Sex-specific molecular signature of mouse podocytes in homeostasis and in response to pharmacological challenge with rapamycin.

BACKGROUND: Sex differences exist in the prevalence and progression of major glomerular diseases. Podocytes are the essential cell-type in the kidney which maintain the physiological blood-urine barrier, and pathological changes in podocyte homeostasis are critical accelerators of impairment of kidney function. However, sex-specific molecular signatures of podocytes under physiological and stress conditions remain unknown. This work aimed at identifying sexual dimorphic molecular signatures of podocytes under physiological condition and pharmacologically challenged homeostasis with mechanistic target of rapamycin (mTOR) inhibition. mTOR is a crucial regulator involved in a variety of physiological and pathological stress responses in the kidney and inhibition of this pathway may therefore serve as a general stress challenger to get fundamental insights into sex differences in podocytes. METHODS: The genomic ROSAmT/mG-NPHS2 Cre mouse model was used which allows obtaining highly pure podocyte fractions for cell-specific molecular analyses, and vehicle or pharmacologic treatment with the mTOR inhibitor rapamycin was performed for 3 weeks. Subsequently, deep RNA sequencing and proteomics were performed of the isolated podocytes to identify intrinsic sex differences. Studies were supplemented with metabolomics from kidney cortex tissues. RESULTS: Although kidney function and morphology remained normal in all experimental groups, RNA sequencing, proteomics and metabolomics revealed strong intrinsic sex differences in the expression levels of mitochondrial, translation and structural transcripts, protein abundances and regulation of metabolic pathways. Interestingly, rapamycin abolished prominent sex-specific clustering of podocyte gene expression and induced major changes only in male transcriptome. Several sex-biased transcription factors could be identified as possible upstream regulators of these sexually dimorphic responses. Concordant to transcriptomics, metabolomic changes were more prominent in males. Remarkably, high number of previously reported kidney disease genes showed intrinsic sexual dimorphism and/or different response patterns towards mTOR inhibition. CONCLUSIONS: Our results highlight remarkable intrinsic sex-differences and sex-specific response patterns towards pharmacological challenged podocyte homeostasis which might fundamentally contribute to sex differences in kidney disease susceptibilities and progression. This work provides rationale and an in-depth database for novel targets to be tested in specific kidney disease models to advance with sex-specific treatment strategies.

The global burden of chronic kidney diseases is rapidly increasing and is projected to become the fifth most common cause of years of life lost worldwide by 2040. Sexual dimorphism in kidney diseases and transplantation is well known, yet sex-specific therapeutic strategies are still missing. One reason is the lack of knowledge due to the lack of inclusion of sex as a biological variable in study designs. This work aimed at identification of molecular signatures of male and female podocytes, gate-keepers of the glomerular filtration barrier. Like cardiomyocytes, podocytes are terminally differentiated cells which are highly susceptible towards pathological challenges. Podocytes are the decisive cell-type of the kidney to maintain the physiological blood-urine barrier, and disturbances of their homeostasis critically accelerate kidney function impairment. By help of a genomic mouse model, highly purified podocytes were obtained from male and female mice with and without pharmacological challenge of the mechanistic target of rapamycin (mTOR) signaling pathway which is known to be deregulated in major kidney diseases. Deep RNA sequencing, proteomics and metabolomics revealed strong intrinsic sex differences in the expression levels of mitochondrial, translation and structural transcripts, protein abundances and regulation of metabolic pathways which might fundamentally contribute to sex differences in kidney disease susceptibilities and progression. Remarkably, high number of previously reported kidney disease genes showed so far unknown intrinsic sexual dimorphism and/or different response patterns towards mTOR inhibition. Our work provides an in-depth database for novel targets to be tested in kidney disease models to advance with sex-specific treatment strategies.

Autoantibodies from patients with kidney allograft vasculopathy stimulate a proinflammatory switch in endothelial cells and monocytes mediated via GPCR-directed PAR1-TNF-α signaling.

Non-HLA-directed regulatory autoantibodies (RABs) are known to target G-protein coupled receptors (GPCRs) and thereby contribute to kidney transplant vasculopathy and failure. However, the detailed underlying signaling mechanisms in human microvascular endothelial cells (HMECs) and immune cells need to be clarified in more detail. In this study, we compared the immune stimulatory effects and concomitant intracellular and extracellular signaling mechanisms of immunoglobulin G (IgG)-fractions from kidney transplant patients with allograft vasculopathy (KTx-IgG), to that from patients without vasculopathy, or matched healthy controls (Con-IgG). We found that KTx-IgG from patients with vasculopathy, but not KTx-IgG from patients without vasculopathy or Con-IgG, elicits HMEC activation and subsequent upregulation and secretion of tumor necrosis factor alpha (TNF-α) from HMECs, which was amplified in the presence of the protease-activated thrombin receptor 1 (PAR1) activator thrombin, but could be omitted by selectively blocking the PAR1 receptor. The amount and activity of the TNF-α secreted by HMECs stimulated with KTx-IgG from patients with vasculopathy was sufficient to induce subsequent THP-1 monocytic cell activation. Furthermore, AP-1/c-FOS, was identified as crucial transcription factor complex controlling the KTx-IgG-induced endothelial TNF-α synthesis, and mircoRNA-let-7f-5p as a regulatory element in modulating the underlying signaling cascade. In conclusion, exposure of HMECs to KTx-IgG from patients with allograft vasculopathy, but not KTx-IgG from patients without vasculopathy or healthy Con-IgG, triggers signaling through the PAR1-AP-1/c-FOS-miRNA-let7-axis, to control TNF-α gene transcription and TNF-α-induced monocyte activation. These observations offer a greater mechanistic understanding of endothelial cells and subsequent immune cell activation in the clinical setting of transplant vasculopathy that can eventually lead to transplant failure, irrespective of alloantigen-directed responses.

Expanded Hemodialysis ameliorates uremia-induced impairment of vasculoprotective KLF2 and concomitant proinflammatory priming of endothelial cells through an ERK/AP1/cFOS-dependent mechanism.

Aims: Expanded hemodialysis (HDx) therapy with improved molecular cut-off dialyzers exerts beneficial effects on lowering uremia-associated chronic systemic microinflammation, a driver of endothelial dysfunction and cardiovascular disease (CVD) in hemodialysis (HD) patients with end-stage renal disease (ESRD). However, studies on the underlying molecular mechanisms are still at an early stage. Here, we identify the (endothelial) transcription factor Krüppel-like factor 2 (KLF2) and its associated molecular signalling pathways as key targets and regulators of uremia-induced endothelial micro-inflammation in the HD/ESRD setting, which is crucial for vascular homeostasis and controlling detrimental vascular inflammation. Methods and results: First, we found that human microvascular endothelial cells (HMECs) and other typical endothelial and kidney model cell lines (e.g. HUVECs, HREC, and HEK) exposed to uremic serum from patients treated with two different hemodialysis regimens in the Permeability Enhancement to Reduce Chronic Inflammation II (PERCI-II) crossover clinical trial - comparing High-Flux (HF) and Medium Cut-Off (MCO) membranes - exhibited strongly reduced expression of vasculoprotective KLF2 with HF dialyzers, while dialysis with MCO dialyzers led to the maintenance and restoration of physiological KLF2 levels in HMECs. Mechanistic follow-up revealed that the strong downmodulation of KLF2 in HMECs exposed to uremic serum was mediated by a dominant engagement of detrimental ERK instead of beneficial AKT signalling, with subsequent AP1-/c-FOS binding in the KLF2 promoter region, followed by the detrimental triggering of pleiotropic inflammatory mediators, while the introduction of a KLF2 overexpression plasmid could restore physiological KLF2 levels and downmodulate the detrimental vascular inflammation in a mechanistic rescue approach. Conclusion: Uremia downmodulates vasculoprotective KLF2 in endothelium, leading to detrimental vascular inflammation, while MCO dialysis with the novel improved HDx therapy approach can maintain physiological levels of vasculoprotective KLF2.

Nailfold Videocapillaroscopy for Non-Invasive Assessment of Microcirculation and Prognostic Correlation with Endothelial Dysfunction, Cardiovascular Risk Factors, and Non-HLA Antibodies in Heart Transplant Recipients: A Pilot Study.

Early identification of allograft vasculopathy and the concomitant elimination of adverse risk factors is essential for improving the long-term prognosis of heart transplant (HTx) recipients with underlying cardiovascular disease (CVD). The major aim of this pilot study was to conduct a non-invasive imaging evaluation of the HTx patient microcirculation by employing nailfold video-capillaroscopy (NVC) in a well-characterized patient and control cohort, and to correlate these data with endothelial cell function, accompanied by studies of traditional cardiovascular risk factors and non-HLA antibodies in HTx recipients. Ten patients undergoing HTx (mean age of 38 ± 14 years) were recruited for the study and compared to a control group of 12 well-matched healthy volunteers (mean age 35 ± 5 years) with normal body mass index (BMI). Detailed medical records were collected from all individuals. NVC was performed using CapillaryScope 200 MEDL4N microscope. For functional readout and correlation analysis, endothelial cell network formation in conjunction with measurements of patient serum levels of vascular endothelial growth factor (VEGF) and non-HLA autoantibodies directed against the angiotensin II type-1-receptor (anti-AT1R-Ab), endothelin-1 type-A-receptor (anti-ETAR-Ab), protease-activated receptor-1 (anti-PAR-1-Ab), and VEGF-A (anti-VEGF-A-Ab) were studied. Our NVC analysis found that the average apical loop diameter of nailfold capillaries was significantly increased in HTx recipients (p = 0.001). In addition, HTx patients with more prominent changes in capillaroscopic patterns were characterized by the presence of traditional cardiovascular risk factors, and HTx patients had increased levels of anti-AT1R-ab, anti-ETAR-ab, and anti-VEGF-A-Ab (p = 0.017, p = 0.025, and p = 0.003, respectively). Capillary diameters most strongly correlated with elevated serum levels of troponin T and triglycerides (R = 0.69, p = 0.028 and R = 0.81, p = 0.004, respectively). In conclusion, we found that an abnormal NVC pattern in HTx patients is associated with traditional CVD risk factors and that NVC is a useful non-invasive tool to conveniently monitor changes in the microvasculature of HTx patients.

Sanqi Oral Solution Ameliorates Renal Ischemia/Reperfusion Injury via Reducing Apoptosis and Enhancing Autophagy: Involvement of ERK/mTOR Pathways.

Ischemia-reperfusion (I/R) induced acute kidney injury (AKI) is a significant health problem with high morbidity and mortality, yet prophylaxis strategies and effective drugs are limited. Sanqi oral solution (SQ) is a formulated medicine widely used in clinical settings to treat various renal diseases via enriching qi and activating blood circulation while its role on I/R-AKI remains unclear. Herein, by establishing rat I/R-AKI models, we intended to investigate the effect of SQ on the prevention of I/R-AKI and explore its underlying mechanisms. We demonstrated that SQ treatment significantly attenuated renal dysfunction of I/R-AKI, alleviated histological damages, inhibited renal apoptosis, and enhanced autophagy. Further investigation proved that SQ could significantly inhibit the activation of ERK and mTOR signaling pathways. Moreover, its renoprotective effect can be abolished by autophagy inhibitor 3-methyladenine (3-MA). Collectively, our results suggest that SQ exerts renoprotective effects on renal I/R injury via reducing apoptosis and enhancing autophagy, which are associated with regulating ERK/mTOR pathways.

Cirrhosis has no impact on therapeutic responses of entecavir for chronic hepatitis B.

OBJECTIVE: As the efficacy of a direct antiviral agent is reduced in cirrhotic chronic hepatitis C patients, prolonged duration of treatment or addition of ribavirin is recommended to improve the rates of sustained virological response. However, the impact of cirrhosis on the efficacy of antiviral treatment for chronic hepatitis B (CHB) remained unclear. PATIENTS AND METHODS: This retrospective cohort study screened entecavir (ETV)-treated CHB patients in Taipei Tzu Chi Hospital from January 2007 till October 2014. The diagnosis of cirrhosis was made on the basis of clinical/imaging or histologic findings. The primary endpoints were hepatitis B e antigen (HBeAg) loss in HBeAg-positive patients and undetectable hepatitis B virus (HBV) DNA in the overall study population. Initial virological response is defined as undetectable HBV DNA at 1-year ETV treatment. RESULTS: A total of 381 (262 men; mean age: 49.6±12.9 years) CHB patients were recruited for the final analysis. Of these, 138 were cirrhotic. In 143 HBeAg-positive patients, there was no difference in the rates of 1- and 2-year HBeAg loss between cirrhotic and noncirrhotic patients (P=0.226 and 0.729, respectively). In the overall population, the rate of 1-year undetectable HBV DNA was higher in patients with cirrhosis than those without cirrhosis (76.1 vs. 64.2%, P=0.016). The rate of 2-year undetectable HBV DNA was not different between these two groups. Using multivariate logistic regression analysis, baseline HBV DNA levels (P=0.006) and HBeAg status (P=0.007), were associated with initial virological response, but not cirrhosis. CONCLUSION: Therapeutic responses of ETV are not decreased in cirrhotic CHB patients. Thus, cirrhotic CHB patients can be treated with ETV without the need for dose adjustment.

Fibrosis index based on four factors better predicts advanced fibrosis or cirrhosis than aspartate aminotransferase/platelet ratio index in chronic hepatitis C patients.

BACKGROUND/PURPOSE: Liver biopsy is the gold standard to determine the severity of hepatic fibrosis despite its risk and invasiveness. The aspartate aminotransferase/platelet ratio index (APRI) could noninvasively predict the severity of hepatic fibrosis in chronic hepatitis C (CHC) patients. Whether fibrosis index based on four factors (FIB-4) could better predict the severity of hepatic fibrosis than APRI in CHC patients remains inconclusive. METHODS: This retrospective study enrolled 1473 CHC patients (784 men and 689 women) with liver biopsy and clinical data including age, aspartate aminotransferase, alanine aminotransferase, and platelet count. FIB-4 and APRI were calculated with a formula using the four clinical parameters. Hepatic fibrosis was staged using the Metavir classification system. RESULTS: The areas under the receiver operating characteristics of FIB-4 for the diagnosis of significant fibrosis (≥ F2), advanced fibrosis (≥ F3), and cirrhosis (F4) were 0.816 [95% confidence interval (CI), 0.795-0.836], 0.827 (95% CI, 0.806-0.849), and 0.849 (95% CI, 0.830-0.867), respectively, compared with those of APRI-0.799 (95% CI, 0.778-0.819), 0.791 (95% CI, 0.770-0.812), and 0.802 (95% CI, 0.781-0.922). In addition, the areas under the receiver operating characteristics of FIB-4 were significantly greater than those of APRI for patients with advanced fibrosis and cirrhosis, respectively (p < 0.0001). CONCLUSION: FIB-4 could predict hepatic fibrosis in CHC patients. By adding two parameters (age and alanine aminotransferase), FIB-4 better predicts advanced fibrosis and cirrhosis than APRI in CHC patients.

Different risk factors between reflux symptoms and mucosal injury in gastroesophageal reflux disease.

Gastroesophageal reflux disease (GERD) is diagnosed based on typical symptoms in clinical practice. It can be divided into two groups using endoscopy: erosive and nonerosive reflux disease (NERD). This study aims to determine the risk factors of reflux symptoms and mucosal injury. This was a two-step case-control study derived from a cohort of 998 individuals having the data of reflux disease questionnaire (RDQ) and endoscopic findings. Those with minor reflux symptoms were excluded. The first step compared symptomatic GERD patients with healthy controls. The 2(nd) step compared patients with erosive esophagitis with healthy controls. In this study, the prevalence of symptomatic GERD and erosive esophagitis were 163 (16.3%) and 166 (16.6%), respectively. A total of 507 asymptomatic individuals without mucosal injury of the esophagus on endoscopy were selected as healthy controls. Compared with healthy controls, multivariate analyses showed that symptomatic GERD patients had a higher prevalence of hypertriglyceridemia [odds ratio (OR), 1.83; 95% confidence interval (CI) 1.13-2.96] and obesity (OR, 1.85; 95% CI 1.08-3.02). By contrast, male sex (OR, 2.24; 95% CI 1.42-3.52), positive Campylo-like organism (CLO) test (OR, 0.56; 95% CI 0.37-0.84), and hiatus hernia (OR, 14.36; 95% CI 3.05-67.6) were associated with erosive esophagitis. In conclusion, obesity and hypertriglyceridemia were associated with reflux symptoms. By contrast, male sex, negative infection of Helicobacter pylori, and hiatus hernia were associated with mucosal injury. Our results suggested that risk factors of reflux symptoms or mucosal injury might be different in GERD patients. The underlying mechanism awaits further studies to clarify.

Factors affecting the diagnostic accuracy of ultrasonography in assessing the severity of hepatic steatosis.

BACKGROUND/PURPOSE: Ultrasonography has long been recognized as a useful tool for detecting hepatic steatosis in clinical practice. However, whether it can assess the severity of hepatic steatosis and which factors affect its diagnostic accuracy remain unclear. METHODS: A total of 171 patients with various causes of hepatitis undergoing liver biopsies were retrospectively reviewed. The clinical, serologic data and ultrasonographical findings were recorded. Hepatic steatosis was graded as negative, mild, moderate, or severe by ultrasonography and histology. Histology was used as gold standard and the agreement rates were calculated. RESULTS: Our data showed that the agreement rate of ultrasonography was 61.4% in assessing the severity of hepatic steatosis and 74.3% in diagnosing hepatic steatosis compared with histology (crude kappa=0.46 vs. 0.46). Using univariate analyses, body mass index and histology activity index score were associated with the agreement in assessing the severity of hepatic steatosis (p=0.008 and 0.035), whereas Ishak fibrosis score had a trend association (p=0.066). Multivariate analyses indicated that age, body mass index, and Ishak fibrosis score could affect the agreement (odds ratio=0.72, 0.89, and 1.41; 95% confidence interval=0.54-0.97, 0.83-0.97, and 1.1-1.8). CONCLUSION: Ultrasonography could assess the severity of hepatic steatosis with moderate accuracy. Obese patients are difficult ultrasonographically. In addition, age and hepatic fibrosis could affect the performance of ultrasonography in assessing the severity of hepatic steatosis.

Baseline hepatitis B surface antigen quantitation can predict virologic response in entecavir-treated chronic hepatitis B patients.

BACKGROUND/PURPOSE: Several anti-viral drugs are approved for the treatment of hepatitis B virus (HBV) infection. However, whether quantitative hepatitis B surface antigen (qHBsAg) can predict the therapeutic response during long-term entecavir treatment remains unclear. METHODS: Fifty-five chronic hepatitis B (CHB) patients who received entecavir for more than 2 years were enrolled. The serum qHBsAg level was measured by HBsAg II quant immunoassay. A significant decline in the qHBsAg level was defined as > 1 log reduction from baseline to 6 months of entecavir treatment. RESULTS: Of the 55 patients (41 males and 14 females with a mean age of 48.3 ± 11.4 years), 23 patients were positive for hepatitis B e antigen (HBeAg). The median treatment period was 34 months, and ranged from 26 months to 43 months. A total of 288 serum samples were used to determine the qHBsAg levels. At year 3 of entecavir therapy, one (1.8%) patient had HBsAg seroclearance. A high qHBsAg level was defined as greater than 10,000 IU/mL. Patients with a high baseline qHBsAg level had a lower rate of virologic response at year 1 (37.5% vs. 89.7%, p < 0.001) and year 2 (56.2% vs. 94.9%, p = 0.001). In this study population, 14.5% had a significant decline of the qHBsAg level. A significant decline could not predict HBeAg loss in HBeAg-positive or virologic response in all patients. CONCLUSION: The baseline serum qHBsAg level can predict virologic response in entecavir-treated CHB patients. However, a significant decline in the qHBsAg level cannot predict serologic or virologic response of entecavir treatment.

Severity of fatty liver on ultrasound correlates with metabolic and cardiovascular risk.

Non-alcoholic fatty liver disease is associated with an increased risk of metabolic and cardiovascular diseases. Whether the severity of fatty liver on ultrasound correlates with metabolic or cardiovascular risk remains unclear. A total of 1000 people receiving health examinations were enrolled, and 126 were excluded due to the presence of HBsAg, anti-HCV, known hepatic disorders or alcohol use (>140 g/wk). Significant fatty liver consisted of moderate and severe fatty liver on ultrasound. The definition of central obesity was modified to a waist circumference of >90 cm in men and >80 cm in women. Framingham risk score was used to estimate the risk of cardiovascular disease. A total of 874 subjects (485 women and 388 men with a mean age of 52.07 ± 11.68 years) were included in the final analysis. By using logistic regression analyses stratified by gender, the odds ratio for the prevalence of diabetes mellitus, metabolic syndrome and risk of cardiovascular disease increased with increasing fatty liver status in both genders (p ≤ 0.001). The difference was not only present between individuals with fatty liver vs. non-fatty liver but also between the mild fatty liver and significant fatty liver groups (p < 0.05). In conclusion, the severity of fatty liver on ultrasound could be useful for the risk stratification of metabolic syndrome, diabetes mellitus and cardiovascular disease in clinical practice.

Male sex, hiatus hernia, and Helicobacter pylori infection associated with asymptomatic erosive esophagitis.

BACKGROUND AND AIMS: Asymptomatic erosive esophagitis (AEE) is an easily forgotten subgroup of gastroesophageal reflux disease due to its lack of warning symptoms, despite having the risk of developing complications, such as bleeding, stricture, or even esophageal adenocarcinoma. METHODS: A total of 2843 potentially eligible patients were screened at the health management center of Buddhist Tzu Chi General Hospital. A total of 1001 patients responded to the survey and gave informed consent; 998 patients who completed the reflux disease diagnostic questionnaire were enrolled. Of them, 594 patients who had no reflux symptoms were included for final analysis. The presence and severity of erosive esophagitis was graded according to the Los Angeles classification. Active infection of Helicobacter pylori (H. pylori) was determined by the Campylo-like organism (CLO) test during endoscopies. RESULTS: A total of 14.5% (86/594) of asymptomatic patients had endoscopic findings of erosive esophagitis. In the univariate analysis, male sex and hiatus hernia were significantly associated with AEE. Positive CLO tests had a trend association. Based on the multivariate analysis, male sex (odds ratio [OR]: 2.32, 95% confidence interval [CI]: 1.35-3.98), hiatus hernia (OR: 4.48, 95% CI: 2.35-89.17), and positive CLO test (OR: 0.57, 95% CI: 0.34-0.95) were associated with AEE, as compared to the healthy controls. CONCLUSIONS: AEE is not a rare condition, and constitutes 14.5% of the asymptomatic population. Male sex, hiatus hernia, and H. pylori infection are factors associated with AEE. These findings are not only helpful in identifying such asymptomatic patients, but also provide information to improve understanding of the relationship between H. pylori infection, reflux symptoms, and erosive esophagitis.

Increased incidence of gastroesophageal reflux disease in patients with chronic hepatitis B virus infection.

BACKGROUND: Although chronic liver disease is associated with gastroesophageal reflux disease (GERD), the impact of chronic hepatitis B virus (HBV) infection on this association remains unclear. We thus aimed to evaluate the relationship between chronic HBV infection and GERD. METHODS: In this prospective population-based study, 1,001 adult subjects who underwent an upper gastrointestinal endoscopic examination in a health check-up and completed a gastroesophageal reflux questionnaire were consecutively enrolled. Endoscopic findings were classified according to the Los Angeles classification. Hepatitis B surface antigen was used as a marker of HBV infection. Univariate and multivariate approaches were used to evaluate the effects of chronic HBV infection on GERD. RESULTS: Chronic HBV infection was associated with heartburn sensation [odds ratio (OR) 1.27, 95% confidence interval 1.01-1.61, P = 0.037], and erosive esophagitis (adjusted OR 1.75, 1.03-2.97, P = 0.037). Although male gender is a risk factor of erosive esophagitis, further analyses stratified by gender and aspartate aminotransferase to platelet ratio index (APRI) showed that chronic HBV infection was associated with erosive esophagitis in female subjects (adjusted OR 2.70, 1.14-6.39, P = 0.024) and those with APRI of more than 0.3 (adjusted OR 3.94, 1.73-8.96, P = 0.001). Moreover, higher serum aspartate aminotransferase (AST) and triglyceride (TG) levels were risk factors of erosive esophagitis in patients with chronic HBV infection. CONCLUSIONS: Our findings indicate a close association between chronic HBV infection and GERD, especially in female subjects and those with higher APRI levels. Moreover, HBV carriers with higher AST or TG levels have higher incidence of erosive esophagitis. The interactions between chronic HBV infection and GERD need further studies. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12072-010-9184-4) contains supplementary material, which is available to authorized users.