Pathological and Transcriptome Changes in the ReninAAV db/db uNx Model of Advanced Diabetic Kidney Disease Exhibit Features of Human Disease

The ReninAAV db/db uNx model of diabetic kidney disease (DKD) exhibits hallmarks of advanced human disease, including progressive elevations in albuminuria and serum creatinine, loss of glomerular filtration rate, and pathological changes. Microarray analysis of renal transcriptome changes were more similar to human DKD when compared to db/db eNOS−/− model. The model responds to treatment with arterial pressure lowering (lisinopril) or glycemic control (rosiglitazone) at early stages of disease. We hypothesized the ReninAAV db/db uNx model with advanced disease would have residual disease after treatment with lisinopril, rosiglitazone, or combination of both. To test this, ReninAAV db/db uNx mice with advanced disease were treated with lisinopril, rosiglitazone, or combination of both for 10 weeks. All treatment groups showed significant lowering of urinary albumin to creatinine ratio compared to baseline; however, only combination group exhibited lowering of serum creatinine. Treatment improved renal pathological scores compared to baseline values with residual disease evident in all treatment groups when compared to db/m controls. Gene expression analysis by TaqMan supported pathological changes with increased fibrotic and inflammatory markers. The results further validate this model of DKD in which residual disease is present when treated with agents to lower arterial pressure and glycemic control

Grape Seed Extract as an Adjunct for Modulating Colon Carcinogenesis

154 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2007.Colon cancer is one of the leading causes of cancer deaths in the United States and increasing in incidence globally. Grape seed extract (GSE) is a nutritional supplement with claims as a chemopreventive compound. Preliminary experimental data suggests that dietary GSE may be protective against colorectal cancer. In preliminary experiments, we identified candidate protein biomarkers expressed differently in human colonic tumors compared with non-cancerous colonic tissue. In Western blot and immunohistochemical staining of human colonic tumors compared with non-cancerous colonic tissue, the tumors exhibited elevated expression of beta-catenin and iNOS and decreased expression of PPARgamma, and increased expression of COX-2 by immunohistochemistry. To carry out experimental analysis of dietary GSE in an in vivo model of human colon cancer, azoxymethane (AOM) was used to induce pre-neoplastic colonic lesions in male Fischer 344 rats. These pre-neoplastic lesions are termed aberrant crypt foci (ACF). Both ACF and ACF with multiplicity (ACF with four or more aberrant crypts within a focus) are putative pre-neoplastic lesions with the potential for development into colonic adenomas or carcinomas. Utilizing three dietary doses of GSE, low (0.005%), mid (0.01%) and high (0.05%) in AOM-induced rats, it was found that the low and mid dose groups had the greatest reduction in ACF and ACF with multiplicity compared to the high dose and positive control (AOM only) groups. PPARgamma, a putative colonic enterocyte cellular differentiation marker, was evaluated by Western blot and observed to be elevated in the mid dose group, consistent with its suggested role in colonic differentiation. Microarray analysis revealed repression of several genes involved in lipid metabolism. Evaluating the effects of dietary GSE in short term studies yielded interesting proteomic and genomic trends. It was found that at an early model of AOM effects on rats fed GSE, two weeks post-AOM, dietary GSE protected colonic enterocytes against AOM damage by reduced expression of beta-catenin and COX-2. Genetic alterations in the GSE groups included up-regulation of apoptotic inducers and lamin B1 and down-regulation of lipid metabolism. In rats GSE three weeks post-AOM, it was found that GSE protected the enterocytes against severe AOM damage that resulted in a large increase in apoptosis in the rats receiving AOM only. Genetically, in this three weeks post-AOM model, GSE up-regulated cellular ubiquitination and sensitivity to natural killer-cell mediated destruction and down-regulated cGMP activity. Dietary GSE alone (without AOM exposure) did not alter proteins differently than the negative control (rats receiving neither AOM or dietary GSE) and genomically down-regulated Jun oncogene. In conclusion, GSE protects against AOM induced colonic pre-neoplastic lesions and proteomic and genomic alterations consistent with protection against development of colonic tumors.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Grape Seed Extract as an Adjunct for Modulating Colon Carcinogenesis

154 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2007.Colon cancer is one of the leading causes of cancer deaths in the United States and increasing in incidence globally. Grape seed extract (GSE) is a nutritional supplement with claims as a chemopreventive compound. Preliminary experimental data suggests that dietary GSE may be protective against colorectal cancer. In preliminary experiments, we identified candidate protein biomarkers expressed differently in human colonic tumors compared with non-cancerous colonic tissue. In Western blot and immunohistochemical staining of human colonic tumors compared with non-cancerous colonic tissue, the tumors exhibited elevated expression of beta-catenin and iNOS and decreased expression of PPARgamma, and increased expression of COX-2 by immunohistochemistry. To carry out experimental analysis of dietary GSE in an in vivo model of human colon cancer, azoxymethane (AOM) was used to induce pre-neoplastic colonic lesions in male Fischer 344 rats. These pre-neoplastic lesions are termed aberrant crypt foci (ACF). Both ACF and ACF with multiplicity (ACF with four or more aberrant crypts within a focus) are putative pre-neoplastic lesions with the potential for development into colonic adenomas or carcinomas. Utilizing three dietary doses of GSE, low (0.005%), mid (0.01%) and high (0.05%) in AOM-induced rats, it was found that the low and mid dose groups had the greatest reduction in ACF and ACF with multiplicity compared to the high dose and positive control (AOM only) groups. PPARgamma, a putative colonic enterocyte cellular differentiation marker, was evaluated by Western blot and observed to be elevated in the mid dose group, consistent with its suggested role in colonic differentiation. Microarray analysis revealed repression of several genes involved in lipid metabolism. Evaluating the effects of dietary GSE in short term studies yielded interesting proteomic and genomic trends. It was found that at an early model of AOM effects on rats fed GSE, two weeks post-AOM, dietary GSE protected colonic enterocytes against AOM damage by reduced expression of beta-catenin and COX-2. Genetic alterations in the GSE groups included up-regulation of apoptotic inducers and lamin B1 and down-regulation of lipid metabolism. In rats GSE three weeks post-AOM, it was found that GSE protected the enterocytes against severe AOM damage that resulted in a large increase in apoptosis in the rats receiving AOM only. Genetically, in this three weeks post-AOM model, GSE up-regulated cellular ubiquitination and sensitivity to natural killer-cell mediated destruction and down-regulated cGMP activity. Dietary GSE alone (without AOM exposure) did not alter proteins differently than the negative control (rats receiving neither AOM or dietary GSE) and genomically down-regulated Jun oncogene. In conclusion, GSE protects against AOM induced colonic pre-neoplastic lesions and proteomic and genomic alterations consistent with protection against development of colonic tumors.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Concordant changes of plasma and kidney microRNA in the early stages of acute kidney injury: time course in a mouse model of bilateral renal ischemia-reperfusion.

BACKGROUND: Acute kidney injury (AKI) is a syndrome characterized by the rapid loss of the kidney excretory function and is strongly associated with increased early and long-term patient morbidity and mortality. Early diagnosis of AKI is challenging; therefore we profiled plasma microRNA in an effort to identify potential diagnostic circulating markers of renal failure. The goal of the present study was to investigate the dynamic relationship of circulating and renal microRNA profiles within the first 24 hours after bilateral ischemia-reperfusion kidney injury in mice. METHODOLOGY/PRINCIPAL FINDINGS: Bilateral renal ischemia was induced in C57Bl/6 mice (n = 10 per group) by clamping the renal pedicle for 27 min. Ischemia-reperfusion caused highly reproducible, progressive, concordant elevation of miR-714, miR-1188, miR-1897-3p, miR-877*, and miR-1224 in plasma and kidneys at 3, 6 and 24 hours after acute kidney injury compared to the sham-operated mice (n = 5). These dynamics correlated with histologic findings of kidney injury and with a conventional plasma marker of renal dysfunction (creatinine). Pathway analysis revealed close association between miR-1897-3p and Nucks1 gene expression, which putative downstream targets include genes linked to renal injury, inflammation and apoptosis. CONCLUSIONS/SIGNIFICANCE: Systematic profiling of renal and plasma microRNAs in the early stages of experimental AKI provides the first step in advancing circulating microRNAs to the level of promising novel biomarkers

Correlation of plasma miR-1897-3p, miR-1188, and miR-714 with plasma creatinine.

<p>Correlation of plasma miR-1897-3p, miR-1188, and miR-714 with plasma creatinine.</p

MicroRNA expression confirmation by real-time PCR in the samples from individual animals.

<p>Eleven microRNA species were selected on the basis of concordant and discordant changes in the kidney and plasma as detected in the pooled samples. The changes in five microRNAs were confirmed in the samples from individual animals. Bars on graphs represent fold changes from the time-matched sham-operated animals. MicroRNA expression levels form the sham-operated animals (not shown on graphs) are set to 1. Panel A shows miR-714 changes in kidney and plasma. Panel B shows miR-1188. Panel C shows miR-1897-3p. Panel D shows miR-877*. Panel E shows miR-1224. Two sample t tests were performed on sham and ischemic groups at each timepoint, then p values were adjusted with Bonferroni correction. # = p<0.05; ## = p<0.01; ### = p<0.001.</p

Changes of plasma creatinine in the time course of renal ischemia-reperfusion.

<p>Creatinine is a conventional circulating marker that is elevated when the kidney excretory function is impaired. In the sham-operated mice, plasma creatinine levels were at or below detection, while they were significantly increased in the animals that underwent bilateral renal ischemia-reperfusion. Plasma creatinine levels progressively grew over the time of reperfusion. Time points on the graph represent the time of reperfusion.</p

Distribution of average histopathology score.

<p>Kidney sections from the mice that underwent sham operation or bi-lateral renal ischemia –reperfusion were stained with hematoxilin-eosin (as represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093297#pone-0093297-g001" target="_blank">Figure 1</a>). These sections were evaluated in a semi-quantitative manner for the signs of tissue injury. Degeneration, necrosis and vacuolation were not detected in the sham-operated animals while they increased in the kidneys with ischemia-reperfusion. Morphological signs of kidney injury progressively accumulated in accordance with reperfusion time. D/N = degeneration/necrosis score; Vac = vacuolation score.</p

Nucks1 expression in the kidneys of mice with renal ischemia-reperfusion.

<p>Nucks1 gene expression was down-regulated in the injured kidneys at all time points and reached significance at 3 and 24 hours after reperfusion.</p