Dual soluble epoxide hydrolase inhibitor/PPAR-γ agonist attenuates renal fibrosis.

Renal fibrosis is a contributor to chronic kidney disease and an important predictor of long-term prognosis. We developed a dual soluble epoxide hydrolase inhibitor-PPAR-γ agonist (sEHi/PPAR-γ), RB394, and investigated its ability to attenuate renal fibrosis in a mouse unilateral ureteral obstruction (UUO) model. RB394 efficacy was compared to an sEH inhibitor (sEHi), a PPAR-γ agonist rosiglitazone (Rosi), or their combination (sEHi + Rosi). All interventional treatments were administrated in drinking water 3 days after UUO induction surgery and continued for 7 days. UUO mice developed renal fibrosis with higher collagen formation and RB394 significantly attenuated fibrosis (P < 0.05). Renal expression of α-smooth muscle actin (α-SMA) was elevated in UUO mice and all treatments except sEHi significantly attenuated renal α-SMA expression. Renal mRNA expression fibrotic and fibrosis regulators were higher in UUO mice and RB394 and sEHi + Rosi treatments attenuated their expression. Renal inflammation was evident in UUO mice with increased infiltration of CD45 and F4/80 positive cells. RB394 and sEHi + Rosi treatments attenuated renal inflammation in UUO mice. UUO mice had renal tubular and vascular injury. Renal tubular and vascular injuries were attenuated to a greater extent by RB394 and sEHi + Rosi than sEHi or Rosi treatment alone. Renal mRNA expression of oxidative stress markers were significantly higher in UUO mice (P < 0.05). RB394 and sEHi + Rosi attenuated expression of oxidative stress markers to a greater extent than other interventional treatments (P < 0.05). These findings demonstrate that RB394 can attenuate renal fibrosis by reducing renal inflammation, oxidative stress, tubular injury, and vascular injury. In conclusion, RB394 demonstrates exciting potential as a therapeutic for renal fibrosis and chronic kidney disease.

Role of the cytochrome P-450/ epoxyeicosatrienoic acids pathway in the pathogenesis of renal dysfunction in cirrhosis.

Background: Hepatorenal syndrome (HRS) is a life-threatening complication of advanced liver cirrhosis that is characterized by hemodynamic alterations in the kidney and other vascular beds. Cytochrome P(CYP)-450 enzymes metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acids. These eicosanoids regulate blood pressure, vascular tone and renal tubular sodium transport under both physiological and pathophysiological states. Methods: Experiments were performed to investigate the role of the CYP system in the pathogenesis of renal dysfunction during cirrhosis. Rats underwent bile duct ligation (BDL) or sham surgery and were studied at 2, 4 and 5 weeks post-surgery. In additional experiments, post-BDL rats were treated with three daily intraperitoneal doses of either the selective epoxygenase inhibitor N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide (MSPPOH) or a vehicle, starting on Day 22 after surgery. Results: BDL led to progressive renal dysfunction that was associated with reduced renal cortical perfusion but without any overt histologic changes, consistent with HRS. CYP isoform enzyme expression was significantly altered in BDL rats. In the kidney, CYP2C23 expression was upregulated at both the mRNA and protein levels in BDL rats, while CYP2C11 was downregulated. Histologically, the changes in CYP2C23 and CYP2C11 expression were localized to the renal tubules. EET production was increased in the kidneys of BDL rats as assessed by urinary eicosanoid levels. Finally, treatment with the selective epoxygenase inhibitor MSPPOH significantly reduced renal function and renal cortical perfusion in BDL rats, suggesting a homeostatic role for epoxygenase-derived eicosanoids. Conclusions: The CYP/EET pathway might represent a novel therapeutic target for modulating renal dysfunction in advanced cirrhosis.

The epoxyeicosatrienoic acid analog PVPA ameliorates cyclosporine-induced hypertension and renal injury in rats.

The introduction of calcineurin inhibitors (CNI) into clinical practice in the late 1970s transformed organ transplantation and led to significant improvement in acute rejection episodes. However, despite their significant clinical utility, the use of these agents is hampered by the development of hypertension and nephrotoxicity, which ultimately lead to end-stage kidney disease and overt cardiovascular outcomes. There are currently no effective agents to treat or prevent these complications. Importantly, CNI-free immunosuppressive regimens lack the overall efficacy of CNI-based treatments and put patients at risk of allograft rejection. Cytochrome P-450 epoxygenase metabolites of arachidonic acid, epoxyeicosatrienoic acids (EETs), have potent vasodilator and antihypertensive properties in addition to many cytoprotective effects, but their effects on CNI-induced nephrotoxicity have not been explored. Here, we show that PVPA, a novel, orally active analog of 14,15-EET, effectively prevents the development of hypertension and ameliorates kidney injury in cyclosporine-treated rats. PVPA treatment reduced proteinuria and renal dysfunction induced by cyclosporine. PVPA inhibited inflammatory cell infiltration into the kidney and decreased renal fibrosis. PVPA also reduced tubular epithelial cell apoptosis, attenuated the generation of reactive oxygen species, and modulated the unfolded protein response that is associated with endoplasmic reticulum stress. Consistent with the in vivo data, PVPA attenuated cyclosporine-induced apoptosis of NRK-52E cells in vitro. These data indicate that the cytochrome P-450/EET system offers a novel therapeutic strategy to treat or prevent CNI-induced nephrotoxicity.

Combining patient proteomics and in vitro cardiomyocyte phenotype testing to identify potential mediators of heart failure with preserved ejection fraction.

BACKGROUND: Heart failure with ejection fraction (HFpEF) is a syndrome resulting from several co-morbidities in which specific mediators are unknown. The platelet proteome responds to disease processes. We hypothesize that the platelet proteome will change composition in patients with HFpEF and may uncover mediators of the syndrome. METHODS AND RESULTS: Proteomic changes were assessed in platelets from hospitalized subjects with symptoms of HFpEF (n = 9), the same subjects several weeks later without symptoms (n = 7) and control subjects (n = 8). Mass spectrometry identified 6102 proteins with five scans with peptide probabilities of ≥0.85. Of the 6102 proteins, 165 were present only in symptomatic subjects, 78 were only found in outpatient subjects and 157 proteins were unique to the control group. The S100A8 protein was identified consistently in HFpEF samples when compared with controls. We validated the fining that plasma S100A8 levels are increased in subjects with HFpEF (654 ± 391) compared to controls (352 ± 204) in an external cohort (p = 0.002). Recombinant S100A8 had direct effects on the electrophysiological and calcium handling profile in human induced pluripotent stem cell-derived cardiomyocytes. CONCLUSIONS: Platelets may harbor proteins associated with HFpEF. S100A8 is present in the platelets of subjects with HFpEF and increased in the plasma of the same subjects. We further established a bedside-to-bench translational system that can be utilized as a secondary screen to ascertain whether the biomarkers may be an associated finding or causal to the disease process. S100A8 has been linked with other cardiovascular disease such as atherosclerosis and risk for myocardial infarction, stroke, or death. This is the first report on association of S100A8 with HFpEF.

Comprehensive characterization of glioblastoma tumor tissues for biomarker identification using mass spectrometry-based label-free quantitative proteomics.

Cancer is a complex disease; glioblastoma (GBM) is no exception. Short survival, poor prognosis, and very limited treatment options make it imperative to unravel the disease pathophysiology. The critically important identification of proteins that mediate various cellular events during disease is made possible with advancements in mass spectrometry (MS)-based proteomics. The objective of our study is to identify and characterize proteins that are differentially expressed in GBM to better understand their interactions and functions that lead to the disease condition. Further identification of upstream regulators will provide new potential therapeutic targets. We analyzed GBM tumors by SDS-PAGE fractionation with internal DNA markers followed by liquid chromatography-tandem mass spectrometry (MS). Brain tissue specimens obtained for clinical purposes during epilepsy surgeries were used as controls, and the quantification of MS data was performed by label-free spectral counting. The differentially expressed proteins were further characterized by Ingenuity Pathway Analysis (IPA) to identify protein interactions, functions, and upstream regulators. Our study identified several important proteins that are involved in GBM progression. The IPA revealed glioma activation with z score 2.236 during unbiased core analysis. Upstream regulators STAT3 and SP1 were activated and CTNNα was inhibited. We verified overexpression of several proteins by immunoblot to complement the MS data. This work represents an important step towards the identification of GBM biomarkers, which could open avenues to identify therapeutic targets for better treatment of GBM patients. The workflow developed represents a powerful and efficient method to identify biomarkers in GBM.

Comprehensive serum proteomic analysis in early endometrial cancer.

OBJECTIVE: Endometrial cancer is the most common gynecologic cancer and yet much is still unknown about this disease. Our goal was to identify unique biomarkers of disease by performing a comprehensive proteomic analysis of early stage, low-grade endometrial cancer through analysis of serum collected from patients pre- and post-definitive surgery. METHODS: We used mass spectrometry (MS)-based proteomics to identify serum proteins from these patients. Serum samples from women undergoing hysterectomy with bilateral salpingo-oophorectomy for benign reasons served as control samples for the correlative studies. We then correlated our findings with The Cancer Genome Atlas (TCGA) database for additional confirmation. RESULTS: The Ingenuity Pathway Analysis of proteins that were differentially expressed in endometrial cancer showed increased cell survival and decreased organismal death, the most common hallmarks of cancer. We identified over expression of FAM83D (family with sequence similarity 83, member D) in the serum of patients with early stage low-grade endometrial cancer and verified the same in the endometrial cancer cell lines and patient tumors. We also confirmed our hypothesis that FAM83D may serve as a biomarker for endometrial cancer in a cohort of patients with endometrial cancer from The Cancer Genome Atlas (TCGA) project. CONCLUSION: Comprehensive proteomic analysis is a feasible strategy for potential biomarker identification. Using this technique, FAM83D was identified as a candidate biomarker in early endometrial cancer in our patient samples and was not present in benign control samples. FAM83D has been associated with poor clinical outcomes in several human malignancies. SIGNIFICANCE: Our manuscript describes an alternative approach to comprehensive protein analysis in a model pre and post tumor removal for a sample of patients with early endometrial cancer. The model is innovative and the findings of over expression FAM83D in this population of early cancer may be useful in the study of a disease where there are few biomarkers or targetable therapies.

Identification of a ternary protein-complex as a therapeutic target for K-Ras-dependent colon cancer.

A cancer phenotype is driven by several proteins and targeting a cluster of functionally interdependent molecules should be more effective for therapeutic intervention. This is specifically important for Ras-dependent cancer, as mutated (MT) Ras is non-druggable and targeting its interaction with effectors may be essential for therapeutic intervention. Here, we report that a protein-complex activated by the Ras effector p38γ MAPK is a novel therapeutic target for K-Ras-dependent colon cancer. Unbiased proteomic screening and immune-precipitation analyses identified p38γ interaction with heat shock protein 90 (Hsp90) and K-Ras in K-Ras MT, but not wild-type (WT), colon cancer cells, indicating a role of this complex in Ras-dependent growth. Further experiments showed that this complex requires p38γ and Hsp90 activity to maintain MT, but not WT, K-Ras protein expression. Additional studies demonstrated that this complex is activated by p38γ-induced Hsp90 phosphorylation at S595, which is important for MT K-Ras stability and for K-Ras dependent growth. Of most important, pharmacologically inhibition of Hsp90 or p38γ activity disrupts the complex, decreases K-Ras expression, and selectively inhibits the growth of K-Ras MT colon cancer in vitro and in vivo. These results demonstrated that the p38γ-activated ternary complex is a novel therapeutic target for K-Ras-dependent colon cancer.

Bacillus stearothermophilus PcrA monomer is a single-stranded DNA translocase but not a processive helicase in vitro.

Structural studies of the Bacillus stearothermophilus PcrA protein along with biochemical studies of the single-stranded (ss) DNA translocation activity of PcrA monomers have led to the suggestion that a PcrA monomer possesses processive helicase activity in vitro. Yet definitive studies testing whether the PcrA monomer possesses processive helicase activity have not been performed. Here we show, using single turnover kinetic methods, that monomers of PcrA are able to translocate along ssDNA, in the 3' to 5' direction, rapidly and processively, whereas these same monomers display no detectable helicase activity under the same solution conditions in vitro. The PcrA monomer ssDNA translocation activity, although necessary, is not sufficient for processive helicase activity, and thus the translocase and helicase activities of PcrA are separable. These results also suggest that the helicase activity of PcrA needs to be activated either by self-assembly or through interactions with accessory proteins. This same behavior is displayed by both the Escherichia coli Rep and UvrD monomers. Hence, all three of these SF1 enzymes are ssDNA translocases as monomers but do not display processive helicase activity in vitro unless activated. The fact that the translocase and helicase activities are separable suggests that each activity may be used for different functions in vivo.

Autoinhibition of Escherichia coli Rep monomer helicase activity by its 2B subdomain.

DNA helicases catalyze separation of double-helical DNA into its complementary single strands, a process essential for DNA replication, recombination, and repair. The Escherichia coli Rep protein, a superfamily 1 DNA helicase, functions in DNA replication restart and is required for replication of several bacteriophages. Monomers of Rep do not display helicase activity in vitro; in fact, DNA unwinding requires Rep dimerization. Here we show that removal of the 2B subdomain of Rep to form RepDelta2B activates monomer helicase activity, albeit with limited processivity. Although both full length Rep and RepDelta2B monomers can translocate with 3' to 5' directionality along single-stranded DNA, the 2B subdomain inhibits the helicase activity of full length Rep. This suggests an autoregulatory mechanism for Rep helicase, which may apply to other nonhexameric helicases, whereby helicase activity is regulated by the rotational conformational state of the 2B subdomain; formation of a Rep dimer may relieve autoinhibition by altering the 2B subdomain orientation.