Transcriptome of hypertension-induced left ventricular hypertrophy and its regression by antihypertensive therapies.

Left ventricular hypertrophy (LVH), a common consequence of systemic hypertension associated with poor clinical outcome, is also a potentially reversible condition. Here, we probed the molecular pathways that underpin the development of LVH and their modulation by antihypertensive regimens that reversed LVH. Spontaneously hypertensive rats were studied at 12 (early LVH) and 48 weeks (late LVH), respectively, with normotensive Wistar-Kyoto rats as age-matched controls. Three treatment groups were maintained for 36 weeks on the following regimens: (1) quinapril, (2) doxazosin and quinapril combination, and (3) losartan. Gene expression profiling was performed with Affymetrix microarrays (GeneChip Rat-230A) and primary function-focused average linkage hierarchical cluster analysis. Of the 15 696 gene sequences expressed on the Affymetrix GeneChip Rat-230A, there was significant alteration in the expression of 295 (1.9%) of these transcripts in 'early' LVH and 143 (0.9%) in 'late' LVH. The predominant changes in gene expression were seen in metabolism, cell growth/proliferation, signal transduction, development and muscle contraction/cytoskeleton functional groups. Although sharing many effects on gene expression, the three treatments showed different expression profiles. Despite significant regression of LVH with treatment, 31 LVH-associated transcripts were unchanged by any of the treatment groups. Our data suggest that LVH regression does not normalize the LVH transcriptome. Therefore, regression of hypertension-induced LVH is associated with a distinct gene expression profile, suggesting the effect of both treatment and a previously unknown specific myocardial physiology after regression of LVH.

IHG-1 amplifies TGF-beta1 signaling and is increased in renal fibrosis.

Induced in high glucose-1 (IHG-1) is an evolutionarily conserved gene transcript upregulated by high extracellular glucose concentrations, but its function is unknown. Here, it is reported that the abundance of IHG-1 mRNA is nearly 10-fold higher in microdissected, tubule-rich renal biopsies from patients with diabetic nephropathy compared with control subjects. In the diabetic nephropathy specimens, in situ hybridization localized IHG-1 to tubular epithelial cells along with TGF-beta1 and activated Smad3, suggesting a possible role in the development of tubulointerstitial fibrosis. Supporting this possibility, IHG-1 mRNA and protein expression also increased with unilateral ureteral obstruction. In the HK-2 proximal tubule cell line, overexpression of IHG-1 increased TGF-beta1-stimulated expression of connective tissue growth factor and fibronectin. IHG-1 was found to amplify TGF-beta1-mediated transcriptional activity by increasing and prolonging phosphorylation of Smad3. Conversely, inhibition of endogenous IHG-1 with small interference RNA suppressed transcriptional responses to TGF-beta1. In summary, IHG-1, which increases in diabetic nephropathy, may enhance the actions of TGF-beta1 and contribute to the development of tubulointerstitial fibrosis.

Association of VEGF-1499C-->T polymorphism with diabetic nephropathy in type 1 diabetes mellitus.

Vascular endothelial growth factor (VEGF) is reported to be implicated in the development of diabetic nephropathy. We performed a case-control study to determine if VEGF-2578C-->A, VEGF-1499C-->T, and VEGF-635G-->C single-nucleotide polymorphisms (SNPs) in the VEGF gene are associated with predisposition to diabetic nephropathy in type 1 diabetes. Genomic DNA was obtained from Irish type 1 diabetic individuals with nephropathy (cases, n=242) and those without nephropathy (controls, n=301), in addition to 400 healthy control samples. These samples were genotyped for the three SNPs using TaqMan or Pyrosequencing technology. Chi-squared analyses revealed no significant differences in genotype or allele frequencies in cases versus controls for VEGF-2578C-->A (genotype, P=.58; allele, P=.52) and VEGF-635G-->C (genotype, P=.58; allele, P=.33). However, a positive association with diabetic nephropathy was observed for the VEGF-1499T allele in the Northern Ireland population (P <.001) and subsequently replicated in a separate population from the Republic of Ireland (P <.001; combined, P <.001). Carriage of the VEGF-1499T allele was associated with a twofold excess risk of developing diabetic nephropathy (OR=2.24, 95% CI=1.50-3.36, P <.0001). No significant differences were found between the healthy control population and the type 1 diabetic population. Our results suggest that the VEGF-1499T allele, or an allele in linkage disequilibrium with this allele, is associated with susceptibility to diabetic nephropathy in the Irish population.

Association between haptoglobin gene variants and diabetic nephropathy: haptoglobin polymorphism in nephropathy susceptibility.

BACKGROUND/AIMS: The Hp(1)/Hp(2) DNA polymorphism has previously been implicated in susceptibility to diabetic nephropathy in some but not all studies. In an attempt to clarify these conflicting findings, we conducted a case-control association study in a Caucasian population. METHODS: We recruited 224 and 285 type 1 diabetic patients with (cases) and without (controls) nephropathy, respectively, from 2 centres based in Northern Ireland and the Republic of Ireland. Hp(1)/Hp(2) genotyping was performed using a combination of long-range and multiplex PCR. Allele and genotype frequencies in cases and controls were compared using the chi(2) test. RESULTS: There was a statistically significant increase in the frequency of the Hp(2) allele in cases compared to controls (65.6 vs. 58.6%, OR = 1.35, 95% CI: 1.04-1.76, p = 0.03). The distributions of genotypes were in Hardy-Weinberg equilibrium for both cases and controls, and the overall frequency of the Hp(1) allele was 38.3%, which is similar to that found in other Western European populations. CONCLUSIONS: The results suggest that the Hp(2) allele may confer susceptibility to nephropathy in patients with type 1 diabetes.

ETV4 and Myeov knockdown impairs colon cancer cell line proliferation and invasion.

We have identified novel colorectal cancer-associated genes using NCBI's UNIGENE cDNA libraries. Colon cancer libraries were examined using Digital Differential Display and disease-associated genes were selected. Among these were ETV4 and MYEOV, novel colorectal cancer-associated genes. Samples of matched normal and neoplastic colon were obtained from human subjects and gene expression was quantified using real-time PCR. ETV4 gene expression was significantly increased in colonic neoplasia in comparison to matched normal colonic tissue (p<0.05). Myeov expression was also increased in colon neoplasia in comparison to matched normal tissue. The effect of siRNA-mediated knockdown of ETV4 and Myeov on cell proliferation and invasion was assessed. ETV4 knockdown resulted in a 90% decrease in cell proliferation (p<0.05) and a 67% decrease in cell invasion. Myeov knockdown resulted in a 48% decrease in cell proliferation (p<0.05) and a 36% decrease in cell invasion. These data suggest that ETV4 and Myeov may provide novel targets for therapeutic intervention.

Resequencing of the characterised CTGF gene to identify novel or known variants, and analysis of their association with diabetic nephropathy.

Connective tissue growth factor (CTGF) has been implicated in the pathogenesis of diabetic nephropathy; however, to date there have been no reports of genomic analysis on this gene. The CTGF gene was comprehensively screened using WAVE (dHPLC) technology and direct capillary sequencing. Single nucleotide polymorphisms (SNPs) with minor allele frequencies greater than 5% were further investigated in an Irish, type 1 diabetic population. The case-control collection consisted of 272 diabetics with nephropathy and 367 non-nephropathic diabetic controls who were genotyped using TaqMan and Pyrosequencing technologies. Ten SNPs were identified, of which seven were novel. Four SNPs are located in the promoter, one in exon 2, two in intron 2 and three in the 3' untranslated region. Based on in silico analysis, three SNPs, c.-650G>C, c.-484T>C and c.247G>C, are potentially functional. Subsequent statistical analysis for common SNPs, c.-650G>C, c.-420InsT, c.-220G>C, c.289+94T>C and c.289+98T>C, in the case-control study revealed no significant differences in genotype or allele frequencies. CTGF has emerged as a biological candidate gene for diabetic nephropathy; however, no significant association was detected between common CTGF SNPs and nephropathy in this population.

A genome-wide DNA microsatellite association screen to identify chromosomal regions harboring candidate genes in diabetic nephropathy.

In an effort to accelerate the identification of susceptibility genes in diabetic nephropathy, the first genome-wide fluorescence-based DNA microsatellite (n=6000) association screen was performed, using pools of genomic DNA derived from Irish patients with (cases; n=200) and without (controls; n=200) type 1 diabetic nephropathy. Allele image profiles were generated for 5353 (89.2%) microsatellite markers for both case and control pools. Allele counts (estimated from allele image profiles) were compared in case versus control groups, and empirical P values were generated. Markers then were ranked on the basis of their empirical P values (lowest to highest). Repeat PCR amplification and electrophoresis of pooled samples were performed systematically on ranked markers until the 50 most associated markers with consistent results were identified. DNA samples that composed the pools then were genotyped individually for these markers. Two markers on chromosome 10, D10S558 (Pcorrected=0.005) and D10S1435 (Pcorrected=0.016), revealed statistically significant associations with diabetic nephropathy. An additional four markers (D6S281, D4S2937, D2S291, and D17S515) also are worthy of further investigation. Relevant functional candidate genes have been identified in the vicinity of these markers, demonstrating the feasibility of low-resolution genome-wide microsatellite association screening to identify possible candidate genes for diabetic nephropathy.

Microarray and bioinformatic detection of novel and established genes expressed in experimental anti-Thy1 nephritis.

BACKGROUND: Microarray technology is a powerful tool that can probe the molecular pathogenesis of renal injury. In this present study microarray analysis was used to monitor serial changes in the renal transcriptome of a rat model of mesangial proliferative glomerulonephritis. Administration of anti-Thy1 antibody results in phases of acute mesangial injury (day 2), cell proliferation (day 5), matrix expansion (days 5 and 7), and subsequent healing (day 14). METHODS: Using Affymetrix (RAE230A) microarrays coupled with sequential primary biologic function-focused and secondary "baited" global cluster analysis, a cohort of established and putative novel modulators of mesangial cell turnover was identified. RESULTS: Cluster analysis of proliferative genes identified a number of gene expression profiles. The most striking pattern was increased gene expression at day 5, a cluster that included platelet-derived growth factor (PDGF), cyclins and transforming growth factor-beta (TGF-beta). The gene expression patterns identified by primary focused cluster analysis were used as bioinformatic bait and resulted in the identification of novel families of genes such as the S100 family. The expression of established and novel genes was confirmed using reverse transcription-polymerase chain reaction (RT-PCR). Next, in vivo gene expression was compared to PDGF-stimulated mesangial cells in vitro revealing similar patterns of dysregulation. CONCLUSION: Transcriptomic analysis defined both known and novel molecules involved in mesangial cell proliferation in vitro and in vivo and defined a panel of molecules that are potential contributors to mesangial cell dysfunction in glomerular disease.

Expression of gremlin, a bone morphogenetic protein antagonist, in human diabetic nephropathy.

BACKGROUND: We report the induction of gremlin, a bone morphogenetic protein antagonist, in cultured human mesangial cells exposed to high glucose and transforming growth factor beta (TGF-beta) levels in vitro and kidneys from diabetic rats in vivo. METHODS: Gremlin expression was assessed in human diabetic nephropathy by means of in situ hybridization, immunohistochemistry, and real-time polymerase chain reaction and correlated with clinical and pathological indices of disease. RESULTS: Gremlin was not expressed in normal human adult kidneys. Conversely, abundant gremlin expression was observed in human diabetic nephropathy. Although some gremlin expression was observed in occasional glomeruli, gremlin expression was most prominent in areas of tubulointerstitial fibrosis, where it colocalized with TGF-beta expression. Gremlin messenger RNA levels correlated directly with renal dysfunction, determined by means of serum creatinine level, but not with proteinuria level. There was a strong correlation between gremlin expression and tubulointerstitial fibrosis score. CONCLUSION: In aggregate, these results indicate that the developmental gene gremlin reemerges in the context of tubulointerstitial fibrosis in diabetic nephropathy and suggests a role for TFG-beta as an inducer of gremlin expression in this context.

Potentiation of glucocorticoid activity in hypoxia through induction of the glucocorticoid receptor.

Tissue hypoxia is intimately associated with chronic inflammatory disease and may signal to the resolution of inflammatory processes. Glucocorticoid signaling through the glucocorticoid receptor (GR) represents a clinically important endogenous anti-inflammatory pathway. Microarray analysis reveals that the GR is transcriptionally up-regulated by hypoxia in human renal proximal tubular epithelial cells. Hypoxic up-regulation of the GR was confirmed at the level of promoter activity, mRNA, and protein expression. Furthermore, functional potentiation of glucocorticoid activity in hypoxia was observed as an enhancement of dexamethasone-induced glucocorticoid response element promoter activity and enhanced dexamethasone-mediated inhibition of IL-1beta-stimulated IL-8 expression and hypoxia-induced vascular endothelial growth factor expression. Knockdown of enhanced GR gene expression in hypoxia using specific GR small inhibitory RNA (siRNA) resulted in an attenuation of the enhanced glucocorticoid sensitivity. A role for the hypoxia-inducible transcription factor, HIF-1alpha, in the regulation of GR expression and the associated potentiation of glucocorticoid activity in hypoxia was also demonstrated. These results reveal a novel signaling aspect responsible for the incorporation of hypoxic and glucocorticoid stimuli, which we hypothesize to be an important co-operative pathway for the control of gene expression observed in complex tissue microenvironments in inflamed states.

Role of alpha-adducin DNA polymorphisms in the genetic predisposition to diabetic nephropathy.

BACKGROUND: There is substantial evidence for genetic susceptibility to diabetic nephropathy. In particular, genes that predispose to hypertension in the general population may confer susceptibility to nephropathy in patients with diabetes. A Gly460Trp variant in the alpha-adducin gene has been associated with essential hypertension. Our aim was to screen the alpha-adducin gene for polymorphisms and to determine if any variants predisposed patients with diabetes to nephropathy. A secondary objective was to assess for association between the Gly460Trp variant and hypertension. METHODS: The exons of the alpha-adducin gene were resequenced in 30 individuals. Selected variants were then genotyped in 155 patients with type 1 diabetes and nephropathy (cases) and 216 persons with type 1 diabetes but no evidence of nephropathy (controls) from Northern Ireland and in 95 cases and 118 controls from the Irish Republic. RESULTS: Eleven polymorphisms were detected, of which six were novel and three caused amino-acid substitutions. The Gly460Trp and a novel Ser617Cys polymorphism were in strong linkage disequilibrium (D' = 0.98). Neither the genotype nor allele frequencies for the Gly460Trp polymorphism (P = 0.89 and 0.93 respectively) or the Ser617Cys polymorphism (P = 0.46 and 0.76) were significantly different between cases and controls when the Northern Ireland and Irish Republic sample groups were combined. Carriage of the 460Trp allele was not significantly associated with systolic or diastolic blood pressure in either the cases (P = 0.48 and 0.06, respectively) or in the controls (P = 0.50 and 0.94, respectively). CONCLUSIONS: Variation in the alpha-adducin gene does not play a major role in the development of nephropathy in persons with type 1 diabetes in the Irish population. Furthermore, the Gly460Trp variant was not associated with hypertension in this population.

Association between variation in the actin-binding gene caldesmon and diabetic nephropathy in type 1 diabetes.

Dysfunction of the actin cytoskeleton is a key event in the pathogenesis of diabetic nephropathy. We previously reported that certain cytoskeletal genes are upregulated in mesangial cells exposed to a high extracellular glucose concentration. One such gene, caldesmon, lies on chromosome 7q35, a region linked to nephropathy in family studies, making it a candidate susceptibility gene for diabetic nephropathy. We screened all exons, untranslated regions, and a 5-kb region upstream of the gene for variation using denaturing high-performance liquid chromatography technology. An A>G single nucleotide polymorphism (SNP) at position -579 in the promoter region was associated with nephropathy in a case-control study using 393 type 1 diabetic patients from Northern Ireland (odds ratio [OR] 1.38, 95% CI 1.02-1.86, P = 0.03). A similar trend was found in an independent sample from a second center. When the sample groups were combined (n = 606), the association between the -579G allele and nephropathy remained significant (OR 1.35, 1.07-1.70, P = 0.01). The haplotype structure in the surrounding 7-kb region was determined. No single haplotype was more strongly associated with nephropathy than the -579A>G SNP. These results suggest a role for the caldesmon gene in susceptibility to diabetic nephropathy in type 1 diabetes.

Sequential extracellular matrix-focused and baited-global cluster analysis of serial transcriptomic profiles identifies candidate modulators of renal tubulointerstitial fibrosis in murine adriamycin-induced nephropathy.

Transcriptome analysis using microarray technology represents a powerful unbiased approach for delineating pathogenic mechanisms in disease. Here molecular mechanisms of renal tubulointerstitial fibrosis (TIF) were probed by monitoring changes in the renal transcriptome in a glomerular disease-dependent model of TIF (adriamycin nephropathy) using Affymetrix (mu74av2) microarray coupled with sequential primary biological function-focused and secondary "baited"-global cluster analysis of gene expression profiles. Primary cluster analysis focused on mRNAs encoding matrix proteins and modulators of matrix turnover as classified by Onto-Compare and Gene Ontology and identified both molecules and pathways already implicated in the pathogenesis of TIF (e.g. transforming growth factor beta1-CTGF-fibronectin-1 pathway) and novel TIF-associated genes (e.g. SPARC and Matrilin-2). Specific gene expression patterns identified by primary extracellular matrix-focused cluster analysis were then used as bioinformatic bait in secondary global clustering, with which to search the renal transcriptome for novel modulators of TIF. Among the genes clustering with ECM proteins in the latter analysis were endoglin, clusterin, and gelsolin. In several notable cases (e.g. claudin-1 and meprin-1beta) the pattern of gene expression identified in adriamycin nephropathy in vivo was replicated during transdifferentiation of renal tubule epithelial cells to a fibroblast-like phenotype in vitro on exposure to transforming growth factor-beta and epidermal growth factor suggesting a role in fibrogenesis. The further exploration of these complex gene networks should shed light on the core molecular pathways that underpin TIF in renal disease.

Lipoxins inhibit Akt/PKB activation and cell cycle progression in human mesangial cells.

Lipoxins (LX) are endogenously produced eicosanoids with a spectrum of bioactions that suggest anti-inflammatory, pro-resolution roles for these agents. Mesangial cell (MC) proliferation plays a pivotal role in the pathophysiology of glomerular inflammation and is coupled to sclerosis and tubulointerstitial fibrosis. We have previously reported that LXA4 acts through a specific G-protein-coupled-receptor (GPCR) to modulate MC proliferation in response to the proinflammatory mediators LTD4 and platelet-derived growth factor (PDGF). Further investigations revealed that these effects were mediated by modulation of receptor tyrosine kinase activity. Here we have explored the underlying mechanisms and report inhibition of growth factor (PDGF; epithelial growth factor) activation of Akt/PKB by LXA4. LXA4 (10 nmol/L) modulates PDGF-induced (10 ng/ml, 24 hours) decrements in the levels of cyclin kinase inhibitors p21Cip1 and p27Kip1. PDGF-induced increases in CDK2-cyclin E complex formation are also inhibited by LXA4. The potential of LXA4 as an anti-inflammatory therapeutic is compromised by its degradation; this has been circumvented by synthesis of stable analogs. We report that 15-(R/S)-methyl-LXA4 and 16-phenoxy-LXA4 mimic the native compound with respect to modulation of cell proliferation and PDGF-induced changes in cell cycle proteins. In vivo, MC proliferation in response to PDGF is associated with TGFbeta1 production and the subsequent development of renal fibrosis. Here we demonstrate that prolonged (24 to 48 hours) exposure to PDGF is associated with autocrine TGFbeta1 production, which is significantly reduced by LXA4. In aggregate these data demonstrate that LX inhibit PDGF stimulated proliferation via modulation of the PI-3-kinase pathway preventing mitogen-elicited G1-S phase progression and suggest the therapeutic potential of LX as anti-fibrotic agents.

Mechanical deformation induced apoptosis in human proximal renal tubular epithelial cells is caspase dependent.

PURPOSE: Ureteral obstruction (UO) results in apoptosis of renal tubular epithelial cells. We postulated that mechanical deformation and inflammation contribute to the cellular loss that occurs as a result of UO and it is mediated through altered heat shock protein 70 (HSP-70) expression and the caspase cascade. MATERIALS AND METHODS: Human HK-2 renal tubular cells were subjected to mechanical stretch. Cell viability and apoptosis were assessed by flow cytometry; HSP-70 and caspase 3 protein expression by Western blotting, and caspase 3 activity by fluorescence substrates. RESULTS: Mechanical stretch caused direct apoptosis induction and it also primed for tumor necrosis factor-alpha induced apoptosis, which was caspase 3 dependent. Although HSP-70 protein expression was increased during mechanical stretch, the protective effects of HSP-70 were only seen after further induction by heat shocking. CONCLUSIONS: Altering HSP-70 expression and manipulating the caspase cell death proteases represent a novel pathway to protect against renal tubular cell apoptosis and the potential for progression to renal failure in UO.

DNA oligonucleotide microarray technology identifies fisp-12 among other potential fibrogenic genes following murine unilateral ureteral obstruction (UUO): modulation during epithelial-mesenchymal transition.

BACKGROUND: Tubulointerstitial inflammation and fibrosis are pathologic hallmarks of end-stage renal disease (ESRD). Here we have used DNA microarray technology to monitor the transcriptomic responses to murine unilateral ureteral obstruction (UUO) with a view to identifying molecular modulators of tubulointerstitial fibrosis. METHODS: Using Affymetrix Mu74Av2 microarrays, gene expression 4 and 10 days postobstruction was investigated relative to control contralateral kidneys. Candidate profibrogenic genes were further investigated in epithelial cells undergoing epithelial to mesenchymal transition (EMT) in vitro. RESULTS: mRNA levels for 1091 gene/EST sequences, of a total of 12,488 displayed on the microarray, were altered twofold or greater by days 4 and 10 postobstruction compared to contralateral control kidneys. Genes were categorised into functional groups, including modulators of cytoskeletal and extracellular matrix metabolism, cell growth, signalling, and transcription/translational events. Among the potentially profibrogenic genes, whose mRNA levels were increased after UUO, were fibroblast-inducible secreted protein (fisp-12), the murine homologue of connective tissue growth factor (CTGF), collagen XVIIIalpha1, secreted protein acidic and rich in cysteine (SPARC), and src-suppressed C-kinase substrate (SSeCKS). A sustained increase in fisp-12 mRNA level was observed during EMT induced by transforming growth factor-beta1 (TGF-beta1) and epidermal growth factor (EGF). CONCLUSION: Altered gene expression in murine UUO has been demonstrated. Increased expression of fisp-12, SPARC, and SSeCKS has been shown in response to TGF-beta1 treatment and during EMT, suggesting that these genes may offer potential therapeutic targets against tubulointerstitial fibrosis.

Identification of Naf1/ABIN-1 among TNF-alpha-induced expressed genes in human synoviocytes using oligonucleotide microarrays.

The cytokine tumor necrosis factor alpha (TNF-alpha) is a critical effector of the pathogenesis of rheumatoid arthritis (RA). We used oligonucleotide microarray (OM) analysis to assess TNF-alpha-modulated gene expression in cultured primary human synoviocytes in vitro. Genes identified include cytokines and inflammatory mediators, extracellular matrix and adhesion molecules, cell cycle and proliferation related proteins, transcription related proteins, and apoptotic mediators. OM identified 1185 differentially expressed genes in TNF-alpha-treated synoviocytes. The regulation of Nef-associated factor-1 (Naf1), an A20-binding, nuclear factor kappa B (NFkappaB) inhibitory protein was probed further given its putative role as an endogenous brake for the expression of some TNF-alpha-driven genes. Naf1 mRNA levels were higher in synovial biopsies from patients with active RA and seronegative arthropathy than in those from patients with osteoarthritis. These findings underscore the value of transcriptome analysis in cytokine-activated synoviocyte cultures in vitro as a means of identifying disease-associated genes in human arthritis, and implicate Naf1 as a potential modulator of TNF-alpha bioactivity in RA.

Digital extractor: analysis of digital differential display output.

Digital Extractor is a program for the high-throughput processing of data sets derived from digital differential display-based comparisons of EST libraries. These comparisons can be utilized to identify discrete subsets of genes whose expression is restricted to distinct tissue types. The program facilitates these investigations by permitting parallel annotation of genes identified as being differentially expressed.

Gremlin - a putative pathogenic player in progressive renal disease.

Progressive renal fibrosis is the end process of renal injury leading to kidney failure. Current therapies for chronic renal failure aim to slow this process but fail to halt its progression. As the mechanisms involved in glomerulosclerosis and tubulointerstitial fibrosis are unravelled, potential treatments for this growing clinical problem should emerge. Gremlin, a developmental regulator of bone morphogenetic proteins (BMPs), has recently been implicated in processes such as glomerulosclerosis, tubulointerstitial fibrosis and cellular hypertrophy, and may represent a novel therapeutic target in progressive renal diseases.

Modification of the transcriptomic response to renal ischemia/reperfusion injury by lipoxin analog.

BACKGROUND: Lipoxins are lipoxygenase-derived eicosanoids with anti-inflammatory and proresolution bioactivities in vitro and in vivo. We have previously demonstrated that the stable synthetic LXA4 analog 15-epi-16-(FPhO)-LXA4-Me is renoprotective in murine renal ischemia/reperfusion injury, as gauged by lower serum creatinine, attenuated leukocyte infiltration, and reduced morphologic tubule injury. METHODS: We employed complementary oligonucleotide microarray and bioinformatic analyses to probe the transcriptomic events that underpin lipoxin renoprotection in this setting. RESULTS: Microarray-based analysis identified three broad categories of genes whose mRNA levels are altered in response to ischemia/reperfusion injury, including known genes previously implicated in the pathogenesis of ischemia/reperfusion injury [e.g., intercellular adhesion molecule-1 (ICAM-1), p21, KIM-1], known genes not previously associated with ischemia/reperfusion injury, and cDNAs representing yet uncharacterized genes. Characterization of expressed sequence tags (ESTs) displayed on microarrays represents a major challenge in studies of global gene expression. A bioinformatic annotation pipeline successfully annotated a large proportion of ESTs modulated during ischemia/reperfusion injury. The differential expression of a representative group of these ischemia/reperfusion injury-modulated genes was confirmed by real-time polymerase chain reaction. Prominent among the up-regulated genes were claudin-1, -3, and -7, and ADAM8. Interestingly, the former response was claudin-specific and was not observed with other claudins expressed by the kidney (e.g., claudin-8 and -6) or indeed with other components of the renal tight junctions (e.g., occludin and junctional adhesion molecule). Noteworthy among the down-regulated genes was a cluster of transport proteins (e.g., aquaporin-1) and the zinc metalloendopeptidase meprin-1 beta implicated in renal remodeling. CONCLUSION: Treatment with the lipoxin analog 15-epi-16-(FPhO)-LXA4-Me prior to injury modified the expression of many differentially expressed pathogenic mediators, including cytokines, growth factors, adhesion molecules, and proteases, suggesting a renoprotective action at the core of the pathophysiology of acute renal failure (ARF). Importantly, this lipoxin-modulated transcriptomic response included many genes expressed by renal parenchymal cells and was not merely a reflection of a reduced renal mRNA load resulting from attenuated leukocyte recruitment. The data presented herein suggest a framework for understanding drivers of kidney injury in ischemia/reperfusion and the molecular basis for renoprotection by lipoxins in this setting.