Renal α-Klotho Research Articles

AMP-dependent kinase stimulates the expression of αKlotho.

Renal αKlotho along with fibroblast growth factor 23 regulates phosphate and vitamin D metabolism. Its cleavage yields soluble Klotho controlling intracellular processes. αKlotho has anti-inflammatory and antioxidant effects and is nephro- and cardioprotective. AMP-dependent kinase (AMPK) is a nephro- and cardioprotective energy sensor. Given that both αKlotho and AMPK have beneficial effects in similar organs, we studied whether AMPK regulates αKlotho gene expression in Madin-Darby canine kidney, normal rat kidney 52E, and human kidney 2 cells. Using quantitative real-time PCR and western blotting, we measured αKlotho expression upon pharmacological manipulation or siRNA-mediated knockdown of AMPKα. AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) enhanced αKlotho expression, an effect reduced in the presence of AMPK inhibitor compound C or siRNA targeting AMPK catalytic α-subunits (α1 and α2). Similarly, AMPK activators metformin and phenformin upregulated αKlotho transcripts. Taken together, our results suggest that AMPK is a powerful inducer of αKlotho and could thereby contribute to the development of future therapeutic interventions.

Association between renal α-klotho and renal pathology among patients with chronic kidney disease.

This study was designed to investigate whether renal α-klotho levels are associated with renal pathology. This is the first report on patients with chronic kidney disease (CKD). We conducted a retrospective observational study. A total of 65 CKD patients were enrolled. Serum and renal biopsy samples were collected. Estimated glomerular filtration rate (eGFR) was examined by biochemical test. And α-klotho expressions were assessed by RT-PCR and immunohistochemistry. In addition, detailed microscopic findings were reviewed. Renal α-klotho levels are associated positively with eGFR, and negatively with renal pathology, including interstitial fibrosis, inflammatory cell infiltration, and tubular atrophy. The renal α-klotho is related to renal pathology.

Phosphate Restriction Prevents Metabolic Acidosis and Curbs Rise in FGF23 and Mortality in Murine Folic Acid-Induced AKI.

Patients with AKI suffer a staggering mortality rate of approximately 30%. Fibroblast growth factor 23 (FGF23) and phosphate (P i ) rise rapidly after the onset of AKI and have both been independently associated with ensuing morbidity and mortality. This study demonstrates that dietary P i restriction markedly diminished the early rise in plasma FGF23 and prevented the rise in plasma P i , parathyroid hormone, and calcitriol in mice with folic acid-induced AKI (FA-AKI). Furthermore, the study provides evidence for P i -sensitive osseous Fgf23 mRNA expression and reveals that P i restriction mitigated calciprotein particles (CPPs) formation, inflammation, acidosis, cardiac electrical disturbances, and mortality in mice with FA-AKI. These findings suggest that P i restriction may have a prophylactic potential in patients at risk for AKI. In AKI, plasma FGF23 and P i rise rapidly and are independently associated with disease severity and outcome. The effects of normal (NP) and low (LP) dietary P i were investigated in mice with FA-AKI after 3, 24, and 48 hours and 14 days. After 24 hours of AKI, the LP diet curbed the rise in plasma FGF23 and prevented that of parathyroid hormone and calcitriol as well as of osseous but not splenic or thymic Fgf23 mRNA expression. The absence of Pth prevented the rise in calcitriol and reduced the elevation of FGF23 in FA-AKI with the NP diet. Furthermore, the LP diet attenuated the rise in renal and plasma IL-6 and mitigated the decline in renal α -Klotho. After 48 hours, the LP diet further dampened renal IL-6 expression and resulted in lower urinary neutrophil gelatinase-associated lipocalin. In addition, the LP diet prevented the increased formation of CPPs. Fourteen days after AKI induction, the LP diet group maintained less elevated plasma FGF23 levels and had greater survival than the NP diet group. This was associated with prevention of metabolic acidosis, hypocalcemia, hyperkalemia, and cardiac electrical disturbances. This study reveals P i -sensitive FGF23 expression in the bone but not in the thymus or spleen in FA-AKI and demonstrates that P i restriction mitigates CPP formation, inflammation, acidosis, and mortality in this model. These results suggest that dietary P i restriction could have prophylactic potential in patients at risk for AKI.

Rapamycin downregulates α-klotho in the kidneys of female rats with normal and reduced renal function.

Both mTOR and α-klotho play a role in the pathophysiology of renal disease, influence mineral metabolism and participate in the aging process. The influence of mTOR inhibition by rapamycin on renal α-klotho expression is unknown. Rats with normal (controls) and reduced (Nx) renal function were treated with rapamycin, 1.3 mg/kg/day, for 22 days. The experiments were conducted with rats fed 0.6% P diet (NP) and 0.2% P diet (LP). Treatment with rapamycin promoted phosphaturia in control and Nx rats fed NP and LP. A decrease in FGF23 was identified in controls after treatment with rapamycin. In rats fed NP, rapamycin decreased mRNA α-klotho/GADPH ratio both in controls, 0.6±0.1 vs 1.1±0.1, p = 0.001, and Nx, 0.3±0.1 vs 0.7±0.1, p = 0.01. At the protein level, a significant reduction in α-klotho was evidenced after treatment with rapamycin both by Western Blot: 0.6±0.1 vs 1.0±0.1, p = 0.01, in controls, 0.7±0.1 vs 1.1±0.1, p = 0.02, in Nx; and by immunohistochemistry staining. Renal α-klotho was inversely correlated with urinary P excretion (r = -0.525, p = 0.0002). The decrease in α-klotho after treatment with rapamycin was also observed in rats fed LP. In conclusion, rapamycin increases phosphaturia and down-regulates α-klotho expression in rats with normal and decreased renal function. These effects can be observed in animals ingesting normal and low P diet.

Myocardial Hypertrophy and Fibrosis Are Associated with Cardiomyocyte Beta-Catenin and TRPC6/Calcineurin/NFAT Signaling in Spontaneously Hypertensive Rats with 5/6 Nephrectomy.

Background: Arterial hypertension (AH) is associated with heart and chronic kidney disease (CKD). However, the precise mechanisms of myocardial remodeling (MR) in the settings of CKD remain elusive. We hypothesized that TRPC6, calcineurin/NFAT, and Wnt/β-catenin signaling pathways are involved in the development of MR in the background of CKD and AH. Methods: Early CKD was induced by performing a 5/6 nephrectomy (5/6NE) in spontaneously hypertensive rats (SHR-NE). Sham-operated (SO) SHR (SHR-SO) and Wistar Kyoto (WKY-SO) rats served as controls. Systolic blood pressure (SBP), heart rate, myocardial mass index (MMI), serum creatinine, cardiomyocyte diameter (dCM), myocardial fibrosis (MF), serum and kidney α-Klotho levels, myocardial expression of calcineurin (CaN), TRPC6, and β-catenin were measured two months after 5/6NE or SO. Results: NE-induced kidney dysfunction corresponded to mild-to-moderate human CKD and was associated with an increase in FGF23 and a decrease in renal α-Klotho. The levels of SBP, MMI, dCM, and MF were higher in SHRs compared to WKY-SO as well as in SHR-NE vs. SHR-SO. The MR was associated with increased cardiomyocyte expression of CaN/NFAT and β-catenin along with its intracellular re-distribution. TRPC6 protein levels were substantially elevated in both SHR groups with higher Trpc6 mRNA expression in SHR-NE. Conclusions: The Wnt/β-catenin and TRPC6/CaN/NFAT hypertrophic signaling pathways seem to be involved in myocardial remodeling in the settings of AH and CKD and might be mediated by FGF23 and α-Klotho axis.

Systemic Jak1 activation provokes hepatic inflammation and imbalanced FGF23 production and cleavage.

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.

Lower urinary α-Klotho is associated with lower angiotensin-(1-7) and higher blood pressure in young adults born preterm with very low birthweight.

Early-life factors including preterm birth and VLBW increase the risk of hypertension, but the mechanisms remain poorly understood. Reductions in the anti-aging protein α-klotho are associated with hypertension, possibly due to angiotensin (Ang) II activation, but the mechanisms are incompletely understood and clinical evidence is lacking. The association of α-klotho with the alternative Ang-(1-7) pathway, which counteracts Ang II to lower BP, is undescribed. We hypothesized that lower urinary α-klotho is associated with higher BP and lower urinary Ang-(1-7) in preterm-born VLBW young adults. In a cross-sectional analysis of data from a prospective cohort of 141 preterm-born VLBW young adults, we assessed the associations among urinary α-klotho/creatinine, Ang II/creatinine, Ang-(1-7)/creatinine, Ang II/Ang-(1-7), and BP using generalized linear models adjusted for age and hypertensive pregnancy and conducted a sensitivity analysis in 32 term-born young adults. Among those born preterm, lower α-klotho/creatinine was associated with higher systolic BP (adjusted β (aβ): -2.58mmHg, 95% CI -4.99 to -0.17), lower Ang-(1-7)/creatinine (ln aβ: 0.1, 0.04-0.16), and higher Ang II/Ang-(1-7) (ln aβ: -0.14, -0.21 to -0.07). In term-born participants, α-klotho/creatinine was inversely associated with Ang II/creatinine (ln aβ: -0.15, -0.27 to -0.03) and Ang II/Ang-(1-7) (ln aβ: -0.15, -0.27 to -0.03). In preterm-born young adults with VLBW, lower urinary α-klotho/creatinine was associated with higher SBP, lower urinary Ang-(1-7)/creatinine, and higher urinary Ang II/Ang-(1-7). Reduced renal α-klotho expression could lead to renal Ang-(1-7) suppression as a novel mechanism for the development of hypertension among individuals born preterm with VLBW.

The tripartite interaction of phosphate, autophagy, and αKlotho in health maintenance.

Aging-related organ degeneration is driven by multiple factors including the cell maintenance mechanisms of autophagy, the cytoprotective protein αKlotho, and the lesser known effects of excess phosphate (Pi), or phosphotoxicity. To examine the interplay between Pi, autophagy, and αKlotho, we used the BK/BK mouse (homozygous for mutant Becn1F121A ) with increased autophagic flux, and αKlotho-hypomorphic mouse (kl/kl) with impaired urinary Pi excretion, low autophagy, and premature organ dysfunction. BK/BK mice live longer than WT littermates, and have heightened phosphaturia from downregulation of two key NaPi cotransporters in the kidney. The multi-organ failure in kl/kl mice was rescued in the double-mutant BK/BK;kl/kl mice exhibiting lower plasma Pi, improved weight gain, restored plasma and renal αKlotho levels, decreased pathology of multiple organs, and improved fertility compared to kl/kl mice. The beneficial effects of heightened autophagy from Becn1F121A was abolished by chronic high-Pi diet which also shortened life span in the BK/BK;kl/kl mice. Pi promoted beclin 1 binding to its negative regulator BCL2, which impairs autophagy flux. Pi downregulated αKlotho, which also independently impaired autophagy. In conclusion, Pi, αKlotho, and autophagy interact intricately to affect each other. Both autophagy and αKlotho antagonizes phosphotoxicity. In concert, this tripartite system jointly determines longevity and life span.

Regulation of α-Klotho Expression by Dietary Phosphate During Growth Periods.

Inorganic phosphate (Pi) is an essential nutrient for maintaining various biological functions, particularly during growth periods. Excess intake of dietary Pi increases the secretion of fibroblast growth factor 23 (FGF23) and parathyroid hormone to maintain plasma Pi levels. FGF23 is a potent phosphaturic factor that binds to the α-klotho/FGFR complex in the kidney to promote excretion of Pi into the urine. In addition, excess intake of dietary Pi decreases renal α-klotho expression. Down-regulation or lack of α-klotho induces a premature aging-like phenotype, resulting from hyperphosphatemia, and leading to conditions such as ectopic calcification and osteoporosis. However, it remains unclear what effects dietary Pi has on α-klotho expression at different life stages, especially during growth periods. To investigate this, we used C57BL/6J mice in two life stages during growing period. Weaned (3 weeks old) and periadolescent (7 weeks old) were randomly divided into seven experimental groups and fed with 0.02, 0.3, 0.6, 0.9, 1.2, 1.5, or 1.8% Pi diets for 7days. As a result, elevated plasma Pi and FGF23 levels and decreased renal α-klotho expression were observed in weaned mice fed with a high Pi diet. In addition, a high Pi diet clearly induced renal calcification in the weaned mice. However, in the periadolescent group, renal calcification was not observed, even in the 1.8% Pi diet group. The present study indicates that a high Pi diet in weaned mice has much greater adverse effects on renal α-klotho expression and pathogenesis of renal calcification compared with periadolescent mice.

Low magnesium diet aggravates phosphate-induced kidney injury.

Magnesium is known to protect against phosphate-induced tubular cell injuries in vitro. We investigated in vivo effects of magnesium on kidney injuries and phosphate metabolism in mice exposed to a high phosphate diet. Heminephrectomized mice were maintained on a high phosphate/normal magnesium diet or a high phosphate/low magnesium diet for 6 weeks. We compared renal histology, phosphaturic hormones and renal α-Klotho expression between the two diet groups. High phosphate diet-induced tubular injuries and interstitial fibrosis were remarkably aggravated by the low-magnesium diet. At 1 week after high phosphate feeding when serum creatinine levels were similar between the two groups, the low magnesium diet suppressed not only fecal phosphate excretion but also urinary phosphate excretion, resulting in increased serum phosphate levels. Parathyroid hormone (PTH) levels were not appropriately elevated in the low magnesium diet group despite lower 1,25-dihydroxyvitamin D and serum calcium levels compared with the normal magnesium diet group. Although fibroblast growth factor 23 (FGF23) levels were lower in the low magnesium diet group, calcitriol-induced upregulation of FGF23 could not restore the impaired urinary phosphate excretion. The low magnesium diet markedly downregulated α-Klotho expression in the kidney. This downregulation of α-Klotho occurred even when mice were fed the low phosphate diet. A low magnesium diet aggravated high phosphate diet-induced kidney injuries. Impaired PTH secretion and downregulation of renal α-Klotho were likely to be involved in the blunted urinary phosphate excretion by the low magnesium diet. Increasing dietary magnesium may be useful to attenuate phosphate-induced kidney injury.

Effect of Continuous Intravenous Calcium Loading on Fibroblast Growth Factor 23 in Normal and Uremic Rats.

Fibroblast growth factor 23 (FGF23) is associated with mortality in patients with CKD. However, the mechanisms underlying stimulation of FGF23 remain to be investigated. We examined whether hypercalcemia induced by continuous intravenous (CIV) calcium (Ca) infusion regulates FGF23 levels in normal rats (Normal) and 5/6 nephrectomized uremic rats (Nx). Microinfusion pumps were implanted in the Normal and Nx rats for CIV Ca infusion, and blood, urine, kidney, and tibia were collected. The results showed an increase in serum Ca-stimulated FGF23 independently of serum phosphate (P) and creatinine levels in Normal and Nx rats. FGF23 mRNA from the tibia was also increased by the Ca infusion. Despite high FGF23 levels after Ca infusion, urinary P excretion was decreased. Renal α-Klotho expression was significantly reduced by Ca infusion. These results suggest that intravenous Ca loading might stimulate FGF23 production from bone in normal and uremic rats. Reduction of renal P excretion suggests that the bioactivity of FGF23 is inhibited, and the decrease in renal α-Klotho expression might have a role in this pathological process. In conclusion, CIV Ca loading increased FGF23 in normal and uremic rats, and renal α-Klotho is necessary to maintain the bioactivity of FGF23 as a phosphaturic factor.

The age-related changes of dietary phosphate responsiveness in plasma 1,25-dihydroxyvitamin D levels and renal Cyp27b1 and Cyp24a1 gene expression is associated with renal α-Klotho gene expression in mice.

In this study, we investigated the relationship between age-related changes in renal α-Klotho gene expression, vitamin D metabolism and the responsiveness of dietary phosphate in 1, 2 and 13 month-old mice fed a high phosphate (phosphate 1.2%) diet or low phosphate (phosphate 0.02%) diet for 5 days. We found that 1,25-dihydroxyvitamin D levels in plasma were significantly lower in the high phosphate group than the low phosphate group for 1 and 2 month-old mice, but not 13 month-old mice. In addition, in the high phosphate group plasma 1,25-dihydroxyvitamin D levels were decreased in 2 month-old mice relative to 1 month-old mice, but 13 month-old mice had higher levels than 2 month-old mice. In fact, plasma 1,25-dihydroxyvitamin D levels showed a significant correlation with vitamin D metabolism gene Cyp27b1 and Cyp24a1 mRNA expression in the high phosphate group. Interestingly, renal α-Klotho mRNA and protein levels were significant change with age. Furthermore, α-Klotho mRNA expression showed a significant negative correlation with plasma 1,25-dihydroxyvitamin D levels in the high phosphate group. Our results suggest that age-related alterations in renal α-Klotho expression could affect the responsiveness of dietary phosphate to vitamin D metabolism.

Deregulated Renal Calcium and Phosphate Transport during Experimental Kidney Failure.

Impaired mineral homeostasis and inflammation are hallmarks of chronic kidney disease (CKD), yet the underlying mechanisms of electrolyte regulation during CKD are still unclear. Here, we applied two different murine models, partial nephrectomy and adenine-enriched dietary intervention, to induce kidney failure and to investigate the subsequent impact on systemic and local renal factors involved in Ca2+ and Pi regulation. Our results demonstrated that both experimental models induce features of CKD, as reflected by uremia, and elevated renal neutrophil gelatinase-associated lipocalin (NGAL) expression. In our model kidney failure was associated with polyuria, hypercalcemia and elevated urinary Ca2+ excretion. In accordance, CKD augmented systemic PTH and affected the FGF23-αklotho-vitamin-D axis by elevating circulatory FGF23 levels and reducing renal αklotho expression. Interestingly, renal FGF23 expression was also induced by inflammatory stimuli directly. Renal expression of Cyp27b1, but not Cyp24a1, and blood levels of 1,25-dihydroxy vitamin D3 were significantly elevated in both models. Furthermore, kidney failure was characterized by enhanced renal expression of the transient receptor potential cation channel subfamily V member 5 (TRPV5), calbindin-D28k, and sodium-dependent Pi transporter type 2b (NaPi2b), whereas the renal expression of sodium-dependent Pi transporter type 2a (NaPi2a) and type 3 (PIT2) were reduced. Together, our data indicates two different models of experimental kidney failure comparably associate with disturbed FGF23-αklotho-vitamin-D signalling and a deregulated electrolyte homeostasis. Moreover, this study identifies local tubular, possibly inflammation- or PTH- and/or FGF23-associated, adaptive mechanisms, impacting on Ca2+/Pi homeostasis, hence enabling new opportunities to target electrolyte disturbances that emerge as a consequence of CKD development.

Reduced Renal α-Klotho Expression in CKD Patients and Its Effect on Renal Phosphate Handling and Vitamin D Metabolism

Renal α-Klotho (α-KL) plays a fundamental role as a co-receptor for fibroblast growth factor 23 (FGF23), a phosphaturic hormone and regulator of 1,25(OH)2 vitamin D3 (1,25VitD3). Disruption of FGF23-α-KL signaling is thought to be an early hallmark of chronic kidney disease (CKD) involving reduced renal α-KL expression and a reciprocal rise in serum FGF23. It remains unclear, however, whether the rise in FGF23 is related to the loss of renal α-KL. We evaluated α-KL expression in renal biopsy samples and measured levels of several parameters of mineral metabolism, as well as soluble α-KL (sKL), in serum and urinary samples from CKD patients (n = 236). We found that although renal α-KL levels were significantly reduced and serum FGF23 levels were significantly elevated in early and intermediate CKD, serum phosphate levels remained within the normal range. Multiple regression analysis showed that the increases in FGF23 were significantly associated with reduced renal function and elevated serum phosphate, but were not associated with loss of renal α-KL. Moreover, despite falling renal α-KL levels, the increase in FGF23 enhanced urinary fractional excretion of phosphate and reduced serum 1,25VitD3 levels in early and intermediate CKD, though not in advanced CKD. Serum sKL levels also fell significantly over the course of CKD, and renal α-KL was a significant independent determinant of sKL. These results demonstrate that FGF23 levels rise to compensate for renal failure-related phosphate retention in early and intermediate CKD. This enables FGF23-α-KL signaling and a neutral phosphate balance to be maintained despite the reduction in α-KL. In advanced CKD, however, renal α-KL declines further. This disrupts FGF23 signaling, and serum phosphate levels significantly increase, stimulating greater FGF23 secretion. Our results also suggest the serum sKL concentration may be a useful marker of renal α-KL expression levels.

Decreased renal α-Klotho expression in early diabetic nephropathy in humans and mice and its possible role in urinary calcium excretion

Hypercalciuria is one of the early manifestations of diabetic nephropathy. We explored here the role of α-Klotho, a protein expressed predominantly in distal convoluted tubules that has a role in calcium reabsorption. We studied 31 patients with early diabetic nephropathy and compared them with 31 patients with IgA nephropathy and 7 with minimal change disease. Renal α-Klotho expression was significantly lower and urinary calcium excretion (UCa/UCr) significantly higher in diabetic nephropathy than in IgA nephropathy or minimal change disease. Multiple regression analyses indicated that α-Klotho mRNA was inversely correlated with calcium excretion. We next measured these parameters in a mouse model of streptozotocin (STZ)-induced diabetic nephropathy, characterized by glomerular hyperfiltration, as seen in early diabetic nephropathy. We also confirmed a reduction of renal α-Klotho mRNA down to almost 50% and enhanced calcium excretion in mice with STZ-induced diabetic nephropathy in comparison with nondiabetic mice. Hypercalciuria was exacerbated in heterozygous α-Klotho knockout mice in comparison with wild-type mice, each with STZ-induced diabetic nephropathy. Thus, α-Klotho expression was decreased in distal convoluted tubules in diabetic nephropathy in humans and mice. Renal loss of α-Klotho may affect urinary calcium excretion in early diabetic nephropathy.