α-Klotho expressison in human tissue

Context: α-Klotho has emerged as a powerful regulator of the aging process. To-date, the expression profile of α-Klotho in human tissues is unknown and its existence in some human tissue types is subject to much controversy. Objective: This is the first study to characterize system-wide tissue expression of transmembrane α-Klotho in humans. We have employed next generation targeted proteomic analysis using Parallel Reaction Monitoring (PRM) in parallel with conventional antibody-based methods to determine the expression and spatial distribution of human α-Klotho expression in health. Results: The distribution of α-Klotho in human tissues from various organ systems, including arterial, epithelial, endocrine, reproductive and neuronal tissues was first identified by immunohistochemistry. Kidney tissues showed strong α-Klotho expression, while liver did not reveal a detectable signal. These results were next confirmed by western blotting of both whole tissues and primary cells. To validate our antibody-based results, α-Klotho expressing tissues were subjected to PRM mass spectrometry identifying peptides specific for the full length, transmembrane α-Klotho isoform. Conclusions: The data presented confirms α-Klotho expression in the kidney tubule and in artery, and provides evidence of α-Klotho expression across organ systems and cell-types that have not previously been described in humans.K.L. received a Genzyme-Sanofi Fellowship in Nephrology grant. T.F.H. is funded by the NIHR award to the Cambridge Biomedical Research Centre and by NIHR grant 14/49/147. The Cambridge Aorta Study is funded by the British Heart Foundation.This is the author accepted manuscript. The final version is available from the Endocrine Society via http://dx.doi.org/10.1210/jc.2015-1800

α-Klotho Expression in Human Tissues.

CONTEXT: α-Klotho has emerged as a powerful regulator of the aging process. To date, the expression profile of α-Klotho in human tissues is unknown, and its existence in some human tissue types is subject to much controversy. OBJECTIVE: This is the first study to characterize systemwide tissue expression of transmembrane α-Klotho in humans. We have employed next-generation targeted proteomic analysis using parallel reaction monitoring in parallel with conventional antibody-based methods to determine the expression and spatial distribution of human α-Klotho expression in health. RESULTS: The distribution of α-Klotho in human tissues from various organ systems, including arterial, epithelial, endocrine, reproductive, and neuronal tissues, was first identified by immunohistochemistry. Kidney tissues showed strong α-Klotho expression, whereas liver did not reveal a detectable signal. These results were next confirmed by Western blotting of both whole tissues and primary cells. To validate our antibody-based results, α-Klotho-expressing tissues were subjected to parallel reaction monitoring mass spectrometry (data deposited at ProteomeXchange, PXD002775) identifying peptides specific for the full-length, transmembrane α-Klotho isoform. CONCLUSIONS: The data presented confirm α-Klotho expression in the kidney tubule and in the artery and provide evidence of α-Klotho expression across organ systems and cell types that has not previously been described in humans.K.L. received a Genzyme-Sanofi Fellowship in Nephrology grant. T.F.H. is funded by the NIHR award to the Cambridge Biomedical Research Centre and by NIHR grant 14/49/147. The Cambridge Aorta Study is funded by the British Heart Foundation.This is the author accepted manuscript. The final version is available from the Endocrine Society via http://dx.doi.org/10.1210/jc.2015-1800

Nonapnea Sleep Disorders and the Risk of Acute Kidney Injury: A Nationwide Population-Based Study

Abstract Nonapnea sleep disorders (NASDs) and associated problems, which are highly prevalent in patients with kidney diseases, are associated with unfavorable medical sequelae. Nonetheless, whether NASDs are associated with acute kidney injury (AKI) development has not been thoroughly analyzed. We examined the association between NASD and AKI. We conducted a population-based study by using 1,000,000 representative data from the Taiwan National Health Insurance Research Database for the period from January 1, 2000, to December 31, 2010. We studied the incidence and risk of AKI in 9178 newly diagnosed NASD patients compared with 27,534 people without NASD matched according to age, sex, index year, urbanization level, region of residence, and monthly income at a 1:3 ratio. The NASD cohort had an adjusted hazard ratio (hazard ratio [HR]; 95% confidence interval [CI] = 1.15–2.63) of subsequent AKI 1.74-fold higher than that of the control cohort. Older age and type 2 diabetes mellitus were significantly associated with an increased risk of AKI (P < 0.05). Among different types of NASDs, patients with insomnia had a 120% increased risk of developing AKI (95% CI = 1.38–3.51; P = 0.001), whereas patients with other sleep disorders had a 127% increased risk of subsequent AKI (95% CI = 1.07–4.80; P = 0.033). Men with NASDs were at a high risk of AKI (P < 0.05). This nationwide population-based cohort study provides evidence that patients with NASDs are at higher risk of developing AKI than people without NASDs

Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is estimated to contribute to 19- 25% of all clinical cases of acute kidney injury (AKI) in critically ill patients. AKI detection has historically relied on metrics such as serum creatinine (sCr) or blood urea nitrogen (BUN) which are demonstrably inadequate in full assessment of nephrotoxicity in the early phase of renal dysfunction. Currently, there is no robust diagnostic method to accurately detect hemodynamic alteration in the early phase of AKI while such alterations might actually precede the rise in serum biomarker levels. Such early detection can help clinicians make an accurate diagnosis and help in in decision making for therapeutic strategy. Rats were treated with Cisplatin to induce AKI. Nephrotoxicity was assessed for six days using high-frequency sonography, sCr measurement and upon histopathology of kidney. Hemodynamic evaluation using 2D and Color-Doppler images were used to serially study nephrotoxicity in rats, using the sonography. Our data showed successful drug-induced kidney injury in adult rats by histological examination. Color-Doppler based sonographic assessment of AKI indicated that resistive-index (RI) and pulsatile-index (PI) were increased in the treatment group; and peak-systolic velocity (mm/s), end-diastolic velocity (mm/s) and velocity-time integral (VTI, mm) were decreased in renal arteries in the same group. Importantly, these hemodynamic changes evaluated by sonography preceded the rise of sCr levels. Sonography-based indices such as RI or PI can thus be useful predictive markers of declining renal function in rodents. From our sonography-based observations in the kidneys of rats that underwent AKI, we showed that these noninvasive hemodynamic measurements may consider as an accurate, sensitive and robust method in detecting early stage kidney dysfunction. This study also underscores the importance of ethical issues associated with animal use in research

Molecular Phenotyping and Mechanisms of Myocardial Fibrosis in Advanced Chronic Kidney Disease

Key Points: * Myocardial fibrosis in hearts from patients with CKD is characterized by increased trimeric tensile collagen type I and decreased elastic collagen type III compared with hearts from hypertensive or healthy donors, suggesting a unique fibrotic phenotype. * Myocardial fibrosis in CKD is driven by alterations in extracellular matrix proteostasis, including dysregulation of metalloproteinases and cross-linking enzymes. * CKD-associated mineral stressors uniquely induce a fibronectin-independent mechanism of fibrillogenesis characterized by formation of trimeric collagen compared with proinflammatory/fibrotic cytokines. Background: Myocardial fibrosis is a major life-limiting problem in CKD. Despite this, the molecular phenotype and metabolism of collagen fibrillogenesis in fibrotic hearts of patients with advanced CKD have been largely unstudied. Methods: We analyzed explanted human left ventricular (LV) heart tissues in a three-arm cross-sectional cohort study of deceased donor patients on hemodialysis (HD, n=18), hypertension with preserved renal function (HTN, n=8), and healthy controls (CON, n=17), ex vivo. RNA-seq and protein analysis was performed on human donor hearts and cardiac fibroblasts treated with mineral stressors (high phosphate and high calcium). Further mechanistic studies were performed using primary cardiac fibroblasts, in vitro treated with mineral stressors, proinflammatory and profibrotic cytokines. Results: Of the 43 donor participants, there was no difference in age (P > 0.2), sex (P > 0.8), or body mass index (P > 0.1) between the groups. Hearts from the HD group had extensive fibrosis (P < 0.01). All LV tissues expressed only the trimeric form of collagen type I. HD hearts expressed increased collagen type I (P < 0.03), elevated collagen type I:III ratio (P < 0.05), and decreased MMP1 (P < 0.05) and MMP2 (P < 0.05). RNA-seq revealed no significant differential gene expression of extracellular matrix proteins of interest in HD hearts, but there was significant upregulation of LH2, periostin, α-SMA, and TGF-β1 gene expression in mineral stressor–treated cardiac fibroblasts. Both mineral stressors (P < 0.009) and cytokines (P < 0.03) increased collagen type I:III ratio. Mineral stressors induced trimeric collagen type I, but cytokine treatment induced only dimeric collagen type I in cardiac fibroblasts. Mineral stressors downregulated fibronectin (P < 0.03) and MMP2 zymogen (P < 0.01) but did not significantly affect expression of periostin, MMP1, or cross-linking enzymes. TGF-β upregulated fibronectin (P < 0.01) and periostin (P < 0.02) only. Conclusions: Myocardial fibrosis in advanced CKD hearts is characterized by increased trimeric collagen type I and dysregulated collagen metabolism, and is differentially regulated by components of uremia

Myocardial Cytoskeletal Adaptations in Advanced Kidney Disease

Background The myocardial cytoskeleton functions as the fundamental framework critical for organelle function, bioenergetics and myocardial remodeling. To date, impairment of the myocardial cytoskeleton occurring in the failing heart in patients with advanced chronic kidney disease has been largely undescribed. Methods and Results We conducted a 3‐arm cross‐sectional cohort study of explanted human heart tissues from patients who are dependent on hemodialysis (n=19), hypertension (n=10) with preserved renal function, and healthy controls (n=21). Left ventricular tissues were subjected to pathologic examination and next‐generation RNA sequencing. Mechanistic and interference RNA studies utilizing in vitro human cardiac fibroblast models were performed. Left ventricular tissues from patients undergoing hemodialysis exhibited increased myocardial wall thickness and significantly greater fibrosis compared with hypertension patients (P<0.05) and control (P<0.01). Transcriptomic analysis revealed that the focal adhesion pathway was significantly enriched in hearts from patients undergoing hemodialysis. Hearts from patients undergoing hemodialysis exhibited dysregulated components of the focal adhesion pathway including reduced β‐actin (P<0.01), β‐tubulin (P<0.01), vimentin (P<0.05), and increased expression of vinculin (P<0.05) compared with controls. Cytoskeletal adaptations in hearts from the hemodialysis group were associated with impaired mitochondrial bioenergetics, including dysregulated mitochondrial dynamics and fusion, and loss of cell survival pathways. Mechanistic studies revealed that cytoskeletal changes can be driven by uremic and metabolic abnormalities of chronic kidney disease, in vitro. Furthermore, focal adhesion kinase silencing via interference RNA suppressed major cytoskeletal proteins synergistically with mineral stressors found in chronic kidney disease in vitro. Conclusions Myocardial failure in advanced chronic kidney disease is characterized by impairment of the cytoskeleton involving disruption of the focal adhesion pathway, mitochondrial failure, and loss of cell survival pathways