Objective: Linkages of renin gene polymorphisms with hypertension have been implicated in several populations with contrasting results. Present study aims to assess the pattern of renin gene polymorphisms in Bangladeshi hypertensive individuals.

Background: Prevalence of chronic kidney disease (CKD) is increasing along with obesity. Hyperinsulinemia is often associated with obesity and in turn linked with CKD. Rodent studies show that a high fat diet induces hyperinsulinemia, accelerates renal lipogenesis (as determined by the expressions of fatty acid synthase (FAS) and Acetyl CoA Carboxylase (ACC), increases renal lipid accumulation and causes renal injury. We previously showed that transgenic expression of E4orf1 (E4), a virus‐derived protein, directly improves glycemic control and reduces endogenous insulin response to a glucose challenge in mice. Using this model, we tested the hypothesis that “ Improvement in glycemic control and hyperinsulinemia in E4 mice will be accompanied by reduction in renal lipogenesis and lipid accumulation along with renal injury ”. Methods: Experiment 1: Twenty‐nine week old control (C57BL6/J, n=8) and E4orf1 transgenic (E4, n=6) mice were fed a chow‐doxycycline diet for 6 weeks, followed by 10 weeks of a high fat‐doxycycline diet (60% kcal). Experiment 2: Fifty week old E4orf1 positive mice (E4; n=3) or E4orf1 negative littermates (control; n=4) were fed same high fat doxycycline diet for 20 weeks. In both experiments, E4 protein was inducibly expressed in the adipose tissue upon doxycycline ingestion in the E4 transgenic mice. As expected, E4 expression significantly improved glycemic control and reduced hyperinsulinemia as presented elsewhere. For this study, the protein expression of FAS and ACC and triglycerides (TG) accumulation was determined in the kidneys from these mice. Moreover, mRNA levels of kidney injury markers like fibronectin, type IV collagen, PA‐1, MCP‐1, TGF‐β, IL‐18, HAVCR/KIM‐1 and VEGF were also determined by RT‐PCR. Results: Kidney weights normalized to total body weight did not differ between groups. Renal expression of FAS and ACC as determined by Western blot was significantly reduced ( p <0.005 and p <0.05 for FAS and ACC respectively) in E4 group compared to control. The amount of TG normalized to kidney weight was significantly lower for the E4 group compared to control ( p <0.05). Moreover, no statistically significant changes were found in mRNA levels for markers of kidney injury between E4 and control mice. Conclusion: E4 reduces lipogenesis and lipid accumulation in the kidneys, which may be linked to the improvement in hyperinsulinemia induced by E4. This is the first report for the role of E4 in influencing renal metabolism. More investigation is required to determine if the lowering of renal lipid accumulation by E4 protects from HFD‐induced renal injury in future studies. US and International patents received for the use of E4. Western blot analysis for FAS and ACC protein in high fat fed mice kidney. (A) and (C) represent data for experiment 1. (B) and (C) represent data for experiment 2. Data is presented as mean±SEM. * p <0.05 and ** p <0.005 calculated by Student’s t test for unpaired data. Figure 1 TG concentration in high fat fed mice kidney. (A) represents data for experiment 1 and (B) represents data for experiment 2. Data is presented as mean±SEM. * p <0.05 by calculated Student’s t test for unpaired data. Figure 2

Cell-free RNAs (cfRNAs) are promising analytes as non-invasive biomarkers and have even greater potential if tied in with metabolomics. Plasma is an optimal source for cfRNAs but is often derived from a variety of anticoagulants. Plasma obtained in heparin is suitable for metabolomics but is difficult to utilize for qPCR-based downstream analysis. In the present study, we aimed to develop a simple, time-efficient, and cost-effective heparinase protocol, followed by library preparation and sequencing of human plasma cfRNAs drawn and stored in heparin at −80 °C for several years. Blood was collected in CPT™ sodium heparin tubes from patients with chronic HCV infection (NCT02400216) at the National Institutes of Health (NIH) Clinical Center. Plasma cfRNAs were treated with heparinase I and used for library preparation and next-generation sequencing (NGS). Heparinase treatment maintained RNA integrity and allowed for successful library preparation for all the study subjects even with 7 ng of cfRNAs as starting material. The classification report derived from Pavian R package v1.2.0 showed no artificial reads. The abundance of chordate over microbial reads suggests no addition of experimental error through heparinase I treatment. We report a novel and practical approach to heparinase treatment for human plasma collected and frozen in sodium heparin for several years. This is an effective demonstration of utilizing heparin plasma for NGS and downstream transcriptomic research, which could then be integrated with metabolomics from the same samples, maximizing efficiency and minimizing blood draws.

Non-alcoholic fatty liver disease (NAFLD) covers a broad spectrum of liver diseases ranging from steatosis to cirrhosis. There are limited data on prevention of hepatic steatosis or its progression to liver disease. Here, we tested if either transgenic (Tg) doxycycline-induced expression in adipose tissue of E4orf1 (E4), an adenoviral protein, or dietary fat restriction attenuated hepatic steatosis or its progression in mice. Twelve to fourteen-week-old TgE4 mice (E4 group) and control mice were exposed to a 60% (Kcal) high fat diet (HFD) for 20 weeks, while another group of mice on HFD for 10 weeks were switched to a chow diet (chow group) for another 10 weeks. Glycemic control was determined at weeks 10 and 20. Tissues were collected for gene and protein analysis at sacrifice. Compared to control, diet reversal significantly reduced body weight in the chow group, whereas E4 expression attenuated weight gain, despite HFD. E4 mice evinced significantly improved glucose clearance, lower endogenous insulin secretion, reduced serum triglycerides, attenuated hepatic steatosis and inflammation. Interestingly, in spite of weight loss and lower liver fat, chow mice showed significant upregulation of hepatic genes involved in lipid metabolism. Despite HFD, E4 prevents hepatic lipid accumulation and progression of hepatic steatosis, while diet reversal maintains hepatic health, but is unable to improve molecular changes.

Mitochondria and peroxisomes are critical for hepatic energy metabolism, lipid homeostasis, and reactive oxygen species (ROS) detoxification. In chronic hepatitis C virus (HCV) infection, continuous injury leads to cirrhosis; however, the spatial arrangement and reversibility of organelle dysfunction remain poorly understood. This study aimed to examine the zonal distribution of mitochondrial and peroxisomal injury in liver biopsies and elucidate the role of circulating cell-free mitochondrial DNA (ccfDNA) in patients with chronic HCV and cirrhosis following antiviral therapy. We employed advanced microscopy imaging and transcriptomic analysis of liver biopsies and quantified ccf-mtDNA as a noninvasive marker of mitochondrial injury in the peripheral blood of these patients. Transcriptomic data revealed alterations in mitochondrial and peroxisomal pathway alterations in HCV-infected patients. The imaging data displayed distinct zone-specific patterns of organelle damage. Following viral removal, significant improvement in mitochondrial and peroxisomal protein expression were noted, indicating partial recovery of organelle integrity following viral clearance; whether this reflects true subcellular regeneration or an early stage of a longer recovery process remains to be determined. Additionally, we showed that ccf-mtDNA quantitatively reflects intrahepatic mitochondrial dysfunction, indicating its potential as a diagnostic biomarker in therapeutic approaches. These findings indicate that organelle injury in chronic HCV is spatially patterned, disease severity-dependent, and partially reversible following antiviral therapy.