Phosphate in Cardiovascular Disease: From New Insights Into Molecular Mechanisms to Clinical Implications.

Hyperphosphatemia is a common feature in patients with impaired kidney function and is associated with increased risk of cardiovascular disease. This phenomenon extends to the general population, whereby elevations of serum phosphate within the normal range increase risk; however, the mechanism by which this occurs is multifaceted, and many aspects are poorly understood. Less than 1% of total body phosphate is found in the circulation and extracellular space, and its regulation involves multiple organ cross talk and hormones to coordinate absorption from the small intestine and excretion by the kidneys. For phosphate to be regulated, it must be sensed. While mostly enigmatic, various phosphate sensors have been elucidated in recent years. Phosphate in the circulation can be buffered, either through regulated exchange between extracellular and cellular spaces or through chelation by circulating proteins (ie, fetuin-A) to form calciprotein particles, which in themselves serve a function for bulk mineral transport and signaling. Either through direct signaling or through mediators like hormones, calciprotein particles, or calcifying extracellular vesicles, phosphate can induce various cardiovascular disease pathologies: most notably, ectopic cardiovascular calcification but also left ventricular hypertrophy, as well as bone and kidney diseases, which then propagate phosphate dysregulation further. Therapies targeting phosphate have mostly focused on intestinal binding, of which appreciation and understanding of paracellular transport has greatly advanced the field. However, pharmacotherapies that target cardiovascular consequences of phosphate directly, such as vascular calcification, are still an area of great unmet medical need.

C-terminal fragment of fibroblast growth factor 23 improves heart function in murine models of high intact fibroblast growth factor 23.

Cardiovascular disease (CVD) is the major cause of death in chronic kidney disease (CKD) and is associated with high circulating fibroblast growth factor (FGF)23 levels. It is unresolved whether high circulating FGF23 is a mere biomarker or pathogenically contributes to cardiomyopathy. It is also unknown whether the C-terminal FGF23 peptide (cFGF23), a natural FGF23 antagonist proteolyzed from intact FGF23 (iFGF23), retards CKD progression and improves cardiomyopathy. We addressed these questions in three murine models with high endogenous FGF23 and cardiomyopathy. First, we examined wild-type (WT) mice with CKD induced by unilateral ischemia-reperfusion and contralateral nephrectomy followed by a high-phosphate diet. These mice were continuously treated with intraperitoneal implanted osmotic minipumps containing either iFGF23 protein to further escalate FGF23 bioactivity, cFGF23 peptide to block FGF23 signaling, vehicle, or scrambled peptide as negative controls. Exogenous iFGF23 protein given to CKD mice exacerbated pathological cardiac remodeling and CKD progression, whereas cFGF23 treatment improved heart and kidney function, attenuated fibrosis, and increased circulating soluble Klotho. WT mice without renal insult placed on a high-phosphate diet and homozygous Klotho hypomorphic mice, both of whom develop moderate CKD and clear cardiomyopathy, were treated with cFGF23 or vehicle. Mice treated with cFGF23 in both models had improved heart and kidney function and histopathology. Taken together, these data indicate high endogenous iFGF23 is not just a mere biomarker but pathogenically deleterious in CKD and cardiomyopathy. Furthermore, attenuation of FGF23 bioactivity by cFGF23 peptide is a promising therapeutic strategy to protect the kidney and heart from high FGF23 activity.NEW & NOTEWORTHY There is a strong correlation between cardiovascular morbidity and high circulating fibroblast growth factor 23 (FGF23) levels, but causality was never proven. We used a murine chronic kidney disease (CKD) model to show that intact FGF23 (iFGF23) is pathogenic and contributes to both CKD progression and cardiomyopathy. Blockade of FGF23 signaling with a natural proteolytic product of iFGF23, C-terminal FGF23, alleviated kidney and cardiac histology, and function in three separate murine models of high endogenous FGF23.

α-Klotho is a non-enzymatic molecular scaffold for FGF23 hormone signalling.

The ageing suppressor α-klotho binds to the fibroblast growth factor receptor (FGFR). This commits FGFR to respond to FGF23, a key hormone in the regulation of mineral ion and vitamin D homeostasis. The role and mechanism of this co-receptor are unknown. Here we present the atomic structure of a 1:1:1 ternary complex that consists of the shed extracellular domain of α-klotho, the FGFR1c ligand-binding domain, and FGF23. In this complex, α-klotho simultaneously tethers FGFR1c by its D3 domain and FGF23 by its C-terminal tail, thus implementing FGF23-FGFR1c proximity and conferring stability. Dimerization of the stabilized ternary complexes and receptor activation remain dependent on the binding of heparan sulfate, a mandatory cofactor of paracrine FGF signalling. The structure of α-klotho is incompatible with its purported glycosidase activity. Thus, shed α-klotho functions as an on-demand non-enzymatic scaffold protein that promotes FGF23 signalling.

Disruption of the beclin 1-BCL2 autophagy regulatory complex promotes longevity in mice.

Autophagy increases the lifespan of model organisms; however, its role in promoting mammalian longevity is less well-established1,2. Here we report lifespan and healthspan extension in a mouse model with increased basal autophagy. To determine the effects of constitutively increased autophagy on mammalian health, we generated targeted mutant mice with a Phe121Ala mutation in beclin 1 (Becn1F121A/F121A) that decreases its interaction with the negative regulator BCL2. We demonstrate that the interaction between beclin 1 and BCL2 is disrupted in several tissues in Becn1 F121A/F121A knock-in mice in association with higher levels of basal autophagic flux. Compared to wild-type littermates, the lifespan of both male and female knock-in mice is significantly increased. The healthspan of the knock-in mice also improves, as phenotypes such as age-related renal and cardiac pathological changes and spontaneous tumorigenesis are diminished. Moreover, mice deficient in the anti-ageing protein klotho 3 have increased beclin 1 and BCL2 interaction and decreased autophagy. These phenotypes, along with premature lethality and infertility, are rescued by the beclin 1(F121A) mutation. Together, our data demonstrate that disruption of the beclin 1-BCL2 complex is an effective mechanism to increase autophagy, prevent premature ageing, improve healthspan and promote longevity in mammals.

Author Correction: Disruption of the beclin 1-BCL2 autophagy regulatory complex promotes longevity in mice.

In this Letter, the graphs in Fig. 2a and c were inadvertently the same owing to a copy and paste error from the original graphs in Prism. The Source Data files containing the raw data were correct. Fig. 2c has been corrected online.

The Effects of Acid on Calcium and Phosphate Metabolism.

A variety of changes in mineral metabolism aiming to restore acid-base balance occur in acid loading and metabolic acidosis. Phosphate plays a key role in defense against metabolic acidosis, both as an intracellular and extracellular buffer, as well as in the renal excretion of excess acid in the form of urinary titratable acid. The skeleton acts as an extracellular buffer in states of metabolic acidosis, as the bone matrix demineralizes, leading to bone apatite dissolution and the release of phosphate, calcium, carbonate, and citrate into the circulation. The renal handling of calcium, phosphate and citrate is also affected, with resultant hypercalciuria, hyperphosphaturia and hypocitraturia.

VEGFR2 insufficiency enhances phosphotoxicity and undermines Klotho's protection against peritubular capillary rarefaction and kidney fibrosis.

Vascular endothelial growth factor (VEGF) and its cognate receptor (VEGFR2) system are crucial for cell functions associated with angiogenesis and vasculogenesis. Klotho contributes to vascular health maintenance in the kidney and other organs in mammals, but it is unknown whether renoprotection by Klotho is dependent on VEGF/VEGFR2 signaling. We used heterozygous VEGFR2-haploinsufficient (VEGFR2+/-) mice resulting from heterozygous knockin of green fluorescent protein in the locus of fetal liver kinase 1 encoding VEGFR2 to test the interplay of Klotho, phosphate, and VEGFR2 in kidney function, the vasculature, and fibrosis. VEGFR2+/- mice displayed downregulated VEGF/VEGFR2 signaling in the kidney, lower density of peritubular capillaries, and accelerated kidney fibrosis, all of which were also found in the homozygous Klotho hypomorphic mice. High dietary phosphate induced higher plasma phosphate, greater peritubular capillary rarefaction, and more kidney fibrosis in VEGFR2+/- mice compared with wild-type mice. Genetic overexpression of Klotho significantly attenuated the elevated plasma phosphate, kidney dysfunction, peritubular capillary rarefaction, and kidney fibrosis induced by a high-phosphate diet in wild-type mice but only modestly ameliorated these changes in the VEGFR2+/- background. In cultured endothelial cells, VEGFR2 inhibition reduced free VEGFR2 but enhanced its costaining of an endothelial marker (CD31) and exacerbated phosphotoxicity. Klotho protein maintained VEGFR2 expression and attenuated high phosphate-induced cell injury, which was reduced by VEGFR2 inhibition. In conclusion, normal VEGFR2 function is required for vascular integrity and for Klotho to exert vascular protective and antifibrotic actions in the kidney partially through the regulation of VEGFR2 function.NEW & NOTEWORTHY This research paper studied the interplay of vascular endothelial growth factor receptor type 2 (VEGFR2), high dietary phosphate, and Klotho, an antiaging protein, in peritubular structure and kidney fibrosis. Klotho protein was shown to maintain VEGFR2 expression in the kidney and reduce high phosphate-induced cell injury. However, Klotho cytoprotection was attenuated by VEGFR2 inhibition. Thus, normal VEGFR2 function is required for vascular integrity and Klotho to exert vascular protective and antifibrotic actions in the kidney.

Prediction of Mortality and Major Adverse Kidney Events in Critically Ill Patients With Acute Kidney Injury.

RATIONALE & OBJECTIVE: Risk prediction tools for assisting acute kidney injury (AKI) management have focused on AKI onset but have infrequently addressed kidney recovery. We developed clinical models for risk stratification of mortality and major adverse kidney events (MAKE) in critically ill patients with incident AKI. STUDY DESIGN: Multicenter cohort study. SETTING & PARTICIPANTS: 9,587 adult patients admitted to heterogeneous intensive care units (ICUs; March 2009 to February 2017) who experienced AKI within the first 3 days of their ICU stays. PREDICTORS: Multimodal clinical data consisting of 71 features collected in the first 3 days of ICU stay. OUTCOMES: (1) Hospital mortality and (2) MAKE, defined as the composite of death during hospitalization or within 120 days of discharge, receipt of kidney replacement therapy in the last 48 hours of hospital stay, initiation of maintenance kidney replacement therapy within 120 days, or a ≥50% decrease in estimated glomerular filtration rate from baseline to 120 days from hospital discharge. ANALYTICAL APPROACH: Four machine-learning algorithms (logistic regression, random forest, support vector machine, and extreme gradient boosting) and the SHAP (Shapley Additive Explanations) framework were used for feature selection and interpretation. Model performance was evaluated by 10-fold cross-validation and external validation. RESULTS: One developed model including 15 features outperformed the SOFA (Sequential Organ Failure Assessment) score for the prediction of hospital mortality, with areas under the curve of 0.79 (95% CI, 0.79-0.80) and 0.71 (95% CI, 0.71-0.71) in the development cohort and 0.74 (95% CI, 0.73-0.74) and 0.71 (95% CI, 0.71-0.71) in the validation cohort (P < 0.001 for both). A second developed model including 14 features outperformed KDIGO (Kidney Disease: Improving Global Outcomes) AKI severity staging for the prediction of MAKE: 0.78 (95% CI, 0.78-0.78) versus 0.66 (95% CI, 0.66-0.66) in the development cohort and 0.73 (95% CI, 0.72-0.74) versus 0.67 (95% CI, 0.67-0.67) in the validation cohort (P < 0.001 for both). LIMITATIONS: The models are applicable only to critically ill adult patients with incident AKI within the first 3 days of an ICU stay. CONCLUSIONS: The reported clinical models exhibited better performance for mortality and kidney recovery prediction than standard scoring tools commonly used in critically ill patients with AKI in the ICU. Additional validation is needed to support the utility and implementation of these models. PLAIN-LANGUAGE SUMMARY: Acute kidney injury (AKI) occurs commonly in critically ill patients admitted to the intensive care unit (ICU) and is associated with high morbidity and mortality rates. Prediction of mortality and recovery after an episode of AKI may assist bedside decision making. In this report, we describe the development and validation of a clinical model using data from the first 3 days of an ICU stay to predict hospital mortality and major adverse kidney events occurring as long as 120 days after hospital discharge among critically ill adult patients who experienced AKI within the first 3 days of an ICU stay. The proposed clinical models exhibited good performance for outcome prediction and, if further validated, could enable risk stratification for timely interventions that promote kidney recovery.

In vivo evidence for therapeutic applications of beclin 1 to promote recovery and inhibit fibrosis after acute kidney injury.

Autophagy regulator beclin 1 activity determines the severity of kidney damage induced by ischemia reperfusion injury, but its role in kidney recovery and fibrosis are unknown and its therapeutic potentials have not been tested. Here, we explored beclin 1 effects on kidney fibrosis in three models of acute kidney injury (AKI)-ischemia reperfusion injury, cisplatin kidney toxicity, and unilateral ureteric obstruction in mouse strains with three levels of beclin 1 function: normal (wild type), low (heterozygous global deletion of beclin 1, Becn1+/-), and high beclin 1 activity (knockin gain-of-function mutant Becn1, Becn1FA). Fourteen days after AKI induction, heterozygous mice had more, but knockin mice had less kidney fibrosis than wild-type mice did. One day after ischemia reperfusion injury, heterozygous pan-kidney tubular Becn1 null mice had more severe kidney damage than homozygous distal tubular Becn1 null mice did, which was similar to the wild-type mice, implying that proximal tubular beclin 1 protects the kidney against ischemia reperfusion injury. By 14 days, both pan-kidney heterozygous Becn1 null and distal tubular homozygous Becn1 null mice had poorer kidney recovery than wild-type mice did. Injection of beclin 1 peptides increased cell proliferation in kidney tubules in normal mice. Beclin 1 peptides injection either before or after (2-5 days) ischemia reperfusion injury protected the kidney from injury and suppressed kidney fibrosis. Thus, both endogenous beclin 1 protein expression in kidney tubules and exogenous beclin 1 peptides are kidney protective via attenuation of acute kidney damage, promotion of cell proliferation, and inhibition of kidney fibrosis, consequently improving kidney recovery post-AKI. Hence, exogenous beclin 1 peptide may be a potential new therapy for AKI.

Strategies to lower fibroblast growth factor 23 bioactivity.

Fibroblast growth factor 23 (FGF23) is a circulating hormone derived from the bone whose release is controlled by many factors and exerts a multitude of systemic actions. There are congenital and acquired disorders of increased and decreased FGF23 levels. In chronic kidney disease (CKD), elevations of FGF23 levels can be 1000-fold above the upper physiological limit. It is still debated whether this high FGF23 in CKD is a biomarker or causally related to morbidity and mortality. Data from human association studies support pathogenicity, while experimental data are less robust. Knowledge of the biology and pathobiology of FGF23 has generated a plethora of means to reduce FGF23 bioactivity at many levels that will be useful for therapeutic translations. This article summarizes these approaches and addresses several critical questions that still need to be answered.

Assessment of a modified renal angina index for AKI prediction in critically ill adults.

BACKGROUND: The renal angina index (RAI) is a useful tool for risk stratification of acute kidney injury (AKI) in critically ill children. We evaluated the performance of a modified adult RAI (mRAI) for the risk stratification of AKI in critically ill adults. METHODS: We used two independent intensive care unit (ICU) cohorts: 13 965 adult patients from the University of Kentucky (UKY) and 4789 from University of Texas Southwestern (UTSW). The mRAI included: diabetes, presence of sepsis, mechanical ventilation, pressor/inotrope use, percentage change in serum creatinine (SCr) in reference to admission SCr (ΔSCr) and fluid overload percentage within the first day of ICU admission. The primary outcome was AKI Stage ≥2 at Days 2-7. Performance and reclassification metrics were determined for the mRAI score compared with ΔSCr alone. RESULTS: The mRAI score outperformed ΔSCr and readjusted probabilities to predict AKI Stage ≥2 at Days 2-7: C-statistic: UKY 0.781 versus 0.708 [integrated discrimination improvement (IDI) 2.2%] and UTSW 0.766 versus 0.696 (IDI 1.8%) (P < 0.001 for both). In the UKY cohort, only 3.3% of patients with mRAI score <10 had the AKI event, while 16.4% of patients with mRAI score of ≥10 had the AKI event (negative predictive value 96.8%). Similar findings were observed in the UTSW cohort as part of external validation. CONCLUSIONS: In critically ill adults, the adult mRAI score determined within the first day of ICU admission outperformed changes in SCr for the prediction of AKI Stage ≥2 at Days 2-7 of ICU stay. The mRAI is a feasible tool for AKI risk stratification in adult patients in the ICU.

Serum IL-17 levels are higher in critically ill patients with AKI and associated with worse outcomes.

BACKGROUND: Interleukin-17 (IL-17) antagonism in rats reduces the severity and progression of AKI. IL-17-producing circulating T helper-17 (TH17) cells is increased in critically ill patients with AKI indicating that this pathway is also activated in humans. We aim to compare serum IL-17A levels in critically ill patients with versus without AKI and to examine their relationship with mortality and major adverse kidney events (MAKE). METHODS: Multicenter, prospective study of ICU patients with AKI stage 2 or 3 and without AKI. Samples were collected at 24-48 h after AKI diagnosis or ICU admission (in those without AKI) [timepoint 1, T1] and 5-7 days later [timepoint 2, T2]. MAKE was defined as the composite of death, dependence on kidney replacement therapy or a reduction in eGFR of ≥ 30% from baseline up to 90 days following hospital discharge. RESULTS: A total of 299 patients were evaluated. Patients in the highest IL-17A tertile (versus lower tertiles) at T1 had higher acuity of illness and comorbidity scores. Patients with AKI had higher levels of IL-17A than those without AKI: T1 1918.6 fg/ml (692.0-5860.9) versus 623.1 fg/ml (331.7-1503.4), p < 0.001; T2 2167.7 fg/ml (839.9-4618.9) versus 1193.5 fg/ml (523.8-2198.7), p = 0.006. Every onefold higher serum IL-17A at T1 was independently associated with increased risk of hospital mortality (aOR 1.35, 95% CI: 1.06-1.73) and MAKE (aOR 1.26, 95% CI: 1.02-1.55). The highest tertile of IL-17A (vs. the lowest tertile) was also independently associated with higher risk of MAKE (aOR 3.03, 95% CI: 1.34-6.87). There was no effect modification of these associations by AKI status. IL-17A levels remained significantly elevated at T2 in patients that died or developed MAKE. CONCLUSIONS: Serum IL-17A levels measured by the time of AKI diagnosis or ICU admission were differentially elevated in critically ill patients with AKI when compared to those without AKI and were independently associated with hospital mortality and MAKE.

Phosphate and Cellular Senescence.

Cellular senescence is one type of permeant arrest of cell growth and one of increasingly recognized contributor to aging and age-associated disease. High phosphate and low Klotho individually and synergistically lead to age-related degeneration in multiple organs. Substantial evidence supports the causality of high phosphate in cellular senescence, and potential contribution to human aging, cancer, cardiovascular, kidney, neurodegenerative, and musculoskeletal diseases. Phosphate can induce cellular senescence both by direct phosphotoxicity, and indirectly through downregulation of Klotho and upregulation of plasminogen activator inhibitor-1. Restriction of dietary phosphate intake and blockage of intestinal absorption of phosphate help suppress cellular senescence. Supplementation of Klotho protein, cellular senescence inhibitor, and removal of senescent cells with senolytic agents are potential novel strategies to attenuate phosphate-induced cellular senescence, retard aging, and ameliorate age-associated, and phosphate-induced disorders.

Physiologic Regulation of Systemic Klotho Levels by Renal CaSR Signaling in Response to CaSR Ligands and pH o.

BACKGROUND: The kidney is the source of sKlotho and kidney-specific loss of Klotho leads to a phenotype resembling the premature multiorgan failure phenotype in Klotho-hypomorphic mice ( kl/kl mice). Klotho and the Ca-sensing receptor (CaSR) are highly expressed in the distal convoluted tubule (DCT). The physiologic mechanisms that regulate sKlotho levels are unknown. METHODS: We measured sKlotho in WT and tubule-specific CaSR -/- (TS-CaSR -/- ) mice treated with calcimimetics, alkali, or acid, and Klotho shed from minced mouse kidneys, and from HEK-293 cells expressing the CaSR and Klotho, in response to calcimimetics, calcilytics, alkalotic and acidic pH, and ADAM protease inhibitors. The CaSR, Klotho, and ADAM10 were imaged in mouse kidneys and cell expression systems using confocal microscopy. RESULTS: The CaSR, Klotho, and ADAM10 colocalize on the basolateral membrane of the DCT. Calcimimetics and HCO 3 increase serum sKlotho levels in WT but not in CaSR -/- mice, and acidic pH suppresses sKlotho levels in WT mice. In minced kidneys and cultured cells, CaSR activation with high Ca, calcimimetics, or alkali increase shed Klotho levels via ADAM10, as demonstrated using the ADAM10 inhibitor GI254023X and siRNA. In cultured cells, the CaSR, Klotho, and ADAM10 form cell surface aggregates that disperse after CaSR activation. CONCLUSIONS: We identify a novel physiologic mechanism for regulation of sKlotho levels by the renal CaSR-ADAM10-Klotho pathway. We show that CaSR activators, including alkali, increase renal CaSR-stimulated Klotho shedding and predict that this mechanism is relevant to the effects of acidosis and alkali therapy on CKD progression.

Constitutive transgenic α-Klotho overexpression enhances resilience to and recovery from murine acute lung injury.

Normal lungs do not express α-Klotho (Klotho) protein but derive cytoprotection from circulating soluble Klotho. It is unclear whether chronic supranormal Klotho levels confer additional benefit. To address this, we tested the age-related effects of modest Klotho overexpression on acute lung injury (ALI) and recovery. Transgenic Klotho-overexpressing (Tg-Kl) and wild-type (WT) mice (2 and 6 mo old) were exposed to hyperoxia (95% O2; 72 h; injury; Hx) then returned to normoxia (21% O2; 24 h; recovery; Hx-R). Control mice were kept in normoxia. Renal and serum Klotho, lung histology, and bronchoalveolar lavage fluid oxidative damage markers were assessed. Effects of hyperoxia on Klotho release were tested in human embryonic kidney cells stably expressing Klotho. A549 lung epithelial cells transfected with Klotho cDNA or vector were exposed to cigarette smoke; lactate dehydrogenase and double-strand DNA breaks were measured. Serum Klotho decreased with age. Hyperoxia suppressed renal Klotho at both ages and serum Klotho at 2 mo of age. Tg-Kl mice at both ages and 2-mo-old WT mice survived Hx-R; 6-mo-old Tg-Kl mice showed lower lung damage than age-matched WT mice. Hyperoxia directly inhibited Klotho expression and release in vitro; Klotho transfection attenuated cigarette smoke-induced cytotoxicity and DNA double-strand breaks in lung epithelial cells. Young animals with chronic high baseline Klotho expression were more resistant to ALI. Chronic constitutive Klotho overexpression in older Tg-Kl animals attenuated hyperoxia-induced lung damage and improves survival and short-term recovery despite an acute reduction in serum Klotho during injury. We conclude that chronic enhancement of Klotho expression increases resilience to ALI.

Spot urinary citrate-to-creatinine ratio is a marker for acid-base status in chronic kidney disease.

Due to multiple compensating mechanisms, the serum bicarbonate concentration is a relatively insensitive marker of acid-base status; especially in chronic kidney disease (CKD). This is a major drawback that impairs the ability to diagnose acid excess or monitor alkali therapy. We postulated that it is more logical to measure the compensatory defense mechanism(s) rather than the defended parameter, which remains normal if the compensation is successful. Therefore, a retrospective cross-sectional study was performed in 1733 stone formers along with a prospective cross-sectional study of 22 individuals with normal kidney function and 50 patients in different stages of CKD. While serum bicarbonate was flat and did not fall below the reference range until near CKD stage 5, citrate excretion (24-hour urinary citrate excretion rate; urinary citrate-to-creatinine ratio, in the retrospective analysis, and spot urinary citrate-to-creatinine ratio in the prospective study) progressively and significantly declined starting from CKD stage 2. Following an acute acid load in 25 participants with a wide range of estimated glomerular filtration rates, the urinary citrate-to-creatinine ratio inversely and significantly associated with acid accumulation, whereas serum bicarbonate did not. We compared changes in serum bicarbonate and urinary citrate-to-creatinine ratio in response to alkali therapy in patients with CKD stage 3 or 4 started on potassium citrate in our kidney stone database. With alkali therapy, there was no change in serum bicarbonate, but the urinary citrate-to-creatinine ratio rose consistently in all patients adherent to potassium citrate therapy. Thus, the urinary citrate-to-creatinine ratio (the defense mechanism) is a potential easily implementable, pragmatic, and a superior parameter to serum bicarbonate (the defended entity) to assess acid-base status, and monitor alkali therapy. Additional studies are needed before a clinical test can be devised.

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.

Serum renin and major adverse kidney events in critically ill patients: a multicenter prospective study.

BACKGROUND: Preliminary studies have suggested that the renin-angiotensin system is activated in critical illness and associated with mortality and kidney outcomes. We sought to assess in a larger, multicenter study the relationship between serum renin and Major Adverse Kidney Events (MAKE) in intensive care unit (ICU) patients. METHODS: Prospective, multicenter study at two institutions of patients with and without acute kidney injury (AKI). Blood samples were collected for renin measurement a median of 2 days into the index ICU admission and 5-7 days later. The primary outcome was MAKE at hospital discharge, a composite of mortality, kidney replacement therapy, or reduced estimated glomerular filtration rate to ≤ 75% of baseline. RESULTS: Patients in the highest renin tertile were more severely ill overall, including more AKI, vasopressor-dependence, and severity of illness. MAKE were significantly greater in the highest renin tertile compared to the first and second tertiles. In multivariable logistic regression, this initial measurement of renin remained significantly associated with both MAKE as well as the individual component of mortality. The association of renin with MAKE in survivors was not statistically significant. Renin measurements at the second time point were also higher in patients with MAKE. The trajectory of the renin measurements between time 1 and 2 was distinct when comparing death versus survival, but not when comparing MAKE versus those without. CONCLUSIONS: In a broad cohort of critically ill patients, serum renin measured early in the ICU admission is associated with MAKE at discharge, particularly mortality.

High-Phosphate Diet Induces Exercise Intolerance and Impairs Fatty Acid Metabolism in Mice.

BACKGROUND: Inorganic phosphate (Pi) is used extensively as a preservative and a flavor enhancer in the Western diet. Physical inactivity, a common feature of Western societies, is associated with increased cardiovascular morbidity and mortality. It is unknown whether dietary Pi excess contributes to exercise intolerance and physical inactivity. METHODS: To determine an association between Pi excess and physical activity in humans, we assessed the relationship between serum Pi and actigraphy-determined physical activity level, as well as left ventricular function by cardiac magnetic resonance imaging, in DHS-2 (Dallas Heart Study phase 2) participants after adjusting for relevant variables. To determine direct effects of dietary Pi on exercise capacity, oxygen uptake, serum nonesterified fatty acid, and glucose were measured during exercise treadmill test in C57/BL6 mice fed either a high-Pi (2%) or normal-Pi (0.6%) diet for 12 weeks. To determine the direct effect of Pi on muscle metabolism and expression of genes involved in fatty acid metabolism, additional studies in differentiated C2C12 myotubes were conducted after subjecting to media containing 1 to 3 mmol/L Pi (pH 7.0) to simulate in vivo phosphate conditions. RESULTS: In participants of the DHS-2 (n=1603), higher serum Pi was independently associated with reduced time spent in moderate to vigorous physical activity ( P=0.01) and increased sedentary time ( P=0.004). There was no association between serum Pi and left ventricular ejection fraction or volumes. In animal studies, compared with the control diet, consumption of high-Pi diet for 12 weeks did not alter body weight or left ventricular function but reduced maximal oxygen uptake, treadmill duration, spontaneous locomotor activity, fat oxidation, and fatty acid levels and led to downregulation of genes involved in fatty acid synthesis, release, and oxidation, including Fabp4, Hsl, Fasn, and Pparγ, in muscle. Similar results were recapitulated in vitro by incubating C2C12 myotubes with high-Pi media. CONCLUSIONS: Our data demonstrate a detrimental effect of dietary Pi excess on skeletal muscle fatty acid metabolism and exercise capacity that is independent of obesity and cardiac contractile function. Dietary Pi may represent a novel and modifiable target to reduce physical inactivity associated with the Western diet.

Beclin 1/Bcl-2 complex-dependent autophagy activity modulates renal susceptibility to ischemia-reperfusion injury and mediates renoprotection by Klotho.

Klotho- and beclin 1-driven autophagy extends life. We examined the role of beclin 1 in modifying acute kidney injury (AKI) and whether beclin 1 mediates Klotho's known renoprotective action in AKI. AKI was induced by ischemia-reperfusion injury in mice with different levels of autophagy activity by genetic manipulation: wild-type (WT) mice with normal beclin 1 expression and function, mice with normal beclin 1 levels but high activity through knockin of gain-of-function mutant beclin 1 (Becn1F121A), mice with low beclin 1 levels and activity caused by heterozygous global deletion of beclin 1 (Becn1+/-), or mice with extremely low beclin 1 activity from knockin of the mutant constitutively active beclin 1 inhibitor Bcl-2 (Bcl2AAA). Klotho was increased by transgenic overexpression (Tg-Kl) or recombinant Klotho protein administration. After ischemia-reperfusion injury, Becn1F121A mice (high autophagy) had milder AKI and Becn1+/- and Bcl2AAA mice (low autophagy) had more severe AKI than WT mice. Tg-Kl mice had milder AKI, but its renoprotection was partially attenuated in Becn1+/-;Tg-Kl mice and was significantly reduced, although not completely abolished, in Bcl2AAA;Tg-Kl mice. Recombinant Klotho protein conferred more renoprotection from AKI in WT mice than in Becn1+/- or Bcl2AAA mice. Klotho reduced beclin 1/Bcl-2 protein complexes and increased autophagy activity, but this effect was less prominent in mice or cells with Bcl2AAA. Transfected Bcl2AAA or Becn1F123A decreased or increased autophagy activity and rendered cells more susceptible or more resistant to oxidative cytotoxicity, respectively. In conclusion, beclin 1 confers renoprotection by activating autophagy. Klotho protects the kidney partially via disruption of beclin 1/Bcl-2 interactions and enhancement of autophagy activity.