Clinical and basic relevance of the phosphate overload on FGF23 renal receptors and chronic kidney disease progression

La enfermedad renal crónica (ERC) es una patología prevalente que se asocia a alteraciones en el metabolismo óseo mineral, alto riesgo cardiovascular y mortalidad. Por ello, es necesaria la realización de investigaciones dirigidas tanto a prevenir como a identificar los mecanismos de progresión de la ERC. El fósforo sérico incrementa con el deterioro de la función renal y a su vez es un factor de riesgo de progresión de ERC. Por otro lado, el factor de crecimiento fibroblástico 23 (FGF23) es una hormona fosfatúrica clave en este contexto ya que se encarga fundamentalmente de la excreción urinaria de fósforo. Por esta razón, el objetivo general de esta tesis es estudiar los mecanismos que subyacen a la resistencia a FGF23 e identificar un biomarcador capaz de predecir la progresión de la ERC. Inicialmente planteamos modelos experimentales animales e in vitro para determinar los mecanismos responsables de la resistencia de FGF23. Para ello, se evaluó el efecto de la administración de FGF23 recombinante en ratas con función renal normal, observándose una reducción significativa de la expresión renal de Klotho. A continuación, se exploró el efecto de FGF23 en ratas con insuficiencia renal que recibían dietas con diferente contenido en fósforo, de forma que la dieta rica en fósforo incrementó los niveles de FGF23 produciendo aumento de su receptor FGFR1 pero disminución del co-receptor Klotho. Tras administrar un anticuerpo anti- FGF23 (que disminuye los niveles de FGF23), la elevación de la fracción de excreción de fósforo en orina se asoció con una reducción de Klotho, responsabilizando al exceso de fósforo como un elemento clave para promover el descenso de Klotho y el aumento de FGF23. Con estos hallazgos, se planteó posteriormente valorar el efecto del calcitriol como estrategia terapéutica para incrementar los niveles de Klotho. Sin embargo, a pesar de aumentar el fósforo sérico, la expresión de Klotho no descendió. Es decir, el calcitriol evitó la reducción de Klotho inducida por fósforo aunque no consiguió incrementar sus niveles. In vitro, se evaluó el papel de la ruta WNT/β- catenina sobre los niveles de Klotho y ante un exceso de fósforo. Comprobamos que la inhibición de la ruta con Dkk1 (Dickkopf) consiguió aumentar los niveles de Klotho descendidos por el elevado fósforo. Por último, se valoró el efecto de calcitriol en la expresión de Klotho en células HEK-293, observando que calcitriol restablece también aquí la expresión de Klotho in vitro. Por tanto, la existencia de una resistencia a FGF23 promovida por la sobrecarga de fósforo, posibilita que el FGF23 aumente excesivamente mientras que la expresión de Klotho disminuye, causando un descenso en la fosfaturia y promoviendo por tanto la hiperfosfatemia y las posteriores calcificaciones vasculares. Por otro lado, a la vista de estos resultados se realizaron estudios clínicos y experimentales para evaluar en qué medida la elevada ingesta de fósforo y el exceso de fósforo en orina pueden servir como marcadores de progresión renal. A través de los siguientes estudios experimentales realizados pudimos observar que, en estadios tempranos de la ERC, la excesiva carga tubular de fósforo es responsable de estés oxidativo, inflamación y daño renal que contribuye a la progresión de la ERC. De la realización de los estudios clínicos se puede concluir que el ratio fósforo/creatinina podría ser un biomarcador útil para estimar la progresión de la ERC en pacientes con estadios iniciales de la enfermedad. Además encontramos que el ratio fósforo/urea en orina de 24h podría ser un marcador de ingesta de fósforo, por lo que podría ser útil evaluando la progresión de la ERC y como guía para el asesoramiento dietético con el que poder controlar la carga de fósforo en los pacientes con ERC en estadios tempranos de la enfermedad.Chronic kidney disease (CKD) is a prevalent pathology that is associated with mineral bone disease (CKD-MBD), high cardiovascular risk and mortality. Therefore, it is necessary to conduct research aimed at both identifying and preventing the mechanisms of progression of CKD. Serum phosphate increases during impaired renal function and in turn is a risk factor for CKD progression. In contrast, fibroblast growth factor 23 (FGF23) is a phosphaturic hormone that is vital in this context since it is primarily responsible for urinary phosphate excretion. For this reason, the general aim of this thesis is to study the mechanisms that be the cause of resistance to FGF23 and identify a talented biomarker of predicting the progression of CKD. In the beginning we propose animal and in vitro experimental models to determine the mechanisms responsible for the resistance of FGF23. For this, the effect of the administration of recombinant FGF23 in rats with normal renal function was evaluated, and a significant reduction in renal expression of Klotho was observed. Next, the effect of FGF23 in rats with renal insufficiency that received diets with different phosphate content was explored; so that the phosphate-rich diet increased FGF23 levels producing an increase in its receptor FGFR1 but a decrease in the coreceptor Klotho. After anti-FGF23 antibody administration (which lowers FGF23 levels), the elevation of the phosphate excretion fraction in urine was associated with a reduction in Klotho, holding excess phosphate responsible as a key element in promoting drop in Klotho and the increase in FGF23. With these findings, it was subsequently proposed to assess the effect of calcitriol as a therapeutic strategy to increase Klotho levels. However, despite increasing serum phosphate, Klotho's expression did not decrease. That is, calcitriol avoided the reduction phosphate-induced of Klotho although it failed to increase its levels. In vitro, the role of the WNT/β- catenin pathway on Klotho levels and with an excess phosphate was evaluated. We verify that the inhibition of the pathway with Dkk1 (Dickkopf) achieved to increase the levels of descended Klotho by the high phosphate. Finally, the effect of calcitriol on the expression of Klotho in HEK-293 cells was assessed, observing that calcitriol also restores here the expression of Klotho in vitro. Therefore, the existence of a resistance to FGF23, promoted by phosphate overload, makes it possible for FGF23 to increase excessively while Klotho's expression decreases, causing a decrease in phosphaturia and thereby promoting hyperphosphatemia and the subsequent vascular calcifications. Then again, in view of these results, clinical and experimental studies were conducted to assess the extent to which high phosphate intake and excess phosphate in urine can serve as markers of renal progression. Through the following experimental studies we were able to observe that, in the early stages of CKD, the excessive phosphate tubular load is responsible for oxidative stress, inflammation and renal damage that contribute to the progression of CKD. From the results of clinical studies, it can be concluded that the hosphate/creatinine ratio could be a useful biomarker to estimate the progression of CKD in patients with initial stages of the disease. Moreover we found that the 24h urine phosphate/urea ratio could be a marker of phosphate intake, so it could be useful in assessing the progression of CKD and as a guide for dietary advice to control the load phosphate in patients with early stages of CKD

Dietary Mg Supplementation Decreases Oxidative Stress, Inflammation, and Vascular Dysfunction in an Experimental Model of Metabolic Syndrome with Renal Failure

whether a dietary Mg supplementation might attenuate vascular dysfunction through the modulation of oxidative stress and inflammation in concurrent MetS and CKD. Methods: A rat model of MetS (Zucker strain) with CKD (5/6 nephrectomy, Nx) was used. Nephrectomized animals were fed a normal 0.1%Mg (MetS+Nx+Mg0.1%) or a supplemented 0.6%Mg (MetS+Nx+Mg0.6%) diet; Sham-operated rats with MetS receiving 0.1%Mg were used as controls. Results: As compared to controls, the MetS+Nx-Mg0.1% group showed a significant increase in oxidative stress and inflammation biomarkers (lipid peroxidation and aortic interleukin-1b and -6 expression) and Endothelin-1 levels, a decrease in nitric oxide and a worsening in uremia and MetS associated pathology as hypertension, and abnormal glucose and lipid profile. Moreover, proteomic evaluation revealed changes mainly related to lipid metabolism and CVD markers. By contrast, in the MetS+Nx+Mg0.6% group, these parameters remained largely similar to controls. Conclusion: In concurrent MetS and CKD, dietary Mg supplementation reduced inflammation and oxidative stress and improved vascular function

Klotho/FGF23 and Wnt Signaling as Important Players in the Comorbidities Associated with Chronic Kidney Disease

Fibroblast Growth Factor 23 (FGF23) and Klotho play an essential role in the regulation of mineral metabolism, and both are altered as a consequence of renal failure. FGF23 increases to augment phosphaturia, which prevents phosphate accumulation at the early stages of chronic kidney disease (CKD). This effect of FGF23 requires the presence of Klotho in the renal tubules. However, Klotho expression is reduced as soon as renal function is starting to fail to generate a state of FGF23 resistance. Changes in these proteins directly affect to other mineral metabolism parameters; they may affect renal function and can produce damage in other organs such as bone, heart, or vessels. Some of the mechanisms responsible for the changes in FGF23 and Klotho levels are related to modifications in the Wnt signaling. This review examines the link between FGF23/Klotho and Wnt/β-catenin in different organs: kidney, heart, and bone. Activation of the canonical Wnt signaling produces changes in FGF23 and Klotho and vice versa; therefore, this pathway emerges as a potential therapeutic target that may help to prevent CKD-associated complications

Magnesium Replacement to Protect Cardiovascular and Kidney Damage? Lack of Prospective Clinical Trials

Patients with advanced chronic kidney disease exhibit an increase in cardiovascular mortality. Recent works have shown that low levels of magnesium are associated with increased cardiovascular and all-cause mortality in hemodialysis patients. Epidemiological studies suggest an influence of low levels of magnesium on the occurrence of cardiovascular disease, which is also observed in the normal population. Magnesium is involved in critical cellular events such as apoptosis and oxidative stress. It also participates in a number of enzymatic reactions. In animal models of uremia, dietary supplementation of magnesium reduces vascular calcifications and mortality; in vitro, an increase of magnesium concentration decreases osteogenic transdifferentiation of vascular smooth muscle cells. Therefore, it may be appropriate to evaluate whether magnesium replacement should be administered in an attempt to reduce vascular damage and mortality in the uremic population In the present manuscript, we will review the magnesium homeostasis, the involvement of magnesium in enzymatic reactions, apoptosis and oxidative stress and the clinical association between magnesium and cardiovascular disease in the general population and in the context of chronic kidney disease. We will also analyze the role of magnesium on kidney function. Finally, the experimental evidence of the beneficial effects of magnesium replacement in chronic kidney disease will be thoroughly described