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author:("comers, radke")
1.  Transcriptome-Based Analysis of Kidney Gene Expression Changes Associated with Diabetes in OVE26 Mice, in the Presence and Absence of Losartan Treatment 
PLoS ONE  2014;9(5):e96987.
Diabetes is among the most common causes of end-stage renal disease, although its pathophysiology is incompletely understood. We performed next-generation sequencing-based transcriptome analysis of renal gene expression changes in the OVE26 murine model of diabetes (age 15 weeks), relative to non-diabetic control, in the presence and absence of short-term (seven-day) treatment with the angiotensin receptor blocker, losartan (n = 3–6 biological replicates per condition). We detected 1438 statistically significant changes in gene expression across conditions. Of the 638 genes dysregulated in diabetes relative to the non-diabetic state, >70% were downregulation events. Unbiased functional annotation of genes up- and down-regulated by diabetes strongly associated (p<1×10−8) with terms for oxidative stress and for endoplasmic reticulum stress/protein folding. Most of the individual gene products up- or down-regulated with diabetes were unaffected by losartan treatment; however, of the gene products dysregulated in diabetes and influenced by losartan treatment, the vast majority of changes were in the direction of amelioration rather than exacerbation of the diabetic dysregulation. This group of losartan-protected genes associated strongly with annotation terms for endoplasmic reticulum stress, heat shock proteins, and chaperone function, but not oxidative stress; therefore, the losartan-unaffected genes suggest avenues for additional therapeutic opportunity in diabetes. Interestingly, the gene product most highly upregulated by diabetes (>52-fold), encoded by the cationic amino acid transporter Slc7a12, and the gene product most highly downregulated by diabetes (>99%) – encoded by the “pseudogene” Gm6300 – are adjacent in the murine genome, are members of the SLC7 gene family, and are likely paralogous. Therefore, diabetes activates a near-total genetic switch between these two paralogs. Other individual-level changes in gene expression are potentially relevant to diabetic pathophysiology, and novel pathways are suggested. Genes unaffected by diabetes alone but exhibiting increased renal expression with losartan produced a signature consistent with malignant potential.
PMCID: PMC4020814  PMID: 24827579
2.  Enhanced phosphorylation of Na-Cl cotransporter in experimental metabolic syndrome – role of insulin 
We investigated the activity of thiazide-sensitive sodium-chloride cotransporter (NCC) in experimental metabolic syndrome (MS) and the role of insulin in NCC activation. Renal responses to NCC inhibitor hydrochlorothiazide (HCTZ), as a measure of NCC activity in vivo were studied in 12-week old Zucker obese rats (ZO), a model of MS, and in lean control animals (ZL), together with renal NCC expression, and molecular markers of NCC activity, such as localization and phosphorylation. Effects of insulin were further studied in mammalian cell lines with inducible and endogenous expression of this molecule. ZO rats displayed marked hyperinsulinemia, but no differences in plasma aldosterone as compared to ZL. In ZO, natriuretic and diuretic responses to NCC inhibition with HCTZ were enhanced compared with ZL, and associated with a decrease in blood pressure (BP). ZO rats displayed enhanced Thr53 NCC phosphorylation and predominant membrane localization of both total and phosphorylated NCC, together with a different profile in expression of SPAK isoforms, and lower expression of WNK4. In vitro, insulin induced NCC phosphorylation, which was blocked by PI3 kinase inhibitor. Insulin-induced reduction in WNK4 expression was also observed, but delayed compared with the time course of NCC phosphorylation. In summary, we report increased NCC activity in hyperinsulinemic rodents in conjunction with SPAK expression profile consistent with NCC activation and reduced WNK4 as well as an ability of insulin to induce NCC stimulatory phosphorylation in vitro. Together, these findings indicate that hyperinsulinemia is an important driving force of NCC activity in MS with possible consequences for BP regulation.
PMCID: PMC3943429  PMID: 22651238
thiazide-sensitive sodium-chloride cotransporter; metabolic syndrome; insulin; Zucker obese rat; WNK4
3.  Microplate-based platform for combined chromatin and DNA methylation immunoprecipitation assays 
BMC Molecular Biology  2011;12:49.
The processes that compose expression of a given gene are far more complex than previously thought presenting unprecedented conceptual and mechanistic challenges that require development of new tools. Chromatin structure, which is regulated by DNA methylation and histone modification, is at the center of gene regulation. Immunoprecipitations of chromatin (ChIP) and methylated DNA (MeDIP) represent a major achievement in this area that allow researchers to probe chromatin modifications as well as specific protein-DNA interactions in vivo and to estimate the density of proteins at specific sites genome-wide. Although a critical component of chromatin structure, DNA methylation has often been studied independently of other chromatin events and transcription.
To allow simultaneous measurements of DNA methylation with other genomic processes, we developed and validated a simple and easy-to-use high throughput microplate-based platform for analysis of DNA methylation. Compared to the traditional beads-based MeDIP the microplate MeDIP was more sensitive and had lower non-specific binding. We integrated the MeDIP method with a microplate ChIP assay which allows measurements of both DNA methylation and histone marks at the same time, Matrix ChIP-MeDIP platform. We illustrated several applications of this platform to relate DNA methylation, with chromatin and transcription events at selected genes in cultured cells, human cancer and in a model of diabetic kidney disease.
The high throughput capacity of Matrix ChIP-MeDIP to profile tens and potentially hundreds of different genomic events at the same time as DNA methylation represents a powerful platform to explore complex genomic mechanism at selected genes in cultured cells and in whole tissues. In this regard, Matrix ChIP-MeDIP should be useful to complement genome-wide studies where the rich chromatin and transcription database resources provide fruitful foundation to pursue mechanistic, functional and diagnostic information at genes of interest in health and disease.
PMCID: PMC3247195  PMID: 22098709
4.  The Increased Activity of Liver Lysosomal Lipase in Nonalcoholic Fatty Liver Disease Contributes to the Development of Hepatic Insulin Resistance 
We tested the hypothesis that TAG accumulation in the liver induced by short-term high-fat diet (HFD) in rats leads to the dysregulation of endogenous TAG degradation by lysosomal lipase (LIPA) via lysosomal pathway and is causally linked with the onset of hepatic insulin resistance. We found that LIPA could be translocated between qualitatively different depots (light and dense lysosomes). In contrast to dense lysosomal fraction, LIPA associated with light lysosomes exhibits high activity on both intracellular TAG and exogenous substrate and prandial- or diet-dependent regulation. On standard diet, LIPA activity was upregulated in fasted and downregulated in fed animals. In the HFD group, we demonstrated an increased TAG content, elevated LIPA activity, enhanced production of diacylglycerol, and the abolishment of prandial-dependent LIPA regulation in light lysosomal fraction. The impairment of insulin signalling and increased activation of PKCε was found in liver of HFD-fed animals. Lipolysis of intracellular TAG, mediated by LIPA, is increased in steatosis probably due to the enhanced formation of phagolysosomes. Consequent overproduction of diacylglycerol may represent the causal link between HFD-induced hepatic TAG accumulation and hepatic insulin resistance via PKCε activation.
PMCID: PMC3163129  PMID: 21904679
5.  High Ambient Glucose Augments Angiotensin II-Induced Proinflammatory Gene mRNA Expression in Human Mesangial Cells: Effects of Valsartan and Simvastatin 
American Journal of Nephrology  2009;30(2):99-111.
Hyperglycemia may potentiate the adverse renal effects of angiotensin II (AII). In the kidney, the major target of AII action is the glomerular mesangial cell, where its hemodynamic and proinflammatory action contributes to renal injury. AII action is mediated by several types of cell receptors. Among those, the AT1 receptor has been best studied using specific AII receptor blockers (ARBs). These agents have emerged as major new modalities in the prevention and amelioration of renal disease where the ARB renoprotective anti-inflammatory properties could be more important than previously appreciated. Like the ARBs, statins may also modulate inflammatory responses that are renoprotective and complement their cholesterol-lowering effects.
The aim of this project was to (i) identify a repertoire of proinflammatory mesangial cell AII-inducible mRNAs; (ii) determine if the AII-induced proinflammatory mRNA responses depend on ambient glucose, and (iii) test the anti-inflammatory effectiveness of an ARB, valsartan, either alone or in combination with a statin, simvastatin.
Using high-density microarrays and real-time PCR we identified several AII-inducible proinflammatory mesangial genes that exhibited augmented mRNA responses in high-glucose milieu. Valsartan blocked the AII-induced mRNA expression of proinflammatory genes (i.e. MCP-1, LIF and COX-2) maintained in normal and high glucose. These observations add to the mounting evidence that ARBs have anti-inflammatory effects in the kidney, a beneficial effect that may be more important in protecting renal function in diabetic patients. While simvastatin inhibited expression of some mRNAs encoding chemokines/cytokines, it enhanced expression of mRNA encoding COX-2, a key mediator of inflammation. Thus, the non-cholesterol effects of statins on inflammatory responses appear complex.
PMCID: PMC2786028  PMID: 19225232
Angiotensin II; Hyperglycemia; Glomerular mesangial cell; Proinflammatory action
6.  Immunohistochemical and functional correlations of renal cyclooxygenase-2 in experimental diabetes 
Journal of Clinical Investigation  2001;107(7):889-898.
Prostaglandins (PGs) generated by the enzyme cyclooxygenase (COX) have been implicated in the pathological renal hemodynamics and structural alterations in diabetes mellitus, but the role of individual COX isoenzymes in diabetic nephropathy remains unknown. We explored COX-1 and COX-2 expression and hemodynamic responses to the COX-1 inhibitor valeryl salicylate (VS) or the COX-2 inhibitor NS398 in moderately hyperglycemic, streptozotocin-diabetic (D) and control (C) rats. Immunoreactive COX-2 was increased in D rats compared with C rats and normalized by improved glycemic control. Acute systemic administration of NS398 induced no significant changes in mean arterial pressure and renal plasma flow in either C or D rats but reduced glomerular filtration rate in D rats, resulting in a decrease in filtration fraction. VS had no effect on renal hemodynamics in D rats. Both inhibitors decreased urinary excretion of PGE2. However, only NS398 reduced excretion of thromboxane A2. In conclusion, we documented an increase in renal cortical COX-2 protein expression associated with a different renal hemodynamic response to selective systemic COX-2 inhibition in D as compared with C animals, indicating a role of COX-2–derived PG in pathological renal hemodynamic changes in diabetes.
PMCID: PMC199567  PMID: 11285308

Results 1-6 (6)