secretion of parathyroid hormone involved in stimulating urinary phosphate excretion. However, we did not find any evidence for a role of the receptor beyond this function.
Asunto(s)
Factor-23 de Crecimiento de Fibroblastos , Riñón , Ratones Noqueados , Hormona Paratiroidea , Fosfatos , Receptores Sensibles al Calcio , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIc , Receptores Sensibles al Calcio/metabolismo , Receptores Sensibles al Calcio/genética , Animales , Hormona Paratiroidea/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/genética , Fosfatos/metabolismo , Riñón/metabolismo , Riñón/efectos de los fármacos , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIc/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIc/genética , Ratones , Reabsorción Renal/efectos de los fármacos , Masculino , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Ratones Endogámicos C57BLRESUMEN
The Calcium-sensing receptor (CaSR) senses extracellular calcium, regulates parathyroid hormone (PTH) secretion, and has additional functions in various organs related to systemic and local calcium and mineral homeostasis. Familial hypocalciuric hypercalcemia type I (FHH1) is caused by heterozygous loss-of-function mutations in the CaSR gene, and is characterized by the combination of hypercalcemia, hypocalciuria, normal to elevated PTH, and facultatively hypermagnesemia and mild bone mineralization defects. To date, only heterozygous Casr null mice have been available as model for FHH1. Here we present a novel mouse FHH1 model identified in a large ENU-screen that carries an c.2579 T > A (p.Ile859Asn) variant in the Casr gene (CasrBCH002 mice). In order to dissect direct effects of the genetic variant from PTH-dependent effects, we crossed CasrBCH002 mice with PTH deficient mice. Heterozygous CasrBCH002 mice were fertile, had normal growth and body weight, were hypercalcemic and hypermagnesemic with inappropriately normal PTH levels and urinary calcium excretion replicating some features of FHH1. Hypercalcemia and hypermagnesemia were independent from PTH and correlated with higher expression of claudin 16 and 19 in kidneys. Likewise, reduced expression of the renal TRPM6 channel in CasrBCH002 mice was not dependent on PTH. In bone, mutations in Casr rescued the bone phenotype observed in Pth null mice by increasing osteoclast numbers and improving the columnar pattern of chondrocytes in the growth zone. In summary, CasrBCH002 mice represent a new model to study FHH1 and our results indicate that only a part of the phenotype is driven by PTH.
Asunto(s)
Hipercalcemia , Hormona Paratiroidea , Receptores Sensibles al Calcio , Animales , Masculino , Ratones , Calcio/metabolismo , Modelos Animales de Enfermedad , Hipercalcemia/genética , Hipercalcemia/metabolismo , Hipercalcemia/congénito , Ratones Endogámicos C57BL , Hormona Paratiroidea/metabolismo , Hormona Paratiroidea/genética , Receptores Sensibles al Calcio/genética , Receptores Sensibles al Calcio/metabolismoRESUMEN
Studies addressing homeostasis of inorganic phosphate (Pi) are mostly restricted to murine models. Data provided by genetically modified mice suggest that renal Pi reabsorption is primarily mediated by the Na+/Pi cotransporter NaPi-IIa/Slc34a1, whereas the contribution of NaPi-IIc/Slc34a3 in adult animals seems negligible. However, mutations in both cotransporters associate with hypophosphatemic syndromes in humans, suggesting major inter-species heterogeneity. Urinary extracellular vesicles (UEV) have been proposed as an alternative source to analyse the intrinsic expression of renal proteins in vivo. Here, we analyse in rats whether the protein abundance of renal Pi transporters in UEV correlates with their renal content. For that, we compared the abundance of NaPi-IIa and NaPi-IIc in paired samples from kidneys and UEV from rats fed acutely and chronically on diets with low or high Pi. In renal brush border membranes (BBM) NaPi-IIa was detected as two fragments corresponding to the full-length protein and to a proteolytic product, whereas NaPi-IIc migrated as a single full-length band. The expression of NaPi-IIa (both fragments) in BBM adapted to acute as well to chronic changes of dietary Pi, whereas adaptation of NaPi-IIc was only detected in response to chronic administration. Both transporters were detected in UEV as well. UEV reflected the renal adaptation of the NaPi-IIa proteolytic fragment (but not the full-length protein) upon chronic but not acute dietary changes, while also reproducing the chronic regulation of NaPi-IIc. Thus, the composition of UEV reflects only partially changes in the expression of NaPi-IIa and NaPi-IIc at the BBM triggered by dietary Pi.
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Vesículas Extracelulares , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa , Animales , Vesículas Extracelulares/metabolismo , Humanos , Riñón/metabolismo , Ratones , Proteínas de Transporte de Fosfato/metabolismo , Fosfatos/metabolismo , Ratas , Sodio/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIc/genéticaRESUMEN
Kidneys are key regulators of phosphate homeostasis. Biallelic mutations of the renal Na+/phosphate cotransporter SLC34A1/NaPi-IIa cause idiopathic infantile hypercalcemia, whereas monoallelic mutations were frequently noted in adults with kidney stones. Genome-wide-association studies identified SLC34A1 as a risk locus for chronic kidney disease. Pathogenic mutations in SLC34A1 are present in 4% of the general population. Here, we characterize a mouse model carrying the 91del7 in-frame deletion, a frequent mutation whose significance remains unclear. Under normal dietary conditions, 12 weeks old heterozygous and homozygous males have similar plasma and urinary levels of phosphate as their wild type (WT) littermates, and comparable concentrations of parathyroid hormone, fibroblast growth factor 23 (FGF-23) and 1,25(OH)2 vitamin D3. Renal phosphate transport, and expression of NaPi-IIa and NaPi-IIc cotransporters, was indistinguishable in the three genotypes. Challenging mice with low dietary phosphate did not result in differences between genotypes with regard to urinary and plasma phosphate. Urinary and plasma phosphate, plasma FGF-23 and expression of cotransporters were similar in all genotypes after weaning. Urinary phosphate and bone mineral density were also comparable in 300 days old WT and mutant mice. In conclusion, mice carrying the 91del7 truncation do not show signs of impaired phosphate homeostasis.
Asunto(s)
Fosfatos , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa , Animales , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Homeostasis , Humanos , Masculino , Ratones , Minerales/metabolismo , Mutación , Fosfatos/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismoRESUMEN
AIMS: Dietary inorganic phosphate (Pi) modulates renal Pi reabsorption by regulating the expression of the NaPi-IIa and NaPi-IIc Pi transporters. Here, we aimed to clarify the role of several Pi-regulatory mechanisms including parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23) and inositol hexakisphosphate kinases (IP6-kinases) in the acute regulation of NaPi-IIa and NaPi-IIc. METHODS: Wildtype (WT) and PTH-deficient mice (PTH-KO) with/without inhibition of FGF23 signalling were gavaged with Pi/saline and examined at 1, 4 and 12 h. RESULTS: Pi-gavage elevated plasma Pi and decreased plasma Ca2+ in both genotypes after 1 h Within 1 h, Pi-gavage decreased NaPi-IIa abundance in WT and PTH-KO mice. NaPi-IIc was downregulated 1 h post-administration in WT and after 4 h in PTH-KO. PTH increased after 1 h in WT animals. After 4 h Pi-gavage, FGF23 increased in both genotypes being higher in the KO group. PTHrp and dopamine were not altered by Pi-gavage. Blocking FGF23 signalling blunted PTH upregulation in WT mice and reduced NaPi-IIa downregulation in PTH-KO mice 4 h after Pi-gavage. Inhibition of IP6-kinases had no effect. CONCLUSIONS: (1) Acute downregulation of renal Pi transporters in response to Pi intake occurs also in the absence of PTH and FGF23 signalling, (2) when FGF23 signalling is blocked, a partial contribution of PTH is revealed, (3) IP6 kinases, intracellular Pi-sensors in yeast and bacteria, are not involved, and (4) Acute Pi does not alter PTHrp and dopamine. Thus, signals other than PTH, PTHrp, FGF23 and dopamine contribute to renal adaption.
Asunto(s)
Fosfatos , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa , Animales , Dopamina/metabolismo , Factores de Crecimiento de Fibroblastos , Riñón/metabolismo , Ratones , Hormona Paratiroidea/metabolismo , Hormona Paratiroidea/farmacología , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Proteína Relacionada con la Hormona Paratiroidea/farmacología , Proteínas de Transporte de Fosfato/metabolismo , Fosfatos/metabolismo , Fosfatos/farmacología , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismoRESUMEN
The ATP6ap2 (Pro)renin receptor protein associates with H+-ATPases which regulate organellar, cellular, and systemic acid-base homeostasis. In the kidney, ATP6ap2 colocalizes with H+-ATPases in various cell types including the cells of the proximal tubule. There, H+-ATPases are involved in receptor-mediated endocytosis of low molecular weight proteins via the megalin/cubilin receptors. To study ATP6ap2 function in the proximal tubule, we used an inducible shRNA Atp6ap2 knockdown rat model (Kd) and an inducible kidney-specific Atp6ap2 knockout mouse model. Both animal lines showed higher proteinuria with elevated albumin, vitamin D binding protein, and procathepsin B in urine. Endocytosis of an injected fluid-phase marker (FITC- dextran, 10 kDa) was normal whereas processing of recombinant transferrin, a marker for receptor-mediated endocytosis, to lysosomes was delayed. While megalin and cubilin expression was unchanged, abundance of several subunits of the H+-ATPase involved in receptor-mediated endocytosis was reduced. Lysosomal integrity and H+-ATPase function are associated with mTOR signaling. In ATP6ap2, KO mice mTOR and phospho-mTOR appeared normal but increased abundance of the LC3-B subunit of the autophagosome was observed suggesting a more generalized impairment of lysosomal function in the absence of ATP6ap2. Hence, our data suggests a role for ATP6ap2 for proximal tubule function in the kidney with a defect in receptor-mediated endocytosis in mice and rats.
Asunto(s)
Túbulos Renales Proximales/metabolismo , ATPasas de Translocación de Protón/metabolismo , Receptores de Superficie Celular/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Endocitosis , Femenino , Lisosomas/metabolismo , Masculino , Ratones Noqueados , Ratas TransgénicasRESUMEN
BACKGROUND: Metabolic acidosis occurs frequently in patients with kidney transplant and is associated with a higher risk for and accelerated loss of graft function. To date, it is not known whether alkali therapy in these patients improves kidney function and whether acidosis and its therapy are associated with altered expression of proteins involved in renal acid-base metabolism. METHODS: We retrospectively collected kidney biopsies from 22 patients. Of these patients, nine had no acidosis, nine had metabolic acidosis [plasma bicarbonate (HCO3- <22 mmol/L) and four had acidosis and received alkali therapy. We performed transcriptome analysis and immunohistochemistry for proteins involved in renal acid-base handling. RESULTS: We found that the expression of 40 transcripts significantly changed between kidneys from non-acidotic and acidotic patients. These genes are mostly involved in proximal tubule (PT) amino acid and lipid metabolism and energy homoeostasis. Three transcripts were fully recovered by alkali therapy: the Kir4.2 potassium channel, an important regulator of PT HCO3- metabolism and transport, acyl-CoA dehydrogenase short/branched chain and serine hydroxymethyltransferase 1, genes involved in beta oxidation and methionine metabolism. Immunohistochemistry showed reduced staining for the PT NBCe1 HCO3- transporter in kidneys from acidotic patients who recovered with alkali therapy. In addition, the HCO3- exchanger pendrin was affected by acidosis and alkali therapy. CONCLUSIONS: Metabolic acidosis in kidney transplant recipients is associated with alterations in the renal transcriptome that are partly restored by alkali therapy. Acid-base transport proteins mostly from PT were also affected by acidosis and alkali therapy, suggesting that the downregulation of critical players contributes to metabolic acidosis in these patients.
Asunto(s)
Acidosis , Trasplante de Riñón , Equilibrio Ácido-Base , Acidosis/etiología , Álcalis , Humanos , Trasplante de Riñón/efectos adversos , Estudios RetrospectivosRESUMEN
BACKGROUND: Phosphate intake has increased in the last decades due to a higher consumption of processed foods. This higher intake is detrimental for patients with chronic kidney disease, increasing mortality and cardiovascular disease risk and accelerating kidney dysfunction. Whether a chronic high phosphate diet is also detrimental for the healthy population is still under debate. METHODS: We fed healthy mature adult mice over a period of one year with either a high (1.2% w/w) or a standard (0.6% w/w) phosphate diet, and investigated the impact of a high phosphate diet on mineral homeostasis, kidney function and bone health. RESULTS: The high phosphate diet increased plasma phosphate, parathyroid hormone (PTH) and calcitriol levels, with no change in fibroblast growth factor 23 levels. Urinary phosphate, calcium and ammonium excretion were increased. Measured glomerular filtration rate was apparently unaffected, while blood urea was lower and urea clearance was higher in animals fed the high phosphate diet. No change was observed in plasma creatinine levels. Blood and urinary pH were more acidic paralleled by higher bone resorption observed in animals fed a high phosphate diet. Total and cortical bone mineral density was lower in animals fed a high phosphate diet and this effect is independent of the higher PTH levels observed. CONCLUSIONS: A chronic high phosphate intake did not cause major renal alterations, but affected negatively bone health, increasing bone resorption and decreasing bone mineral density.
RESUMEN
Fibroblast growth factor 23 (FGF23) is a main regulator of mineral homeostasis. Low and high circulating FGF23 levels are associated with bone, renal, cardiovascular diseases, and increased mortality. Understanding the factors and signaling pathways affecting FGF23 levels is crucial for the management of these diseases and their complications. Here, we show that activation of the Jak1/Stat3 signaling pathway leads to inflammation in liver and to an increase in hepatic FGF23 synthesis, a key hormone in mineral metabolism. This increased synthesis leads to massive C-terminal FGF23 circulating levels, the inactive C-terminal fragment, and increased intact FGF23 levels, the active form, resulting in imbalanced production and cleavage. Liver inflammation does not lead to activation of the calcineurin-NFAT pathway, and no signs of systemic inflammation could be observed. Despite the increase of active intact FGF23, excessive C-terminal FGF23 levels block the phosphaturic activity of FGF23. Therefore, kidney function and renal αKlotho expression are normal and no activation of the MAPK pathway was detected. In addition, activation of the Jak1/Stat3 signaling pathway leads to high calcitriol levels and low parathyroid hormone production. Thus, JAK1 is a central regulator of mineral homeostasis. Moreover, this study also shows that in order to assess the impact of high FGF23 levels on disease and kidney function, the source and the balance in FGF23 production and cleavage are critical.
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Inflamación/metabolismo , Janus Quinasa 1/metabolismo , Hígado/inmunología , Hígado/metabolismo , Animales , Huesos/metabolismo , Línea Celular , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/genética , Células HEK293 , Humanos , Inmunoprecipitación , Inflamación/genética , Janus Quinasa 1/genética , Riñón/metabolismo , Ratones , Factor de Transcripción STAT3/metabolismoRESUMEN
Fine-tuning of salt and acid-base homeostasis is achieved in the renal collecting duct through the action of intercalated and principal cells. Their activity is tightly regulated adapting to changes in systemic acid-base, fluid, or electrolyte status. The relative number of acid or bicarbonate secretory intercalated cells changes in response to acid or alkali loading. Several factors that may induce collecting duct plasticity in response to acid loading have been identified including cell proliferation, Growth Differentiation Factor 15 (Gdf15), hensin (DMBT1), and SDF1 (or CXCL12). Also, the transcription factors Foxi1 and CP2L1, or the Notch2-Jag1 signaling pathway, may play a role. However, little is known about the mechanisms mediating the adaptive response of the collecting duct to alkali loading. Here, we examined in mouse kidney the response of these factors to alkali loading. Mice were left untreated or received NaHCO3 or NaCl over 7 days. Cell proliferation in vivo was monitored by Ki67 labeling or BrdU incorporation and expression of cell markers, and regulatory factors were examined. Foxi1 and GDF15 were upregulated and CP2L1 downregulated during alkali loading. Ki67 staining and BrdU incorporation were frequent in AQP2-positive cells in the NaCl and NaHCO3 groups, but no evidence was found for increased Ki67 or BrdU staining in bicarbonate-secretory cells consistent with a model that AQP2 positive precursor cells may differentiate into intercalated cells. Thus, alkali loading alters the cellular profile of the collecting duct, which may involve cell proliferation and changes in the network of molecules determining the plasticity of the collecting duct.
Asunto(s)
Álcalis/metabolismo , Túbulos Renales Colectores/metabolismo , Equilibrio Ácido-Base/fisiología , Animales , Bicarbonatos/metabolismo , Biomarcadores/metabolismo , Proliferación Celular/fisiología , Regulación hacia Abajo/fisiología , Homeostasis/fisiología , Antígeno Ki-67/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo , Regulación hacia Arriba/fisiologíaRESUMEN
Na+-coupled phosphate cotransporters from the SLC34 and SLC20 families of solute carriers mediate transepithelial transport of inorganic phosphate (Pi). NaPi-IIa/Slc34a1, NaPi-IIc/Slc34a3, and Pit-2/Slc20a2 are all expressed at the apical membrane of renal proximal tubules and therefore contribute to renal Pi reabsorption. Unlike NaPi-IIa and NaPi-IIc, which are rather kidney-specific, NaPi-IIb/Slc34a2 is expressed in several epithelial tissues, including the intestine, lung, testis, and mammary glands. Recently, the expression of NaPi-IIb was also reported in kidneys from rats fed on high Pi. Here, we systematically quantified the mRNA expression of SLC34 and SLC20 cotransporters in kidneys from mice, rats, and humans. In all three species, NaPi-IIa mRNA was by far the most abundant renal transcript. Low and comparable mRNA levels of the other four transporters, including NaPi-IIb, were detected in kidneys from rodents and humans. In mice, the renal expression of NaPi-IIa transcripts was restricted to the cortex, whereas NaPi-IIb mRNA was observed in medullary segments. Consistently, NaPi-IIb protein colocalized with uromodulin at the luminal membrane of thick ascending limbs of the loop of Henle segments. The abundance of NaPi-IIb transcripts in kidneys from mice was neither affected by dietary Pi, the absence of renal NaPi-IIc, nor the depletion of intestinal NaPi-IIb. In contrast, it was highly upregulated in a model of oxalate-induced kidney disease where all other SLC34 phosphate transporters were downregulated. Thus, NaPi-IIb may contribute to renal phosphate reabsorption, and its upregulation in kidney disease might promote hyperphosphatemia.
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Riñón/metabolismo , Insuficiencia Renal Crónica/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIb/metabolismo , Regulación hacia Arriba , Animales , Membrana Celular/metabolismo , Humanos , Masculino , Ratones Endogámicos C57BL , Fosfatos/metabolismo , Ratas Wistar , Sodio/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismoRESUMEN
Fibroblast Growth Factor 23 (FGF23) is a phosphaturic factor causing increased renal phosphate excretion as well as suppression of 1,25 (OH)2-vitamin D3. Highly elevated FGF23 can promote development of rickets and osteomalacia. We and others previously reported that acute application of erythropoietin (EPO) stimulates FGF23 production. Considering that EPO is clinically used as chronic treatment against anemia, we used here the Tg6 mouse model that constitutively overexpresses human EPO in an oxygen-independent manner, to examine the consequences of long-term EPO therapy on mineral and bone metabolism. Six to eight weeks old female Tg6 mice showed elevated intact and C-terminal fragment of FGF23 but normal plasma levels of PTH, calcitriol, calcium and phosphate. Renal function showed moderate alterations with higher urea and creatinine clearance and mild albuminuria. Renal phosphate excretion was normal whereas mild hypercalciuria was found. Renal expression of the key proteins TRPV5 and calbindin D28k involved in active calcium reabsorption was reduced in Tg6 mice. Plasma levels of the bone turnover marker osteocalcin were comparable between groups. However, urinary excretion of deoxypyridinoline (DPD) was lower in Tg6 mice. MicroCT analysis showed reduced total, cortical, and trabecular bone mineral density in femora from Tg6 mice. Our data reveal that chronic elevation of EPO is associated with high FGF23 levels and disturbed mineral homeostasis resulting in reduced bone mineral density. These observations imply the need to study the impact of therapeutically applied EPO on bone mineralization in patients, especially those suffering from chronic kidney disease.
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Calcificación Fisiológica/genética , Eritropoyetina/sangre , Factores de Crecimiento de Fibroblastos/metabolismo , Riñón/metabolismo , Minerales/metabolismo , Aminoácidos/orina , Animales , Densidad Ósea/genética , Calcitriol/sangre , Calcio/sangre , Calcio/orina , Eritropoyetina/genética , Femenino , Factor-23 de Crecimiento de Fibroblastos , Homeostasis/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Osteocalcina/sangre , Fosfatos/sangre , Fosfatos/orina , Insuficiencia Renal Crónica/metabolismoRESUMEN
Fibroblast growth factor 23 (FGF23) is a major endocrine regulator of phosphate and 1,25 (OH)2 vitamin D3 metabolism and is mainly produced by osteocytes. Its production is upregulated by a variety of factors including 1,25 (OH)2 vitamin D3, high dietary phosphate intake, and parathyroid hormone (PTH). Recently, iron deficiency and hypoxia have been suggested as additional regulators of FGF23 and a role of erythropoietin (EPO) was shown. However, the regulation of FGF23 by EPO and the impact on phosphate and 1,25(OH)2 vitamin D3 are not completely understood. Here, we demonstrate that acute administration of recombinant human EPO (rhEPO) to healthy humans increases the C-terminal fragment of FGF23 (C-terminal FGF23) but not intact FGF23 (iFGF23). In mice, rhEPO stimulates acutely (24 h) C-terminal FGF23 but iFGF23 only after 4 days without effects on PTH and plasma phosphate. 1,25 (OH)2 D3 levels and αklotho expression in the kidney decrease after 4 days. rhEPO induced FGF23 mRNA in bone marrow but not in bone, with increased staining of FGF23 in CD71+ erythroid precursors in bone marrow. Chronic elevation of EPO in transgenic mice increases iFGF23. Finally, acute injections of recombinant FGF23 reduced renal EPO mRNA expression. Our data demonstrate stimulation of FGF23 levels in mice which impacts mostly on 1,25 (OH)2 vitamin D3 levels and metabolism. In humans, EPO is mostly associated with the C-terminal fragment of FGF23; in mice, EPO has a time-dependent effect on both FGF23 forms. EPO and FGF23 may form a feedback loop controlling and linking erythropoiesis and mineral metabolism.
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Eritropoyetina/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación hacia Arriba , Adulto , Animales , Médula Ósea/metabolismo , Calcitriol/metabolismo , Células Cultivadas , Retroalimentación Fisiológica , Femenino , Factor-23 de Crecimiento de Fibroblastos , Glucuronidasa/metabolismo , Humanos , Riñón/metabolismo , Proteínas Klotho , Masculino , Ratones , Ratones Endogámicos C57BL , Hormona Paratiroidea/metabolismoRESUMEN
Maintenance of the composition of inner ear fluid and regulation of electrolytes and acid-base homeostasis in the collecting duct system of the kidney require an overlapping set of membrane transport proteins regulated by the forkhead transcription factor FOXI1. In two unrelated consanguineous families, we identified three patients with novel homozygous missense mutations in FOXI1 (p.L146F and p.R213P) predicted to affect the highly conserved DNA binding domain. Patients presented with early-onset sensorineural deafness and distal renal tubular acidosis. In cultured cells, the mutations reduced the DNA binding affinity of FOXI1, which hence, failed to adequately activate genes crucial for normal inner ear function and acid-base regulation in the kidney. A substantial proportion of patients with a clinical diagnosis of inherited distal renal tubular acidosis has no identified causative mutations in currently known disease genes. Our data suggest that recessive mutations in FOXI1 can explain the disease in a subset of these patients.
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Acidosis Tubular Renal/genética , Sordera/genética , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Células Cultivadas , Niño , Consanguinidad , ADN/metabolismo , Sordera/complicaciones , Femenino , Pérdida Auditiva Central/genética , Homocigoto , Humanos , Lactante , Túbulos Renales Distales/metabolismo , Masculino , Mutación Missense , LinajeRESUMEN
Agrin, a multidomain proteoglycan and neurotrypsin, a neuronal serine protease, are important for forming (neuromuscular) synapses. Proteolytical activity of neurotrypsin produces a C-terminal fragment of agrin, termed CAF, of approximately 22 kDA molecular size which also circulates in blood. The presence of CAF in urine suggests either glomerular filtration or secretion into urine. Blood levels of CAF have been identified as a potential novel marker of kidney function. Here we describe that several nephron segments in the mouse kidney express agrin and neutrotrypsin in addition to the localization of both protein in the glomerulum. Agrin mRNA and protein was detected in almost all nephron segments and mRNA abundance was highest in the inner medullary collecting duct. Neurotrypsin mRNA was mostly detected in the thick ascending limb of the loop of Henle, the distal convoluted tubule, and the inner medullary collecting duct. Moreover, we show that the proximal tubule absorbs injected recombinant CAF by a process shared with receptor-mediated and fluid phase endocytosis. Co-injection of CAF with recombinant human transferrin, a substrate of the receptor-mediated endocytic pathway as well as with FITC-labelled dextran (10 kDa), a marker of fluid phase endocytosis, showed partial colocalization of CAF with both markers. Further colocalization of CAF with the lysosomal marker cathepsin B suggested degradation of CAF by the lysosome in the proximal tubule. Thus, the murine kidney expresses agrin and neurotrypsin in nephron segments beyond the glomerulum. CAF is filtered by the glomerulum and is reabsorbed by endocytosis by the proximal tubule. Thus, impaired kidney function could impair glomerular clearance of CAF and thereby increase circulating CAF levels.
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Agrina/metabolismo , Biomarcadores/metabolismo , Túbulos Renales Proximales/fisiología , Riñón/fisiología , Fragmentos de Péptidos/metabolismo , Agrina/genética , Animales , Biomarcadores/sangre , Biomarcadores/orina , Endocitosis , Perfilación de la Expresión Génica/métodos , Tasa de Filtración Glomerular , Humanos , Immunoblotting , Riñón/metabolismo , Túbulos Renales Proximales/metabolismo , Asa de la Nefrona/metabolismo , Asa de la Nefrona/fisiología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Nefronas/metabolismo , Nefronas/fisiología , Fragmentos de Péptidos/genética , Proteoglicanos/genética , Proteoglicanos/metabolismo , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismoRESUMEN
The fibroblast growth factor (FGF23) plasma level is high in cardiac and renal failure and is associated with poor clinical prognosis of these disorders. Both diseases are paralleled by hyperaldosteronism. Excessive FGF23 levels and hyperaldosteronism are further observed in Klotho-deficient mice. The present study explored a putative aldosterone sensitivity of Fgf23 transcription and secretion the putative involvement of the aldosterone sensitive serum & glucocorticoid inducible kinase SGK1, SGK1 sensitive transcription factor NFκB and store operated Ca(2+) entry (SOCE). Serum FGF23 levels were determined by ELISA in mice following sham treatment or exposure to deoxycorticosterone acetate (DOCA) or salt depletion. In osteoblastic UMR106 cells transcript levels were quantified by qRT-PCR, cytosolic Ca(2+) concentration utilizing Fura-2-fluorescence, and SOCE from Ca(2+) entry following store depletion by thapsigargin. As a result, DOCA treatment and salt depletion of mice elevated the serum C-terminal FGF23 concentration. In UMR106 cells aldosterone enhanced and spironolactone decreased SOCE. Aldosterone further increased Fgf23 transcript levels in UMR106 cells, an effect reversed by mineralocorticoid receptor blockers spironolactone and eplerenone, SGK1 inhibitor EMD638683, NFκB-inhibitor withaferin A, and Ca(2+) channel blocker YM58483. In conclusion, Fgf23 expression is up-regulated by aldosterone, an effect sensitive to SGK1, NFκB and store-operated Ca(2+) entry.
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Aldosterona/metabolismo , Señalización del Calcio/fisiología , Calcio/metabolismo , Factores de Crecimiento de Fibroblastos/biosíntesis , FN-kappa B/metabolismo , Osteoblastos/metabolismo , Animales , Células Cultivadas , Femenino , Factor-23 de Crecimiento de Fibroblastos , Masculino , Ratones , Ratones Endogámicos C57BL , Regulación hacia Arriba/fisiologíaRESUMEN
The (Pro)renin receptor (P)RR/Atp6ap2 is a cell surface protein capable of binding and non-proteolytically activate prorenin. Additionally, (P)RR is associated with H(+)-ATPases and alternative functions in H(+)-ATPase regulation as well as in Wnt signalling have been reported. Kidneys express very high levels of H(+)-ATPases which are involved in multiple functions such as endocytosis, membrane protein recycling as well as urinary acidification, bicarbonate reabsorption, and salt absorption. Here, we wanted to localize the (P)RR/Atp6ap2 along the murine nephron, exmaine whether the (P)RR/Atp6ap2 is coregulated with other H(+)-ATPase subunits, and whether acute stimulation of the (P)RR/Atp6ap2 with prorenin regulates H(+)-ATPase activity in intercalated cells in freshly isolated collecting ducts. We localized (P)PR/Atp6ap2 along the murine nephron by qPCR and immunohistochemistry. (P)RR/Atp6ap2 mRNA was detected in all nephron segments with highest levels in the collecting system coinciding with H(+)-ATPases. Further experiments demonstrated expression at the brush border membrane of proximal tubules and in all types of intercalated cells colocalizing with H(+)-ATPases. In mice treated with NH4Cl, NaHCO3, KHCO3, NaCl, or the mineralocorticoid DOCA for 7 days, (P)RR/Atp6ap2 and H(+)-ATPase subunits were regulated but not co-regulated at protein and mRNA levels. Immunolocalization in kidneys from control, NH4Cl or NaHCO3 treated mice demonstrated always colocalization of PRR/Atp6ap2 with H(+)-ATPase subunits at the brush border membrane of proximal tubules, the apical pole of type A intercalated cells, and at basolateral and/or apical membranes of non-type A intercalated cells. Microperfusion of isolated cortical collecting ducts and luminal application of prorenin did not acutely stimulate H(+)-ATPase activity. However, incubation of isolated collecting ducts with prorenin non-significantly increased ERK1/2 phosphorylation. Our results suggest that the PRR/Atp6ap2 may form a complex with H(+)-ATPases in proximal tubule and intercalated cells but that prorenin has no acute effect on H(+)-ATPase activity in intercalated cells.
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Corteza Renal/efectos de los fármacos , Médula Renal/efectos de los fármacos , Túbulos Renales Colectores/efectos de los fármacos , Túbulos Renales Proximales/efectos de los fármacos , ATPasas de Translocación de Protón/genética , Receptores de Superficie Celular/genética , Renina/farmacología , Cloruro de Amonio/farmacología , Animales , Proteínas de Transporte de Anión/genética , Proteínas de Transporte de Anión/metabolismo , Acuaporina 2/genética , Acuaporina 2/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Perros , Regulación de la Expresión Génica , Corteza Renal/citología , Corteza Renal/metabolismo , Médula Renal/citología , Médula Renal/metabolismo , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/metabolismo , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/metabolismo , Células de Riñón Canino Madin Darby , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , ATPasas de Translocación de Protón/metabolismo , Receptores de Superficie Celular/metabolismo , Sistema Renina-Angiotensina/efectos de los fármacos , Transducción de Señal , Bicarbonato de Sodio/farmacología , Cloruro de Sodio/farmacología , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12/genética , Miembro 1 de la Familia de Transportadores de Soluto 12/metabolismo , Miembro 3 de la Familia de Transportadores de Soluto 12/genética , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , Transportadores de SulfatoRESUMEN
UNLABELLED: Fibroblast growth factor (FGF23) plasma levels are elevated in cardiac and renal failure and correlate with poor clinical prognosis of those disorders. Both disorders are associated with inflammation and activation of the inflammatory transcription factor NFκB. An excessive FGF23 level is further observed in Klotho-deficient mice. The present study explored a putative sensitivity of FGF23 expression to transcription factor NFκB, which is known to upregulate Orai1, the Ca(2+) channel accomplishing store-operated Ca(2+) entry (SOCE). In osteoblastic cells (UMR106) and immortalized primary periosteal (IPO) cells, protein abundance was determined by Western blotting, and in UMR106 cells, transcript levels were quantified by RT-PCR, cytosolic Ca(2+) activity utilizing Fura-2-fluorescence, and SOCE from Ca(2+) entry following store depletion by thapsigargin. As a result, UMR106 and IPO cells expressed Ca(2+) channel Orai1. SOCE was lowered by NFκB inhibitor wogonin as well as by Orai1 inhibitors 2-APB and YM58483. UMR106 cell Fgf23 transcripts were increased by stimulation of SOCE and Ca(2+) ionophore ionomycin and decreased by Orai inhibitors 2-APB, YM58483 and SK&F96365, by Orai1 silencing, as well as by NFκB inhibitors wogonin, withaferin A, and CAS 545380-34-5. In conclusion, Fgf23 expression is upregulated by stimulation of NFκB-sensitive, store-operated Ca(2+) entry. KEY MESSAGES: Osteoblast UMR106 and IPO cells express Ca(2+) channel Orai1. Osteoblast store-operated Ca(2+) entry is accomplished by NFκB-sensitive Orai1. Osteoblast Fgf23 transcription is upregulated by increase in the cytosolic Ca(2+) activity. Fgf23 transcription is decreased by Orai inhibitors and Orai1 silencing. Fgf23 transcription is lowered by NFκB inhibitors.
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Factores de Crecimiento de Fibroblastos/metabolismo , Regulación de la Expresión Génica , FN-kappa B/metabolismo , Proteína ORAI1/genética , Animales , Huesos/metabolismo , Calcio/metabolismo , Línea Celular Tumoral , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/sangre , Factores de Crecimiento de Fibroblastos/genética , Espacio Intracelular/metabolismo , Osteoblastos/metabolismo , RatasRESUMEN
BACKGROUND: Diabetes is the leading cause of CKD in the developed world. C-terminal fragment of agrin (CAF) is a novel kidney function and injury biomarker. We investigated whether serum CAF predicts progression of kidney disease in type 2 diabetics. METHODS: Serum CAF levels were measured in 71 elderly patients with diabetic nephropathy using a newly developed commercial ELISA kit (Neurotune®). Estimated glomerular filtration rate (eGFR) and proteinuria in spot urine were assessed at baseline and after 12 months follow up. The presence of end stage renal disease (ESRD) was evaluated after 24 months follow-up. Correlation and logistic regression analyses were carried out to explore the associations of serum CAF levels with GFR, proteinuria, GFR loss and incident ESRD. Renal handling of CAF was tested in neurotrypsin-deficient mice injected with recombinant CAF. RESULTS: We found a strong association of serum CAF levels with eGFR and a direct association with proteinuria both at baseline (r = 0.698, p<0.001 and r = 0. 287, p = 0.02) as well as after 12 months follow-up (r = 0.677, p<0.001 and r = 0.449, p<0.001), respectively. Furthermore, in multivariate analysis, serum CAF levels predicted eGFR decline at 12 months follow-up after adjusting for known risk factors (eGFR, baseline proteinuria) [OR (95%CI) = 4.2 (1.2-14.5), p = 0.024]. In mice, injected CAF was detected in endocytic vesicles of the proximal tubule. CONCLUSION: Serum CAF levels reflect renal function and are highly associated with eGFR and proteinuria at several time points. Serum CAF was able to predict subsequent loss of renal function irrespective of baseline proteinuria in diabetic nephropathy. CAF is likely removed from circulation by glomerular filtration and subsequent endocytosis in the proximal tubule. These findings may open new possibilities for clinical trial design, since serum CAF levels may be used as a selection tool to monitor kidney function in high-risk patients with diabetic nephropathy.