黒尾 誠

Age-related loss of lower extremity muscle strength is pronounced in individuals with chronic kidney disease (CKD). In contrast, an increase in intrarenal flow pulsatility results in initial age-related changes in renal hemodynamics, leading to the development of CKD. To date, it remains unclear whether lower extremity muscle strength determines elevated renal flow pulsatility. This study aimed to determine the association of lower extremity muscle strength and function with intrarenal hemodynamics in individuals with and without CKD. One hundred seventy-six individuals without CKD (aged 63 ± 9 years) and 101 individuals with CKD (aged 66 ± 8 years) were included in this study. Using Doppler ultrasound, the renal resistive index (RI) was measured as a parameter of renal hemodynamics. Knee extensor muscle strength (KES), gait speed (GS), and the 30 s chair stand test (30s-CST) were used to measure lower extremity muscle strength and function. Multivariate analyses showed that GS and 30s-CST scores were independent determinants of renal RI, whereas the KES score was not associated with renal RI in individuals with and without CKD. In the two-way analysis of covariance, renal RI was the highest in individuals with CKD who had lower KES, GS, and 30s-CST scores. Reduced lower extremity muscle strength and function are independent determinants of elevated renal flow pulsatility in individuals with and without CKD.

Abstract Inappropriate activation of intrarenal renin–angiotensin system (RAS) may contribute to the pathogenesis of cardio-renal syndrome (CRS). We aimed to examine the cross-sectional associations of urinary angiotensinogen (AGT) excretion, a biomarker of intrarenal RAS activity, with central (aortic) and renal hemodynamic parameters in middle-aged and older adults, including patients with chronic kidney disease. Aortic and renal hemodynamic parameters were measured using applanation tonometry and duplex ultrasonography in 282 participants. Urinary AGT, liver-type fatty acid-binding protein (L-FABP), and plasma N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels were measured for each participant. Multiple linear regression analyses demonstrated that urinary AGT levels were associated with aortic blood pressures, pulsatile measures of renal blood flow, plasma NT-proBNP and urinary L-FABP levels after adjusting for potential covariates, including age, sex, body mass index, estimated glomerular filtration rate (GFR), and medication use. Additionally, when classified based on GFR stages and urinary AGT levels, plasma NT-proBNP and urinary L-FABP levels increased in participants with lower GFR and higher AGT groups. Our findings suggest that urinary AGT excretion is a shared determinant of central (aortic) and renal hemodynamics in middle-aged and older adults, providing clinical evidence for the potential role of intrarenal RAS activity in the development of CRS.

Calcium phosphate forms particles under excessive urinary excretion of phosphate in the kidney. While the formation of calcium phosphate particles (CaPs) has been implicated in the damage to renal tubular cells and renal dysfunction, clarifying the ultrastructural information and the elemental composition of the small CaPs in the wide areas of kidney tissue has been technically difficult. This study introduces correlative and sequential light as well as electron microscopic CaP observation in the kidney tissue by combining fluorescent staining for CaPs and energy-dispersive X-ray spectroscopy (EDS) in scanning electron microscopy (SEM) on resin sections prepared using high-pressure freezing and freeze substitution. CaPs formed in mouse kidneys under long-term feeding of a high-phosphate diet were clearly visualized on resin sections by fluorescence-conjugated alendronate derivatives and toluidine blue metachromasia. These CaPs were verified by correlative observation with EDS. Furthermore, small CaPs formed in the kidney under short-term feeding were detected using fluorescent probes. The elemental composition of the particles, including calcium and magnesium, was identified following EDS analyses. These results suggest that the correlative microscopy approach is helpful for observing in situ distribution and elemental composition of CaPs in the kidney and contributing to studies regarding CaP formation-associated pathophysiology.

Calciprotein particles (CPPs) are mineral-protein complexes containing solid-phase calcium-phosphate and the serum protein fetuin-A. CPPs are dispersed in the blood as colloids. Previous clinical studies revealed that circulating levels of CPPs were correlated with inflammation and vascular calcification/stiffness in patients with chronic kidney disease (CKD). Measurement of blood CPP levels is challenging because CPPs are unstable and change their physical and chemical properties spontaneously over time in vitro. Several different methods have been developed for quantification of blood CPP levels with different advantages and limitations. We have developed a simple and sensitive assay using a fluorescent probe that bound to calcium-phosphate crystals. This assay may be useful as a clinical test to evaluate the cardiovascular risk and prognosis in CKD patients.

Calciprotein particles (CPPs) are colloids composed of solid-phase calcium-phosphate and serum protein fetuin-A. CPPs form a polydispersed system with different particle size and density. CPPs with specific physical properties can induce calcification and innate immune responses in cultured cells. In hemodialysis patients, blood CPP levels were reported to correlate with vascular calcification and inflammation. However, little is known about relation between these disorders and physical properties of CPPs. Here, we show that the association between physical properties of plasma CPPs and serum levels of inflammatory cytokines/chemokines in 78 hemodialysis out-patients by cross-sectional study. Patients with cardiovascular disease (CVD) had significantly higher high density CPP (H-CPP) levels than patients without CVD but not low density CPP (L-CPP). Seven cytokines/chemokines (EGF, eotaxin, IL-8, IP-10, MCP-1, MIP-1, MIP-1β and TNFα) were detectable in the serum samples from > 95% of the patients. In multivariate regression analysis, H-CPP was positively associated with eotaxin after adjusting for age, gender, smoking, serum phosphate and FGF23. L-CPP was negatively associated with IL-8 after adjusting for age, gender, serum albumin, phosphate and FGF23. High H-CPP levels were associated with pro-inflammatory response, whereas L-CPPs were associated with anti-inflammatory response. CPPs with different physical properties may impact differently on pathophysiology in HD patients.

Fibroblast growth factor-23 (FGF23) is a phosphaturic hormone secreted by the bone in response to dietary phosphate intake. Since the phosphate content in the diet correlates with the protein content, both plant- and animal-based protein intake can increase the serum FGF23 level. However, a higher percentage of energy from plant protein than from animal protein is associated with a lower serum FGF23 level in patients with chronic kidney disease (CKD) in the United States. Since dietary habits differ between Asian and Western populations, we performed a cross-sectional study to determine the association between the percentages of energy from plant and animal proteins and the serum FGF23 level in Japanese CKD patients. In 107 non-dialysis CKD patients (age: 66 ± 9 years; estimated glomerular filtration rate: 56 ± 21 mL/min/1.73 m2), the percentages of energy from plant and animal proteins were assessed using a food frequency questionnaire based on food groups. Venous blood samples were used to measure the serum FGF23, phosphate, 1,25-dihydroxyvitamin D, and intact parathyroid hormone levels. The percentages of energy from plant and animal proteins showed a negative and positive association, respectively, with the serum FGF23 level. Furthermore, isocaloric substitution modeling showed that replacing animal protein with plant protein was associated with a low serum FGF23 level. Our findings suggest that encouraging diets with high plant protein level may prevent an increase in the serum FGF23 level in Japanese CKD patients.

Abstract Calciprotein particles (CPPs) are circulating colloidal mineral-protein complexes containing crystalline and/or non-crystalline (amorphous) calcium-phosphate (CaPi). Serum CPP levels correlate with vascular stiffness and calcification in patients with chronic kidney disease (CKD). In vitro studies showed that CPPs containing crystalline CaPi were more arteriosclerogenic and inflammogenic than CPPs without containing crystalline CaPi. Thus, we hypothesized that not only the quantity but also the quality of CPPs (the phase of CaPi) might affect clinical outcomes. To test this hypothesis, we quantified amorphous CaPi ratio defined as the ratio of the amorphous CaPi amount to the total CaPi amount in serum CPPs from 183 hemodialysis patients and explored its possible correlation with serum parameters associated with prognosis of hemodialysis patients. Multivariate analysis revealed that the amorphous CaPi ratio correlated positively with hemoglobin and negatively with fibroblast growth factor-21 (FGF21), which remained significant after adjusting for the total CaPi amount. Because low hemoglobin and high FGF21 are associated with increased mortality, the present study warrants further studies to determine whether low amorphous CaPi ratio in circulating CPPs may be associated with poor prognosis in hemodialysis patients.

BACKGROUND: The development of aging-induced male-specific disorders is accelerated by impaired renal function. Although aging-induced male-specific disorders are clinically serious complications in individuals with reduced renal function, their practical management strategies remain obscure. The purpose of this cross-sectional study was to investigate the association between daily behavioral and sleep patterns and aging-induced male-specific disorders in individuals with reduced renal function. METHODS: Eighty men with glomerular filtration rate stage 2-4 (age, 67 ± 9 years), sedentary behavior and physical activity were assessed using a triaxial accelerometer. The mean sleep time was calculated from the sleep time during the accelerometer measurement. Sleep quality was assessed using the Pittsburgh Sleep Quality Index (PSQI). Aging-induced male-specific disorders were assessed using the Aging Males' Symptoms Questionnaire (AMS). RESULTS: A lower moderate to vigorous-intensity physical activity (MVPA) time and a higher PSQI score were independently associated with a higher AMS score. Moreover, when the participants were divided into four groups according to the median MVPA values and the PSQI score (more or less than 6 points), the AMS score was the highest in those with a lower MVPA time and a higher PSQI score. In the mediation analysis, the PSQI score did not mediate a correlation between the MVPA time and AMS score. MVPA time also did not mediate a correlation between the PSQI and AMS scores. CONCLUSIONS: Collectively, these findings suggest that increasing MVPA time and improving sleep quality may contribute to attenuating aging-induced male-specific disorders in individuals with reduced renal function.

Sit-stand maneuvers (SSM) have increasingly been used for baroreflex sensitivity (BRS) measurement in physiological research, but it remains unknown as to how many SSM need to be performed to measure BRS and assess its relation with cardiovascular disease (CVD) risk. Therefore, this study aimed to determine 1) the effect of the number of SSM repetitions on BRS and 2) the association between BRS and CVD risk factors. Data were collected from 174 individuals during 5 minutes of spontaneous rest and 5 minutes of repeated SSM at 0.05 Hz (i.e., 15 cycles of 10-second sit and 10-second stand). During SSM, BRS was calculated from the incremental cycles of 3, 6, 9, 12, and 15 SSM using transfer function analysis of heart rate (HR) and systolic blood pressure (SBP). General CVD risk factors, carotid arterial stiffness, and cardiorespiratory fitness were measured. In result, HR and SBP increased during SSM (p<0.05). The BRS remained at a similar level during the resting and SSM conditions, while the coherence function reached its peak after 3 cycles of SSM. BRS with ≥6 cycles of SSM was strongly correlated with age (r=-0.721 to -0.740), carotid distensibility (r=0.625 to 0.629), and cardiorespiratory fitness (r=0.333 to 0.351) (all p<0.001). Multiple regression analysis demonstrated that BRS with ≥6 cycles of SSM explained >60% of the variance in CVD risk factors. Therefore, our findings suggest that repeated SSM significantly strengthens the association between BRS and CVD risk factors. Particularly, BRS with ≥6 cycles of SSM is strongly associated with CVD risk.

Fibroblast growth factor 21 (FGF21) modulates energy metabolism and neuroendocrine stress responses. FGF21 synthesis is increased after environmental or metabolic challenges. Detailed roles of FGF21 in the control of behavioural disturbances under stressful conditions remain to be clarified. Here, we examined the roles of FGF21 in the control of behavioural changes after social defeat stress in male rodents. Central administration of FGF21 increased the number of tyrosine hydroxylase-positive catecholaminergic cells expressing c-Fos protein, an activity marker of neurones, in the nucleus tractus solitarius and area postrema. Double in situ hybridisation showed that some catecholaminergic neurones in the dorsal medulla oblongata expressed β-Klotho, an essential co-receptor for FGF21, in male mice. Social defeat stress increased FGF21 concentrations in the plasma of male mice. FGF21-deficient male mice showed social avoidance in a social avoidance test with C57BL/6J mice (background strain of FGF21-deficient mice) and augmented immobility behaviour in a forced swimming test after social defeat stress. On the other hand, overexpression of FGF21 by adeno-associated virus vectors did not significantly change behaviours either in wild-type male mice or FGF21-deficient male mice. The present data are consistent with the view that endogenous FGF21, possibly during the developmental period, has an inhibitory action on stress-induced depression-like behaviour in male rodents.

Calciprotein particles (CPPs) are nanoparticles composed of calcium phosphate crystals and fetuin-A and have been implicated in diseases associated with inflammation. In the current study, we investigated the molecular mechanisms underlying CPP-induced inflammation in mice. CPPs predominantly upregulated IL-1β and IL-1α and provided priming and activation signals for the NLRP3 inflammasome in murine macrophages. Pharmacological and genetic inhibition of the NLRP3 inflammasome revealed that CPPs induced the release of IL-1β and IL-1α via NLRP3 inflammasome-dependent and -independent mechanisms, respectively. CPPs also induced necrotic cell death, but gasdermin D was dispensable for CPP-induced IL-1β release and necrotic cell death. Although phagocytosis of CPPs was required for CPP-induced IL-1β/α release and necrotic cell death, lysosomal dysfunction and K+ efflux were mainly involved in CPP-induced NLRP3 inflammasome activation and subsequent IL-1β release but not in CPP-induced IL-1α release and necrotic cell death. In vivo experiments showed that CPP administration evoked acute inflammatory responses characterized by neutrophil accumulation via both IL-1β and IL-1α. In particular, CPP-induced neutrophil inflammation was mediated predominantly through an IL-1α-induced CXCL1/CXCR2 signaling pathway. These results provide new insights into the mechanism underlying CPP-induced inflammation and suggest that targeting both IL-1β and IL-1α is necessary to regulate the CPP-induced inflammatory response and to treat CPP-associated inflammatory disorders.

OBJECTIVE: Insufficient physical activity and excessive sedentary behavior can contribute to decreased skeletal muscle strength, which is strongly associated with increased mortality in patients with chronic kidney disease (CKD). However, the potential impact of replacing sedentary behavior with physical activity on skeletal muscle strength remains unclear in these patients. The purpose of this study was to examine the associations of physical activity, sedentary behavior, and skeletal muscle strength in patients with CKD using an isotemporal substitution model to estimate the associations on replacing time from one behavior to another while keeping the total time and other behaviors fixed. METHODS: A total of 108 patients with CKD (mean age = 65 [SD = 9] y; mean estimated glomerular filtration rate = 57 [SD = 22] mL/min/1.73m2) participated in this cross-sectional analysis study. The time spent in sedentary behavior, light-intensity physical activity, and moderate- to vigorous-intensity physical activity (MVPA) were assessed using a triaxial accelerometer. Handgrip strength, isometric knee extension strength, and 30-second chair stand test were used to measure skeletal muscle strength. RESULTS: In multivariate analyses (single-factor and partition models), the time spent in MVPA was beneficially associated with both isometric knee extension strength and 30-second chair stand test. Furthermore, the isotemporal substitution model found that replacing 10 minutes per day of sedentary behavior or light-intensity physical activity with equivalent MVPA time was beneficially associated with both isometric knee extension strength and 30-second chair stand test. CONCLUSIONS: Our cross-sectional findings indicate that MVPA time is beneficially associated with lower extremity muscle strength and that a slight increase in the MVPA time may contribute to maintaining skeletal muscle strength in patients with CKD. IMPACT: Increasing the time spent in MVPA (10 min/day) may be a feasible strategy in patients with CKD, who have a high prevalence of impaired physical function.

In patients with sarcoidosis, dysregulated calcium metabolism is one of the frequently observed complications. However, little attention has been paid to abnormal phosphate metabolism. Herein we present the case of a 42-year-old Japanese man with renal sarcoidosis who developed acute kidney injury due to hypercalcemia and nephrolithiasis. Laboratory data showed hypercalcemia with a normal serum phosphate level and high serum 1,25-hydroxyvitamin D3, fibroblast growth factor 23 (FGF23) and calciprotein particle (CPP) levels. After treatment with oral prednisone and bisphosphonate, the laboratory abnormalities and renal dysfunction were resolved. Thus increases in FGF23 and CPP may indicate disturbed phosphate metabolism in renal sarcoidosis.

Dietary phosphate overload induces chronic kidney disease (CKD), and calciprotein particles (CPPs), a form of nanoparticle comprising calcium phosphate and serum proteins, has been proposed to cause renal toxicity. However, the mechanism of CPP cytotoxicity in renal tubular cells is unknown. Here we show that in renal proximal tubular epithelial HK-2 cells, endocytosed CPPs accumulate in late endosomes/lysosomes (LELs) and increase their luminal pH by ~ 1.0 unit. This results in a decrease in lysosomal hydrolase activity and autophagic flux blockage without lysosomal rupture and reactive oxygen species generation. CPP treatment led to vulnerability to H2O2-induced oxidative stress and plasma membrane injury, probably because of autophagic flux blockage and decreased plasma membrane cholesterol, respectively. CPP-induced disruption of lysosomal homeostasis, autophagy flux and plasma membrane integrity might trigger a vicious cycle, leading to progressive nephron loss.

The World Health Organization has recommended 5 g/day as dietary reference intakes for salt. In Japan, the averages for men and women were 11.0 g/day and 9.3 g/day, respectively. Recently, it was reported that amounts of sodium accumulation in skeletal muscles of older people were significantly higher than those in younger people. The purpose of this study was to investigate whether the risk of sarcopenia with decreased muscle mass and strength was related to the amount of salt intake. In addition, we investigated its involvement with renalase. Four groups based on age and salt intake ("younger low-salt," "younger high-salt," "older low-salt," and "older high-salt") were compared. Stratifying by age category, body fat percentage significantly increased in high-salt groups in both younger and older people. Handgrip strength/body weight and chair rise tests of the older high-salt group showed significant reduction compared to the older low-salt group. However, there was no significant difference in renalase concentrations in plasma. The results suggest that high-salt intake may lead to fat accumulation and muscle weakness associated with sarcopenia. Therefore, efforts to reduce salt intake may prevent sarcopenia.

© 2020 Elsevier Inc. Objective: Circulating levels of fibroblast growth factor 21 (FGF21) increase with advancing age and may lead to the development of cardiometabolic diseases via impaired lipid and glucose metabolism. While physical activity can reduce these risks of cardiometabolic dysfunction, it remains obscure whether circulation FGF21 levels are influenced by physical activity. The purpose of this study was to investigate the relations between daily physical activities and circulating FGF21 levels in middle-aged and older adults. Methods: In this cross-sectional study with 110 middle-aged and 102 older adults, circulating (serum) FGF21 levels were evaluated by enzyme-linked immunosorbent assay, and the time spent in light-intensity physical activity (LPA) and moderate-to-vigorous-intensity physical activity (MVPA) was assessed using a uniaxial accelerometer. Results: Serum FGF21 levels in the older group (158 pg/mL) were significantly higher than those in the middle-aged group (117 pg/mL). When we examined the joint association of age (middle-aged or older) and MVPA (lower or higher than the median) groups, serum FGF21 levels in the older and higher MVPA group (116 pg/mL) were significantly lower than those in the older and lower MVPA group (176 pg/mL). However, there was no difference in serum FGF21 levels between the lower and higher MVPA groups in the middle-aged group. In multivariable liner regression analysis, serum FGF21 levels were independently determined by MVPA time after adjusting for potential covariates in older adults (β = −0.209). Conclusions: These cross-sectional study findings indicate that the time spent in MVPA is an independent determinant of circulating FGF21 levels, and that an age-related increase in serum FGF21 levels may be attenuated by habitually performing MVPA. (250/250 words).

Although disturbed phosphate metabolism frequently accompanies chronic kidney disease (CKD), its causal role in CKD progression remains unclear. It is also not fully understood how excess salt induces organ damage. We here show that urinary phosphate-containing nanoparticles promote kidney injury in salt-sensitive hypertension. In Dahl salt-sensitive rats, salt loading resulted in a significant increase in urinary phosphate excretion without altering serum phosphate levels. An intestinal phosphate binder sucroferric oxyhydroxide attenuated renal inflammation and proteinuria in this model, along with the suppression of phosphaturia. Using cultured proximal tubule cells, we confirmed direct pathogenic roles of phosphate-containing nanoparticles in renal tubules. Finally, transcriptome analysis revealed a potential role of complement C1q in renal inflammation associated with altered phosphate metabolism. These data demonstrate that increased phosphate excretion promotes renal inflammation in salt-sensitive hypertension and suggest a role of disturbed phosphate metabolism in the pathophysiology of hypertensive kidney disease and high salt-induced kidney injury.

Excessive intake of phosphate has been known to induce renal tubular damage and interstitial inflammation, leading to acute kidney injury or chronic kidney disease in rodents and humans. However, sensitive and early biomarkers for phosphate-induced kidney damage remain to be identified. Our previous RNA sequencing analysis of renal gene expression identified interleukin-36α (IL-36α) as a gene significantly upregulated by dietary phosphate load in mice. To determine the time course and dose dependency of renal IL-36α expression induced by dietary phosphate load, we placed mice with or without uninephrectomy on a diet containing either 0.35%, 1.0%, 1.5%, or 2.0% inorganic phosphate for 10 days, 4 weeks, or 8 weeks and evaluated renal expression of IL-36α and other markers of tubular damage and inflammation by quantitative RT-PCR, immunoblot analysis, and immunohistochemistry. We found that IL-36α expression was induced in distal convoluted tubules and correlated with phosphate excretion per nephron. The increase in IL-36α expression was simultaneous with but more robust in amplitude than the increase in tubular damage markers such as Osteopontin and neutrophil gelatinase-associated lipocalin, preceding the increase in expression of other inflammatory cytokines, including transforming growth factor-α, interleukin-1β, and transforming growth factor-β1. We conclude that IL-36α serves as a marker that reflects the degree of phosphate load excreted per nephron and of associated kidney damage.

Fibroblast growth factor-23 (FGF23) is a hormone indispensable for maintaining phosphate homeostasis. In response to phosphate intake, FGF23 is secreted from osteocytes/osteoblasts and acts on the kidney to increase urinary phosphate excretion. However, the mechanism by which these cells sense phosphate intake remains elusive. Calciprotein particles are nanoparticles of calcium-phosphate precipitates bound to serum protein fetuin-A and are generated spontaneously in solution containing calcium, phosphate, and fetuin-A to be dispersed as colloids. In cultured osteoblastic cells, increase in either calcium or phosphate concentration in the medium induced FGF23 expression, which was dependent on calciprotein particle formation. When transition of calcium-phosphate precipitates from the amorphous phase to the crystalline phase was blocked by bisphosphonate, the calciprotein particle size was reduced and FGF23 expression was augmented, suggesting that small calciprotein particles containing amorphous calcium-phosphate precipitates function as a more potent FGF23 inducer than larger calciprotein particles containing crystalline calcium-phosphate precipitates. In mice, bolus phosphate administration by oral gavage transiently increased circulating calciprotein particle levels followed by a modest increase in FGF23 expression and serum FGF23 levels. However, continuous dietary phosphate load induced robust and persistent increase in circulating calciprotein particles and FGF23 levels. We confirmed by in vivo imaging that calciprotein particles injected intravenously extravasated into the bone marrow and were deposited on the inner surface of the bone, indicating that these particles have direct access to osteoblasts. Thus, we propose that osteoblasts induce FGF23 expression and secretion when they sense an increase in extracellular calciprotein particles following phosphate ingestion.

NEW FINDINGS: What is the central question of this study? Does modulating the expression of Klotho affect myogenesis following acute injury of healthy, non-senescent muscle? What is the main finding and its importance? Klotho can accelerate muscle growth following acute injury of healthy, adult mice, which supports the possibility that increased delivery of Klotho could have therapeutic value for improving repair of damaged muscle. ABSTRACT: Skeletal muscle injuries activate a complex programme of myogenesis that can restore normal muscle structure. We tested whether modulating the expression of klotho influenced the response of mouse muscles to acute injury. Our findings show that klotho expression in muscle declines at 3 days post-injury. That reduction in klotho expression coincided with elevated expression of targets of Wnt signalling (Ccnd1; Myc) and increased MyoD+ muscle cell numbers, reflecting the onset of myogenic cell differentiation. klotho expression subsequently increased at 7 days post-injury with elevated expression occurring primarily in inflammatory lesions, which was accompanied by reduced expression of Wnt target genes (Ccnd1: 91%; Myc: 96%). Introduction of a klotho transgene maintained high levels of klotho expression over the course of muscle repair and attenuated the increases in Ccnd1 and Myc expression that occurred at 3 days post-injury. Correspondingly, transgene expression reduced Wnt signalling in Pax7+ cells, reflected by reductions in Pax7+ cells expressing active β-catenin, and reduced the numbers of MyoD+ cells at 3 days post-injury. At 21 days post-injury, muscles in klotho transgenic mice showed increased Pax7+ and decreased myogenin+ cell densities and large increases in myofibre size. Likewise, treating myogenic cells in vitro with Klotho reduced Myod expression but did not affect Pax7 expression. Muscle inflammation was only slightly modulated by increased klotho expression, initially reducing the expression of M2-biased macrophage markers Cd163 and Cd206 at 3 days post-injury and later increasing the expression of pan-macrophage marker F480 and Cd68 at 21 days post-injury. Collectively, our study shows that Klotho modulates myogenesis and that increased expression accelerates muscle growth after injury.

Objective To prolong the health expectancy, it is important to prevent age-related diseases, such as osteoporosis and cerebrovascular disease, which are major causes of a bedridden state. Early predictable biomarkers for these diseases are urgently required in the clinical setting. Three members of the fibroblast growth factor (FGF) family - FGF19, FGF21, and FGF23 - are designated as endocrine FGFs and play crucial roles in various metabolic processes. We tried to clarify the clinical utility of endocrine FGFs as biomarkers for age-related diseases in elderly patients. Methods We examined the serum endocrine FGF levels and analyzed their association with various clinical parameters in 73 outpatients >60 years old as a single-center cross-sectional study. Results In a multivariable linear regression analysis, FGF19 was associated with ALT, a history of cardiovascular disease, and medication with active vitamin D3. FGF21 was associated with the estimated glomerular filtration rate (eGFR), triglyceride level, and hypertension. FGF23 was associated with the eGFR and the serum levels of 1,25-dihydroxy vitamin D3 and TRACP5b. In addition, a receiver operating characteristics analysis revealed that the measurement of FGF21 and FGF23 was useful for detecting chronic kidney disease (CKD) and its complications, including cardiovascular disease and metabolic bone disorder. Conclusion The measurement of FGF21 and FGF23 may be useful for evaluating CKD and its complications. Using serum endocrine FGFs as biomarkers for age-related conditions may help prevent elderly patients from entering a bedridden state.

BACKGROUND: Aggregation of solid-phase calcium-phosphate and fetuin-A form nanoparticles called calciprotein particles (CPP). Serum CPP levels are increased in CKD patients and correlated with vascular stiffness and calcification. In this study, we evaluated effects of lanthanum carbonate (LC) and calcium carbonate (CC) on serum CPP levels in hemodialysis (HD) patients. METHODS: Twenty-four (24) HD patients (50% men, age; 68 ± 12 years, dialysis period; 6.2 ± 4.8 years, Kt/v; 1.74 ± 0.34) were treated with CC during 0-8 weeks and then switched to LC during 9-16 weeks. Blood samples were obtained at 0, 8, 16 weeks. Serum CPP levels (TCPP) were measured by the gel-filtration method. Low-density CPP (LCPP) levels were determined by centrifuging the serum samples at 16,000 g for 2 h and measuring CPP levels in the supernatant. The difference between TCPP and LCPP was defined as the high-density CPP (HCPP) level. We evaluated association of TCPP, LCPP, and HCPP with serum calcium (Ca), phosphorus (P), intact PTH, FGF23, Klotho, fetuin-A, aortic calcification index (ACI), LDL cholesterol, and hs-CRP. RESULTS: TCPP and LCPP levels were significantly decreased after switching CC to LC, whereas Ca and P levels were not changed. HCPP levels were below the lower limit quantification in all patients. The changes in P, Ca × P, LDL cholesterol, but not ACI and the changes in hs-CRP, were correlated with the change in TCPP levels. CONCLUSION: The TCPP levels were significantly decreased after switching CC to LC. Non-calcium-containing phosphate binders may be preferable for lowering CPP levels.

Circulating levels of fibroblast growth factor-21 (FGF21) start increasing in patients with chronic kidney disease (CKD) since early stages during the cause of disease progression. FGF21 is a liver-derived hormone that induces responses to stress through acting on hypothalamus to activate the sympathetic nervous system and the hypothalamus-pituitary-adrenal endocrine axis. However, roles that FGF21 plays in pathophysiology of CKD remains elusive. Here we show in mice that FGF21 is required to survive CKD but responsible for blood pressure dysregulation. When introduced with CKD, Fgf21-/- mice died earlier than wild-type mice. Paradoxically, these Fgf21-/- CKD mice escaped several complications observed in wild-type mice, including augmentation of blood pressure elevating response and activation of the sympathetic nervous system during physical activity and increase in serum noradrenalin and corticosterone levels. Supplementation of FGF21 by administration of an FGF21-expressing adeno-associated virus vector recapitulated these complications in wild-type mice and restored the survival period in Fgf21-/- CKD mice. In CKD patients, high serum FGF21 levels are independently associated with decreased baroreceptor sensitivity. Thus, increased FGF21 in CKD can be viewed as a survival response at the sacrifice of blood pressure homeostasis.

BACKGROUND: Cardiac dysfunction and arrhythmia are common and onerous cardiovascular events in end-stage renal disease (ESRD) patients, especially those on dialysis. Fibroblast growth factor (FGF)-23 is a phosphate-regulating hormone whose levels dramatically increase as renal function declines. Beyond its role in phosphorus homeostasis, FGF-23 may elicit a direct effect on the heart. Whether FGF-23 modulates ventricular cardiac rhythm is unknown, prompting us to study its role on excitation-contraction (EC) coupling. METHODS: We examined FGF-23 in vitro actions on EC coupling in adult rat native ventricular cardiomyocytes using patch clamp and confocal microscopy and in vivo actions on cardiac rhythm using electrocardiogram. RESULTS: Compared with vehicle treatment, FGF-23 induced a significant decrease in rat cardiomyocyte contraction, L-type Ca2+ current, systolic Ca2+ transients and sarcoplasmic reticulum (SR) load and SR Ca2+-adenosine triphosphatase 2a pump activity. FGF-23 induced pro-arrhythmogenic activity in vitro and in vivo as automatic cardiomyocyte extracontractions and premature ventricular contractions. Diastolic spontaneous Ca2+ leak (sparks and waves) was significantly increased by FGF-23 via the calmodulin kinase type II (CaMKII)-dependent pathway related to hyperphosphorylation of ryanodine receptors at the CaMKII site Ser2814. Both contraction dysfunction and spontaneous pro-arrhythmic Ca2+ events induced by FGF-23 were blocked by soluble Klotho (sKlotho). CONCLUSIONS: Our results show that FGF-23 reduces contractility and enhances arrhythmogenicity through intracellular Ca2+ mishandling. Blocking its actions on the heart by improving sKlotho bioavailability may enhance cardiac function and reduce arrhythmic events frequently observed in ESRD.

BACKGROUND: Calciprotein particles (CPPs) have been suggested to be associated with the degree of coronary atherosclerosis, and have also been established as a molecular marker for clinical outcome in patients with chronic kidney disease (CKD). However, there are several concerns with regard to conventional measurement of CPPs. We therefore developed a new CPP measurement system that can detect both smaller and lower-density CPPs. METHODS: We analyzed 71 consecutive patients who underwent percutaneous coronary intervention for acute coronary syndrome (ACS, n=29) and/or stable angina pectoris (AP, n=42) who did not have CKD of stage 4 or greater. CPP measurement was made using an infrared fluorescent bisphosphonate (OsteoSense, PerkinElmer, Waltham, MA, USA) and a gel filtration method. The coronary artery plaque was analyzed by gray-scale intravascular ultrasound (IVUS) and integrated backscatter (IB)-IVUS. RESULTS: The median CPP level (interquartile range) was 40,953 (19,171-74,131) arbitrary units (AU). When we divided the CPP level into quintiles, the total and lipid plaque volume were incrementally higher with increasing quintile from lowest to highest (both p<0.02). After adjustment by age, body mass index, and estimated glomerular filtration rate, which factors were correlated with the above-described plaque components, the top quintile of CPP (>86,751AU) had significantly higher total plaque (263mm3 vs. 161mm3; p=0.001) and lipid plaque volume (156mm3 vs. 89mm3; p<0.001) than the other quintiles. However, these associations were not found for the fibrous or calcified plaque volume. The CPP level was higher in the ACS group than the stable AP group (p=0.02), and the total and lipid plaque volume were also higher in the ACS group than the stable AP group (both p<0.05). CONCLUSIONS: The results suggested that a high CPP level, as measured by the novel assay, is a surrogate marker for coronary atherosclerosis, especially in lipid-rich plaques, contributing to an increased risk of plaque vulnerability.

Medial vascular calcification has emerged as a putative key factor contributing to the excessive cardiovascular mortality of patients with chronic kidney disease (CKD). Hyperphosphatemia is considered a decisive determinant of vascular calcification in CKD. A critical role in initiation and progression of vascular calcification during elevated phosphate conditions is attributed to vascular smooth muscle cells (VSMCs), which are able to change their phenotype into osteo-/chondroblasts-like cells. These transdifferentiated VSMCs actively promote calcification in the medial layer of the arteries by producing a local pro-calcifying environment as well as nidus sites for precipitation of calcium and phosphate and growth of calcium phosphate crystals. Elevated extracellular phosphate induces osteo-/chondrogenic transdifferentiation of VSMCs through complex intracellular signaling pathways, which are still incompletely understood. The present review addresses critical intracellular pathways controlling osteo-/chondrogenic transdifferentiation of VSMCs and, thus, vascular calcification during hyperphosphatemia. Elucidating these pathways holds a significant promise to open novel therapeutic opportunities counteracting the progression of vascular calcification in CKD.

Sepsis-induced organ failure is characterized by a massive inflammatory response and oxidative stress. Acute kidney injury (AKI) occurs in approximately half of patients in septic shock, and the mortality associated with sepsis-induced AKI is unacceptably high. Klotho is a protein expressed by renal cells and has anti-senescence properties. Klotho has also been shown to protect the kidneys in ischemia-reperfusion injury and to have antioxidant properties. To analyze the role of Klotho in sepsis-related organ dysfunction and AKI, we used a cecal ligation and puncture (CLP) model of sepsis in heterozygous Klotho-haploinsufficient mice and their wild-type littermates (CLP- Kl/+ and CLP-WT mice, respectively). In comparison with the CLP-WT mice, CLP- Kl/+ mice showed lower survival, impaired renal function, impaired hepatic function, greater oxidative stress, upregulation of inflammatory pathways (at the systemic and kidney tissue levels), and increased NF-κB activation. It is noteworthy that CLP- Kl/+ mice also showed lower heart-rate variability, less sympathetic activity, impaired baroreflex sensitivity to sodium nitroprusside, and a blunted blood pressure response to phenylephrine. We also demonstrated that sepsis creates a state of acute Klotho deficiency. Given that low Klotho expression exacerbates sepsis and multiple organ dysfunction, Klotho might play a protective role in sepsis, especially in elderly individuals in whom Klotho expression is naturally reduced.

Recent clinical studies indicate that the disturbed phosphate metabolism in chronic kidney disease (CKD) may facilitate kidney injury; nonetheless, the causal role of phosphate in CKD progression remains to be elucidated. Here, we show that intestinal phosphate binding by sucroferric oxyhydroxide (SF) ameliorates renal injury in the rat remnant kidney model. Sprague-Dawley rats received 5/6 nephrectomy (RK) and had a normal chow or the same diet containing SF (RK + SF). RK rats showed increased plasma FGF23 and phosphate levels, which were suppressed by SF administration. Of note, albuminuria in RK rats was significantly ameliorated by SF at both 4 and 8 weeks. SF also attenuated glomerulosclerosis and tubulointerstitial injury. Moreover, several different approaches confirmed the protective effects on podocytes, explaining the attenuation of glomerulosclerosis and albuminuria observed in this study. As a possible mechanism, we found that SF attenuated renal inflammation and fibrosis in RK rats. Interestingly, von Kossa staining of the kidney revealed calcium phosphate deposition in neither RK nor RK + SF rats; however, plasma levels of calciprotein particles were significantly reduced by SF. These data indicate that latent positive phosphate balance accelerates CKD progression from early stages, even when overt ectopic calcification is absent.

The Klotho proteins, αKlotho and βKlotho, are essential components of endocrine fibroblast growth factor (FGF) receptor complexes, as they are required for the high-affinity binding of FGF19, FGF21 and FGF23 to their cognate FGF receptors (FGFRs). Collectively, these proteins form a unique endocrine system that governs multiple metabolic processes in mammals. FGF19 is a satiety hormone that is secreted from the intestine on ingestion of food and binds the βKlotho-FGFR4 complex in hepatocytes to promote metabolic responses to feeding. By contrast, under fasting conditions, the liver secretes the starvation hormone FGF21, which induces metabolic responses to fasting and stress responses through the activation of the hypothalamus-pituitary-adrenal axis and the sympathetic nervous system following binding to the βKlotho-FGFR1c complex in adipocytes and the suprachiasmatic nucleus, respectively. Finally, FGF23 is secreted by osteocytes in response to phosphate intake and binds to αKlotho-FGFR complexes, which are expressed most abundantly in renal tubules, to regulate mineral metabolism. Growing evidence suggests that the FGF-Klotho endocrine system also has a crucial role in the pathophysiology of ageing-related disorders, including diabetes, cancer, arteriosclerosis and chronic kidney disease. Therefore, targeting the FGF-Klotho endocrine axes might have therapeutic benefit in multiple systems; investigation of the crystal structures of FGF-Klotho-FGFR complexes is paving the way for the development of drugs that can regulate these axes.