Ronald P. Brown

Expression, Circulation, and Excretion Profile of MicroRNA-21, -155, and -18a Following Acute Kidney Injury

MicroRNAs (miRNAs) are endogenous noncoding RNA molecules that are involved in post-transcriptional gene silencing. Using global miRNA expression profiling, we found miR-21, -155, and 18a to be highly upregulated in rat kidneys following tubular injury induced by ischemia/reperfusion (I/R) or gentamicin administration. Mir-21 and -155 also showed decreased expression patterns in blood and urinary supernatants in both models of kidney injury. Furthermore, urinary levels of miR-21 increased 1.2-fold in patients with clinical diagnosis of acute kidney injury (AKI) (n = 22) as compared with healthy volunteers (n = 25) (p < 0.05), and miR-155 decreased 1.5-fold in patients with AKI (p < 0.01). We identified 29 messenger RNA core targets of these 3 miRNAs using the context likelihood of relatedness algorithm and found these predicted gene targets to be highly enriched for genes associated with apoptosis or cell proliferation. Taken together, these results suggest that miRNA-21 and -155 could potentially serve as translational biomarkers for detection of AKI and may play a critical role in the pathogenesis of kidney injury and tissue repair process.

Distribution of bisphenol A into tissues of adult, neonatal, and fetal Sprague-Dawley rats.

Bisphenol A (BPA) is an important industrial chemical used in the manufacture of polycarbonate plastic products and epoxy resin-based food can liners. The presence of BPA metabolites in urine of >90% of Americans aged 6-60 suggests ubiquitous and frequent exposure in the range of 0.02-0.2μg/kgbw/d (25th-95th percentiles). The current study used LC/MS/MS to measure placental transfer and concentrations of aglycone (receptor-active) and conjugated (inactive) BPA in tissues from Sprague-Dawley rats administered deuterated BPA (100μg/kg bw) by oral and IV routes. In adult female rat tissues, the tissue/serum concentration ratios for aglycone BPA ranged from 0.7 in liver to 5 in adipose tissue, reflecting differences in tissue perfusion, composition, and metabolic capacity. Following IV administration to dams, placental transfer was observed for aglycone BPA into fetuses at several gestational days (GD), with fetal/maternal serum ratios of 2.7 at GD 12, 1.2 at GD 16, and 0.4 at GD 20; the corresponding ratios for conjugated BPA were 0.43, 0.65, and 3.7. These ratios were within the ranges observed in adult tissues and were not indicative of preferential accumulation of aglycone BPA or hydrolysis of conjugates in fetal tissue in vivo. Concentrations of aglycone BPA in GD 20 fetal brain were higher than in liver or serum. Oral administration of the same dose did not produce measurable levels of aglycone BPA in fetal tissues. Amniotic fluid consistently contained levels of BPA at or below those in maternal serum. Concentrations of aglycone BPA in tissues of neonatal rats decreased with age in a manner consistent with the corresponding circulating levels. Phase II metabolism of BPA increased with fetal age such that near-term fetus was similar to early post-natal rats. These results show that concentrations of aglycone BPA in fetal tissues are similar to those in other maternal and neonatal tissues and that maternal Phase II metabolism, especially following oral administration, and fetal age are critical in reducing exposures to the fetus.

Clinically relevant concentrations of di (2-ethylhexyl) phthalate (DEHP) uncouple cardiac syncytium.

Di(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer found in a variety of polyvinyl chloride (PVC) medical products. The results of studies in experimental animals suggest that DEHP leached from flexible PVC tubing may cause health problems in some patient populations. While the cancerogenic and reproductive effects of DEHP are well recognized, little is known about the potential adverse impact of phthalates on the heart. This study examined the effects of clinically relevant concentrations of DEHP on neonatal rat cardiomyocytes. It was found that application of DEHP to a confluent, synchronously beating cardiac cell network, leads to a marked, concentration-dependent decrease in conduction velocity and asynchronous cell beating. The mechanism behind these changes was a loss of gap junctional connexin-43, documented using Western blot analysis, dye-transfer assay and immunofluorescence. In addition to its effect on electrical coupling, DEHP treatment also affected the mechanical movement of myocyte layers. The latter was linked to the decreased stiffness of the underlying fibroblasts, as the amount of triton-insoluble vimentin was significantly decreased in DEHP-treated samples. The data indicate that DEHP, in clinically relevant concentrations, can impair the electrical and mechanical behavior of a cardiac cell network. Applicability of these findings to human patients remains to be established.

Immunolocalization of Kim-1, RPA-1, and RPA-2 in Kidney of Gentamicin-, Mercury-, or Chromium-Treated Rats: Relationship to Renal Distributions of iNOS and Nitrotyrosine

Immunohistochemical studies for kidney injury molecule-1 (Kim-1), renal papillary antigen-1 (RPA-1), and renal papillary antigen-2 (RPA-2) were conducted to explore their relationship to inducible nitric oxide synthase (iNOS) and nitrotyrosine expression. Male Sprague-Dawley rats were exposed to gentamicin (100 mg/kg/day Gen, sc, for 3 days), mercury (0.25 mg Hg/kg, iv, single dose), or chromium (5 mg Cr/kg, sc, single dose) and kidney tissue was examined 24 hours or 72 hours after the last dose of the nephrotoxicant. Another group of kidneys was evaluated 24 hours after rats were administered 3 daily doses (50, 100, 150, 200, or 300 mg/kg/day) of Gen. Gen- and Cr-treated rats exhibited increased immunoreactivity of Kim-1, RPA-1, and RPA-2 largely in the S1/S2 segments and to a lesser extent in the S3 segments of the proximal tubule of the kidney, whereas Hg-treated rats showed increased immunoreactivity of Kim-1, RPA-1, and RPA-2 in the S3 segments. Up-regulation of Kim-1, RPA-1, and RPA-2 expression correlated with injured tubular epithelial cells and also correlated with immunoreactivity of iNOS and nitrotyrosine. It is possible that iNOS activation with nitrotyrosine production in injured nephron segments may be involved in the induction of Kim-1, RPA-1, and RPA-2 following exposure to nephrotoxicants.

Differences in Immunolocalization of Kim-1, RPA-1, and RPA-2 in Kidneys of Gentamicin-, Cisplatin-, and Valproic Acid-Treated Rats: Potential Role of iNOS and Nitrotyrosine

The present study compared the immunolocalization of Kim-1, renal papillary antigen (RPA)-1, and RPA-2 with that of inducible nitric oxide synthase (iNOS) and nitrotyrosine in kidneys of gentamicin sulfate (Gen)- and cisplatin (Cis)-treated rats. The specificity of acute kidney injury (AKI) biomarkers, iNOS, and nitrotyrosine was evaluated by dosing rats with valproic acid (VPA). Sprague-Dawley (SD) rats were injected subcutaneously (sc) with 100 mg/kg/day of Gen for six or fourteen days; a single intraperitoneal (ip) dose of 1, 3, or 6 mg/kg of Cis; or 650 mg/kg/day of VPA (ip) for four days. In Gen-treated rats, Kim-1 was expressed in the epithelial cells, mainly in the S1/S2 segments but less so in the S3 segment, and RPA-1 was increased in the epithelial cells of collecting ducts (CD) in the cortex. Spatial expression of iNOS or nitrotyrosine with Kim-1 or RPA-1 was detected. In Cis-treated rats, Kim-1 was expressed only in the S3 segment cells, and RPA-1 and RPA-2 were increased in the epithelial cells of medullary CD or medullary loop of Henle (LH), respectively. Spatial expression of iNOS or nitrotyrosine with RPA-1 or RPA-2 was also identified. These findings suggest that peroxynitrite formation may be involved in the pathogenesis of Gen and Cis nephrotoxicity and that Kim-1, RPA-1, and RPA-2 have the potential to serve as site-specific biomarkers for Gen or Cis AKI.

Silver nanoparticles: Significance of physicochemical properties and assay interference on the interpretation of in vitro cytotoxicity studies

Silver nanoparticles (AgNPs) have generated a great deal of interest in the research, consumer product, and medical product communities due to their antimicrobial and anti-biofouling properties. However, in addition to their antimicrobial action, concerns have been expressed about the potential adverse human health effects of AgNPs. In vitro cytotoxicity studies often are used to characterize the biological response to AgNPs and the results of these studies may be used to identify hazards associated with exposure to AgNPs. Various factors, such as nanomaterial size (diameter), surface area, surface charge, redox potential, surface functionalization, and composition play a role in the development of toxicity in in vitro test systems. In addition, the interference of AgNPs with in vitro cytotoxicity assays may result in false negative or false positive results in some in vitro biological tests. The goal of this review is to: 1) summarize the impact of physical-chemical parameters, including size, shape, surface chemistry and aggregate formation on the in vitro cytotoxic effects of AgNPs; and 2) explore the nature of AgNPs interference in in vitro cytotoxicity assays.

Gene expression profiling of DEHP-treated cardiomyocytes reveals potential causes of phthalate arrhythmogenicity

BACKGROUNDnDi-(2-ethylhexyl)-phthalate (DEHP) is a widely used plasticizer that imparts flexibility to polyvinyl chloride. We have recently reported that clinically relevant concentrations of DEHP can affect electrical coupling between cardiac myocytes causing significant rhythm disturbances. The underlying causes for this effect are currently unknown.nnnOBJECTIVESnTo use data on global mRNA expression as a tool to reveal possible pathways leading to arrhythmogenic effects of DEHP.nnnMETHODSnRat neonatal cardiomyocytes were treated with 50 μg/mL DEHP for 72 h. Extracted RNA samples were hybridized onto Affymetrix Rat Gene 1.0 ST arrays. The mRNA expression of a subset of genes was validated by qRT-PCR. In a second set of experiments, cells were treated in a concentration dependent manner to identify genes affected by low DEHP concentrations.nnnRESULTSnDEHP exposure is associated with global changes in mRNA expression, with differentially expressed genes overrepresented in 47 Gene Ontology categories. Modified expression was detected for genes associated with cell electrical activity, calcium handling, adhesion and microtubular transport. For a number of key proteins, including kinesin, TGFβ2, α-tubulin, and α1 & β1 integrins, changes in mRNA levels were confirmed on the level of the protein expression. A number of genes associated with cell adhesion and electrical activity were identified as early DEHP targets as they were affected by concentrations as low as 1 μg/mL.nnnCONCLUSIONSnExposure of neonatal rat cardiomyocytes to clinically relevant DEHP concentrations leads to global changes in mRNA expression. These changes help to explain the arrhythmogenic effects of phthalates on these cells.

Age-related differences in susceptibility to cisplatin-induced renal toxicity

Limited experimental models exist to assess drug toxicity in pediatric populations. We recently reported how a multi‐age rat model could be used for pre‐clinical studies of comparative drug toxicity in pediatric populations. The objective of this study was to expand the utility of this animal model, which previously demonstrated an age‐dependent sensitivity to the classic nephrotoxic compound, gentamicin, to another nephrotoxicant, namely cisplatin (Cis). Sprague–Dawley rats (10, 25, 40 and 80 days old) were injected with a single dose of Cis (0, 1, 3 or 6u2009mgu2009kg−1 i.p.). Urine samples were collected prior and up to 72u2009h after treatment in animals that were ≥ 25 days old. Several serum, urinary and ‘omic’ injury biomarkers as well as renal histopathology lesions were evaluated. Statistically significant changes were noted with different injury biomarkers in different age groups. The order of age‐related Cis‐induced nephrotoxicity was different than our previous study with gentamicin: 80 > 40 > 10 > 25 day‐old vs 10 ≥ 80 > 40 > 25‐day‐old rats, respectively. The increased levels of kidney injury molecule‐1 (Kim‐1: urinary protein/tissue mRNA) provided evidence of early Cis‐induced nephrotoxicity in the most sensitive age group (80 days old). Levels of Kim‐1 tissue mRNA and urinary protein were significantly correlated to each other and to the severity of renal histopathology lesions. These data indicate that the multi‐age rat model can be used to demonstrate different age‐related sensitivities to renal injury using mechanistically distinct nephrotoxicants, which is reflected in measurements of a variety of metabolite, gene transcript and protein biomarkers. Published in 2009 by John Wiley & Sons, Ltd.

Fibrinogen Excretion in the Urine and Immunoreactivity in the Kidney Serves as a Translational Biomarker for Acute Kidney Injury

Fibrinogen (Fg) is significantly up-regulated in the kidney after acute kidney injury (AKI). We evaluated the performance of Fg as a biomarker for early detection of AKI. In rats and mice with kidney tubular damage induced by ischemia/reperfusion (I/R) or cisplatin administration, respectively; kidney tissue and urinary Fg increased significantly and correlated with histopathological injury, urinary kidney injury molecule-1 (KIM-1) and N-acetyl glucosaminidase (NAG) corresponding to the progression and regression of injury temporally. In a longitudinal follow-up of 31 patients who underwent surgical repair of abdominal aortic aneurysm, urinary Fg increased earlier than SCr in patients who developed postoperative AKI (AUC-ROC = 0.72). Furthermore, in a cohort of patients with biopsy-proven AKI (n = 53), Fg immunoreactivity in the tubules and interstitium increased remarkably and was able to distinguish patients with AKI from those without AKI (n = 59). These results suggest that immunoreactivity of Fg in the kidney, as well as urinary excretion of Fg, serves as a sensitive and early diagnostic translational biomarker for detection of AKI.

Urinary biomarkers track the progression of nephropathy in hypertensive and obese rats

AIMSnTo determine whether urinary biomarkers of acute kidney injury can be used to monitor the progression of chronic kidney injury in a rat model of hypertension and obesity.nnnMATERIALS & METHODSnA suite of novel urinary biomarkers were used to track the progression of kidney damage in SHROB and SHR-lean rats.nnnRESULTSnUrinary albumin, NAG, clusterin, osteopontin, RPA-1 and fibrinogen levels were significantly elevated over time and were closely associated with the severity of histopathologically determined nephropathy in both SHROB and SHR-lean rats.nnnCONCLUSIONnUrinary biomarkers, such as albumin, fibrinogen, NAG, clusterin, RPA-1 and osteopontin, may serve as useful tools to track the progression of chronic kidney disease associated with hypertension and obesity.