Anne E. Prada

Identification of Neutrophil Gelatinase-Associated Lipocalin as a Novel Early Urinary Biomarker for Ischemic Renal Injury

Acute renal failure (ARF) secondary to ischemic injury remains a common and potentially devastating problem. A transcriptome-wide interrogation strategy was used to identify renal genes that are induced very early after renal ischemia, whose protein products might serve as novel biomarkers for ARF. Seven genes that are upregulated >10-fold were identified, one of which (Cyr61) has recently been reported to be induced after renal ischemia. Unexpectedly, the induction of the other six transcripts was novel to the ARF field. In this study, one of these previously unrecognized genes was further characterized, namely neutrophil gelatinase-associated lipocalin (NGAL), because it is a small secreted polypeptide that is protease resistant and consequently might be readily detected in the urine. The marked upregulation of NGAL mRNA and protein levels in the early postischemic mouse kidney was confirmed. NGAL protein expression was detected predominantly in proliferating cell nuclear antigen-positive proximal tubule cells, in a punctate cytoplasmic distribution that co-localized with markers of late endosomes. NGAL was easily detected in the urine in the very first urine output after ischemia in both mouse and rat models of ARF. The appearance of NGAL in the urine was related to the dose and duration of renal ischemia and preceded the appearance of other urinary markers such as N-acetyl-beta-D-glucosaminidase and beta2-microglobulin. The origin of NGAL from tubule cells was confirmed in cultured human proximal tubule cells subjected to in vitro ischemic injury, where NGAL mRNA was rapidly induced in the cells and NGAL protein was readily detectable in the culture medium within 1 h of mild ATP depletion. NGAL was also easily detectable in the urine of mice with cisplatin-induced nephrotoxicity, again preceding the appearance of N-acetyl-beta-D-glucosaminidase and beta2-microglobulin. The results indicate that NGAL may represent an early, sensitive, noninvasive urinary biomarker for ischemic and nephrotoxic renal injury.

Nuclear phosphatases and the proteasome in suppression of STAT1 activity in hepatocytes.

IFN-gamma induction of C1 inhibitor (C1INH) is mediated by an IFN-gamma-activated sequence (GAS), via binding of signal transducer and activator of transcription 1 (STAT1). These studies focused on the factors responsible for down-regulation of nuclear STAT1 in hepatocytes, the primary site of synthesis of C1INH. The activity of nuclear STAT1 following stimulation with IFN-gamma was sustained with the phosphatase inhibitor, pervanadate, or the proteasome inhibitor, lactacystin. Pervanadate prolonged STAT1 activation and blocked the inactivation of nuclear STAT1. Binding of ubiquitin to phosphorylated STAT1 was detectable in cells treated with lactacystin. Staurosporine only moderately decreased the prolongation of nuclear phosphorylated STAT1 after pretreatment with pervanadate or lactacystin. An antisense mitogen-activated protein kinase phosphatase (MKP-1) oligonucleotide prolonged the accumulation of phosphorylated STAT1. These data are consistent with the hypothesis that down-regulation of IFN-gamma-mediated nuclear STAT1 binding in hepatocytes involves both dephosphorylation by MKP-1 and degradation via proteolysis by the ubiquitin-dependent proteasome pathway.

Human PRMT5 expression is enhanced during in vitro tubule formation and after in vivo ischemic injury in renal epithelial cells.

Background: The interactions between cells and the extracellular matrix (ECM) are important in the regulation of cell growth and differentiation. Cells cultured in ECM differentiate and develop tubular structures. The kidney has the ability to partially recover function after an ischemic insult through repairing its tubular epithelium. The factors that contribute to tubule formation in vitro may mediate tubule regeneration in the recovery stage of acute tubular necrosis. Methods: RNA purified from cells grown on plastic, on Matrigel and in Matrigel matrix were subjected to differential display analysis to identify the transcripts that are differentially expressed during in vitro tubulogenesis. Results: Protein arginine methyltransferase 5 (PRMT5) expression increased in renal epithelial cells undergoing tubule formation. PRMT5 expression is developmentally regulated and ubiquitously expressed in a variety of adult tissues. We also demonstrated that expression of PRMT5 is enhanced in the renal tubular epithelium of animals subjected to ischemic reperfusion injury (IRI). Conclusion: The role of PRMT5 in the regulation of mitosis, its induction in renal epithelial cells undergoing tubule formation in vitro and its expression in the tubules of the kidneys subjected to IRI suggest that it functions in the regulation of cell growth and differentiation during tubule formation and regeneration.

Normal transcription of the C1 inhibitor gene is dependent upon a polypurine-polypyrimidine region within the promoter.

Analysis of the transcriptional activity of C1 inhibitor (C1INH) promoter reporter constructs with mutations in the R⋅Y region indicate that triplex formation by this region is not a predictor of transcriptional activity and that normal promoter function depends on the interaction of trans acting factors with specific elements within this region. Electrophoretic mobility shift assay (EMSA) of Hep3B nuclear extracts using the wild type promoter probe (nucleotides −98 to −9) yielded four major bands. Incubation of the same extracts with probes lacking the HNF-1 site resulted in the disappearance of one band. Supershift assays indicate that HNF-1α is the only previously identified protein that is present in the EMSA bands. Southwestern blot analysis detected four bands (Mrs ∼130, 75, 65 and 20 kDa). These data suggest that the −98 to −9 region of the C1INH promoter interacts with at least four proteins, one of which is HNF-1α.

Characterization of the Promoter of C1inh Gene • 47

C1inh is a serine protease inhibitor that plays a pivotal role in the regulation of the complement and contact activation pathways. Partial deficiency of C1inh leads to the development of hereditary angioedema which afflicts ≈ 1 in 50,000 people. The C1inh gene is made up of 8 exons and is mapped to human chromosome 11. It is a TATA-less initiator (Inr) driven gene. In the present studies, the effect of various components of the C1inh promoter on its ability to direct transcription were examined. Mutations in the Inr element led to a drop in the transcriptional activity of the reporter gene. Replacement of the Inr with a TATA-box at -30 or addition of a TATA-box at -30 region led to enhanced transcriptional activity. The role of a H-DNA/Core sequence spanning nucleotides -49 to -17 was also examined. The results indicate that this region is capable of down regulating the activity of the promoter in the context of as yet unidentified upstream elements (probably a SP-1 or HNF-1 element). These results indicate that the C1inh promoter is initiator driven and that it is further controlled by an upstream H-DNA region which down regulates the activity of other upstream enhancer elements.