The transcription factor NF-kappaB regulates a wide variety of biological effects in diverse cell types and organs, particularly stress and adaptive responses. Recently, it has become recognized that NF-kappaB and its upstream regulator tumor necrosis factor (TNF)-alpha regulate specific antithetical effects. For instance, in the heart, NF-kappaB has been found to be required for development of late preconditioning against myocardial infarction and yet is critically involved in mediating cell death after ischemia/reperfusion injury. There remains a bias that NF-kappaB is a "general" transcription factor that is activated by a plethora of stimuli, including neurohormonal, pathophysiological, and stress stimuli, and affects regulation of numerous downstream genes. The question has become, how can such a "general" transcription factor be critically involved in mediating specific effects? An emerging hypothesis is that NF-kappaB is part of a complicated signaling network or web, and that different combinatorial interactions between various activated signaling pathway components produce specific outcomes. This idea is supported by the large number of interactions discovered in the past 14 years between NF-kappaB and other signaling pathways at multiple levels. Notwithstanding the complexities of signal-induced activation of NF-kappaB, since it is a transcription factor, specific effects of NF-kappaB activation must be underlain by the activation and/or suppression of distinct subsets of NF-kappaB-dependent genes. At this level, selectivity is conferred by the expression of specific NF-kappaB subunits, their post translational modifications, and by combinatorial interactions between NF-kappaB and other transcription factors and coactivators that form specific enhanceosome complexes in association with particular promoters. These enhanceosome complexes represent another level of signaling integration whereby the activities of multiple upstream pathways converge to impress a distinct pattern of gene expression upon the NF-kappaB-dependent transcriptional network. Understanding how the overall cellular signaling network translates NF-kappaB activation into the regulation of specific subsets of NF-kappaB-dependent genes will lead to a mechanistic understanding of how NF-kappaB mediates diverse and paradoxical biological effects.

Rationale It has been shown that the transcription factor NF-κB is necessary for late phase cardioprotection after ischemic preconditioning (IPC) in the heart, and yet is injurious after ischemia/reperfusion (I/R). However the downstream gene expression programs that underlie the contribution of NF-κB to cardioprotection after late IPC are incompletely understood. Objective To delineate the specific genes that are regulated by NF-κB immediately after a late IPC stimulus and validate the methodology for identification of NF-κB-dependent genes that contribute to cardioprotection. Methods and Results A directed microarray analysis identified 238 genes as up or down regulated in an NF-κB-dependent manner 3.5 h after late IPC. Among these are several genes previously implicated in late IPC. Gene ontological analysis showed that the most significant group of NF-κB-dependent genes are heat shock response genes, including the genes encoding Hsp70.1 and Hsp70.3. Though an Hsp70.1/70.3 double knockout failed to exhibit cardioprotection, late IPC was intact in the Hsp70.1 single knockout. After I/R, the Hsp70.1/70.3 double knockout and the Hsp70.1 single knockout had significantly increased and reduced infarct size, respectively. Conclusions These results delineate the immediate NF-κB-dependent transcriptome after late IPC. One of the major categories of NF-κB-dependent genes induced by late IPC is the heat shock response. The results of infarct studies confirm that Hsp70.3 is protective after IPC. However, though Hsp70.1 and Hsp70.3 are coordinately regulated, their functions are opposing after I/R injury.

The transcription factor Nuclear Factor Kappa B (NF-κB) has been shown to be cardioprotective after permanent coronary occlusion (PO) and late ischemic preconditioning (IPC), and yet it is cell injurious after ischemia/reperfusion (I/R) in the heart. There is limited information regarding NF-κB-dependent cardioprotection, and the NF-κB-dependent genes that contribute to the cardioprotection after PO are completely unknown. The objective of the study was to identify NF-κB-dependent genes that contribute to cardioprotection after PO. Microarray analysis was used to delineate genes that potentially contribute to the NF-κB-dependent cardioprotection by determining the overlap between the set of PO regulated genes and genes regulated by NF-κB, using mice with genetic abrogation of NF-κB activation in the heart. This analysis identified 16 genes as candidates for NF-κB-dependent effects after PO. This set of genes overlaps with, but is significantly different from the set of genes we previously identified as regulated by NF-κB after IPC. The genes encoding heat shock protein 70.3 (hspa1a) and heat shock protein 70.1 (hspa1b) were the most significantly regulated genes after PO and were up-regulated by NF-κB. Results using knockout mice show that Hsp70.1 contributes to NF-κB-dependent cardioprotection after PO and likely underlies, at least in part, the NF-κB-dependent cardioprotective effect. Our previous results show that Hsp70.1 is injurious after I/R injury. This demonstrates that, like NF-κB itself, Hsp70.1 has antithetical effects on myocardial survival and suggests that this may underlie the similar antithetical effects of NF-κB after different ischemic stimuli. The significance of the research is that understanding the gene network regulated by NF-κB after ischemic insult may lead to identification of therapeutic targets more appropriate for clinical development.

Congenital obstructive nephropathy is a common cause of chronic kidney disease and a leading indication for renal transplant in children. The cellular and molecular responses of the kidney to congenital obstruction are incompletely characterized. In this study, we evaluated global transcription in kidneys with graded hydronephrosis in the megabladder (mgb −/−) mouse to better understand the pathophysiology of congenital obstructive nephropathy. Three primary pathways associated with kidney remodeling/repair were induced in mgb −/− kidneys independent of the degree of hydronephrosis. These pathways included retinoid signaling, steroid hormone metabolism, and renal response to injury. Urothelial proliferation and the expression of genes with roles in the integrity and maintenance of the renal urothelium were selectively increased in mgb −/− kidneys. Ngal/Lcn2, a marker of acute kidney injury, was elevated in 36% of kidneys with higher grades of hydronephrosis. Evaluation of Ngalhigh versus Ngallow kidneys identified the expression of several novel candidate markers of renal injury. This study indicates that the development of progressive hydronephrosis in mgb −/− mice results in renal adaptation that includes significant changes in the morphology and potential functionality of the renal urothelium. These observations will permit the development of novel biomarkers and therapeutic approaches to progressive renal injury in the context of congenital obstruction.

In this issue of Molecular Pharmacology, Luo et al. (p. 1953) present a study employing a HIF-1␣/VP16 chimera to investigate the mechanism by which this constitutively active transcription factor activates expression of brain natriuretic peptide (BNP). The results define a functional hypoxia responsive element (HRE) in the promoter of the human BNP gene and demonstrate that this HRE is necessary for HIF-1␣/VP16-induced gene expression in human cardiomyocytes grown under normoxic conditions. Luo et al. also show that a consensus E-box DNA binding sequence is necessary for appropriate BNP

PurposeUrinary stasis is a risk factor for recurrent urinary tract infection (UTI). Homozygous mutant Megabladder (Mgb-/-) mice exhibit incomplete bladder emptying as a consequence of congenital detrusor aplasia. We hypothesize that this predisposes Mgb-/- mice to spontaneous and experimental UTI.Methods Mgb-/-, Mgb+/-, and wild-type female mice underwent serial ultrasound and urine cultures at 4, 6, and 8 weeks to detect spontaneous UTI. Urine bacterial isolates were analyzed by Gram stain and speciated. Bladder stones were analyzed by x-ray diffractometry. Bladders and kidneys were subject to histologic analysis. The pathogenicity of coagulase-negative Staphylococcus (CONS) isolated from Mgb-/- urine was tested by transurethral administration to culture-negative Mgb-/- or wild-type animals. The contribution of urinary stasis to CONS susceptibility was evaluated by cutaneous vesicostomy in Mgb-/- mice.Results Mgb-/- mice develop spontaneous bacteriuria (42%) and struvite bladder stones (31%) by 8 weeks, findings absent in Mgb+/- and wild-type controls. CONS was cultured as a solitary isolate from Mgb-/- bladder stones. Bladders and kidneys from mice with struvite stones exhibit mucosal injury, inflammation, and fibrosis. These pathologic features of cystitis and pyelonephritis are replicated by transurethral inoculation of CONS in culture-negative Mgb-/- females, whereas wild-type animals are less susceptible to CONS colonization and organ injury. Cutaneous vesicostomy prior to CONS inoculation significantly reduces the quantity of CONS recovered from Mgb-/- urine, bladders, and kidneys.ConclusionsCONS is an opportunistic uropathogen in the setting of urinary stasis, leading to enhanced UTI incidence and severity in Mgb-/- mice.

Background Congenital obstructive nephropathy (CON) is a leading cause of pediatric chronic kidney disease (CKD). Despite optimal surgical and medical care, there is a high rate of CKD progression. Better understanding of molecular and cellular changes is needed to facilitate development of improved biomarkers and novel therapeutic approaches in CON. Methods The megabladder (mgb) mouse is an animal model of CKD with impaired bladder emptying, hydronephrosis, and progressive renal injury. In this study, we characterize a particular microRNA, miR-205, whose expression changes with the degree of hydronephrosis in the mgb-/- kidney. Results Expression of miR-205 is progressively increased in the adult mgb-/-mouse with worsening severity of hydronephrosis. miR-205 expression is correlated with altered expression of cytokeratins and uroplakins, which are markers of cellular differentiation in urothelium. We describe the spatial pattern of miR-205 expression, including increased expression in renal urothelium and novel miR-205 expression in medullary collecting duct epithelium in the congenitally obstructed kidney. Conclusion miR-205 is increased with severity of congenital obstructive nephropathy and CKD in the mgb-/- mouse, and may regulate urothelial differentiation.

Nuclear factor‐kappaB (NF‐κB) is a key player in the physiological outcomes of myocardial ischemic events including ischemia‐reperfusion (I/R), permanent occlusion (PO), and the cardioprotection of ischemic preconditioning (IPC). Our lab has demonstrated that NF‐κB plays a protective role in late IPC and after PO, yet has an injurious role after I/R. Such results suggest the transcriptional activation of differential gene sets by each ischemic event. This was confirmed in our lab by a gene microarray analysis comparing I/R, PO, and IPC. Our microarray results also suggested potential cross‐talk of NF‐κB with other transcriptional networks, such as those regulated by HIF‐1α, AP‐1, and HSF‐1. Our aim is to deliver transcription factor decoys and interfering RNAs to the heart in vivo and begin to elucidate the relative role of these gene networks in myocardial ischemia. We have demonstrated nucleic acid delivery to the heart via novel polyglycoamidoamine (PGAA) glycopolymers that act through formation of nanoscale “polyplexes” and enhance cellular uptake via endocytosis resulting in nearly transmural transfection of the myocardium without eliciting cytotoxicity. Our work provides a high‐throughput in vivo approach to the study of the regulation of interlaced gene networks during myocardial ischemic events and will augment the identification of new clinical targets for the treatment of myocardial ischemia. This work was supported by NIH grant R01 HL63034 (WKJ) and NSF award number 0333377.