These findings demonstrate that a novel sHE inhibitor/PPAR-γ agonist molecule targets multiple risk factors of the metabolic syndrome and is a glucose-lowering agent with a strong ability to treat diabetic complications.

Renal fibrosis, which is a critical pathophysiological event in chronic kidney diseases, is associated with renal epithelial-to-mesenchymal transition (EMT). Epoxyeicosatrienoic acids (EETs) are Cyp epoxygenase arachidonic acid metabolites that demonstrate biological actions that result in kidney protection. Herein, we investigated the ability of 14,15-EET and its synthetic analog, EET-A, to reduce kidney fibrosis induced by unilateral ureter obstruction (UUO). C57/BL6 male mice underwent sham or UUO surgical procedures and were treated with 14,15-EET or EET-A in osmotic pump (i.p.) for 10 days following UUO surgery. UUO mice demonstrated renal fibrosis with an 80% higher kidney-collagen positive area and 70% higher α-smooth muscle actin (SMA) positive renal areas compared to the sham group. As a measure of collagen content, kidney hydroxyproline content was also higher in UUO (6.4 ± 0.5 μg/10 mg) compared to sham group (2.5 ± 0.1 μg/10 mg). Along with marked renal fibrosis, UUO mice had reduced renal expression of EET producing Cyp epoxygenase enzymes. Endogenous 14,15-EET or EET-A demonstrated anti-fibrotic action in UUO by reducing kidney-collagen positive area (50–60%), hydroxyproline content (50%), and renal α-SMA positive area (85%). In UUO mice, renal expression of EMT inducers, Snail1 and ZEB1 were higher compared to sham group. Accordingly, renal epithelial marker E-cadherin expression was reduced and mesenchymal marker expression was elevated in the UUO compared to sham mice. Interestingly, EET-A reduced EMT in UUO mice by deceasing renal Snail1 and ZEB1 expression. EET-A treatment also opposed the decrease in renal E-cadherin expression and markedly reduced several prominent renal mesenchymal/myofibroblast markers in UUO mice. Overall, our results demonstrate that EET-A is a novel anti-fibrotic agent that reduces renal fibrosis by decreasing renal EMT.

Cardiac fibrosis is an underlying cause of diastolic dysfunction, contributing to heart failure. Substance P (SP) activation of the neurokinin-1 receptor (NK-1R) contributes to cardiac fibrosis in hypertension. However, based on in vitro experiments, this does not appear to be via direct activation of cardiac fibroblasts. While numerous cells could mediate the fibrotic effects of SP, herein, we investigate mast cells (MC) as a mechanism mediating the fibrotic actions of SP, since MCs are known to play a role in cardiac fibrosis and respond to SP. Spontaneously hypertensive rats (SHR) were treated with the NK-1R antagonist L732138 (5 mg/kg/d) from 8 to 12 weeks of age. L732138 prevented increased MC maturation of resident immature MCs. NK-1R blockade also prevented increased cardiac MC maturation in angiotensin II-infused mice. MC-deficient mice were used to test the importance of MC NK-1Rs to MC activation. MC-deficient mice administered angiotensin II did not develop fibrosis; MC-deficient mice reconstituted with MCs did develop fibrosis. MC-deficient mice reconstituted with MCs lacking the NK-1R also developed fibrosis, indicating that NK-1Rs are not required for MC activation in this setting. In conclusion, the NK-1R causes MC maturation, however, other stimuli are required to activate MCs to cause fibrosis.

Reduced levels of the sensory nerve neuropeptide substance P (SP) have been reported in the diabetic rat heart, the consequence being a loss of cardioprotection in response to ischemic post-conditioning. We considered whether this loss of SP also predisposes the heart to non-ischemic diabetic cardiomyopathy in the form of fibrosis and hypertrophy. We report that diabetic Leprdb/db mice have reduced serum SP and that administration of exogenous replacement SP ameliorated cardiac fibrosis. Cardiac hypertrophy did not occur in Leprdb/db mice. Cardiac fibroblasts exposed to high glucose converted to a myofibroblast phenotype and produced excess extracellular matrix proteins; this was prevented by the presence of SP in the culture media. Cardiac fibroblasts exposed to high glucose produced increased amounts of the receptor for advanced glycation end products, reactive oxygen species and inflammatory cytokines, all of which were prevented by SP. Cultured macrophages assumed an M1 pro-inflammatory phenotype in response to high glucose as indicated by increased TNF-, CCL2, and IL-6. SP promoted a shift to the reparative M2 macrophage phenotype characterized by arginase-1 and IL-10. Leprdb/db mice showed increased left ventricular M1 phenotype macrophages and an increase in the M1/M2 ratio. Replacement SP in Leprdb/db mice restored a favorable M1 to M2 balance. Together these findings indicate that a loss of SP predisposes the diabetic heart to developing fibrosis. The anti-fibrotic actions of replacement SP involve direct effects on cardiac fibroblasts and macrophages to oppose adverse phenotype changes. This study identifies the potential of replacement SP to treat diabetic cardiomyopathy.

Diabetes is a highly prevalent chronic disease worldwide that represents a global healthcare burden. A devastating type 2 diabetes complication is diabetic nephropathy. We developed a novel dual modulator, PTUPB that concurrently acts as a soluble epoxide hydrolase (sEH) inhibitor as well as a cyclooxyganse‐2 inhibitor (sEHi/COXi) to combat diabetic nephropathy. The objective for the current investigation is to determine if PTUPB can treat diabetic nephropathy in obese ZSF1 rats. PTUPB was compared to an angiotensin converting enzyme inhibitor that is the current standard of care for diabetic nephropathy. Obese ZSF‐1 rats at 16 weeks of age received PTUPB (10mg/kg/d i.p.) or enalapril (10mg/kg/d, p/o.) for 8 weeks. Type 2 diabetes was assessed in obese ZSF1 rats. Renal injury was determined using several biochemical, histological, and immunohistological techniques. Obese ZSF1 rats were diabetic with fasting blood glucose (19±0.4 vs. 4.8±0.1 mmol/l in lean ZSF1) . Neither PTUPB nor enalapril treated diabetes and did not reduce hyperglycemia or HbA1c in obese ZSF1 rats. Obese ZSF1 rats developed diabetic nephropathy with elevated albuminuria (49±6 vs. 1.3±0.2 mg/mg creatinine in lean ZSF1), tubular cast formation (cortex, 14±1 vs. 1.0±0.1%; medulla, 22.4±2 vs. 0.9±0.2%), renal fibrosis (cortex, 11±1 vs. 4.4±0.3%; medulla, 10±1.2 vs. 4.0±0.8%) and glomerular injury (3.4±0.6 vs. 0.6±0.08 a.u.) compared to lean ZSF1 lean (all P<0.05). PTUPB treated diabetic nephropathy by reducing albuminuria (by 50%), renal tubular cast formation (by 60% in cortex; 70% in medulla), renal fibrosis (by 50% in cortex; 36% in medulla), and glomerular injury (by 55%) compared to vehicle. Enalapril treatment reduced diabetic nephropathy in obese ZSF1 rats like PTUPB and reduced the renal injury parameters by 30 to 50% compared to vehicle. Diabetic renal injury in obese ZSF1 rats was accompanied by inflammation with elevated urinary MCP‐1 levels (29±2 vs. 5±0.6 ng/mg creatinine in lean ZSF1 rat, P<0.05) and renal infiltration of CD‐68 cells (95±10 vs. 16±1.2 cell/mm2, P<0.05). PTUPB or enalapril treated renal inflammation and reduced MCP‐1 excretion (PTUPB reduced by 50% and enalapril reduced by 20%) and renal CD‐68 positive cell infiltration (by 60% and 50%, respectively by PTUPB or enalapril). These findings demonstrate that PTUPB is not anti‐diabetic but markedly treat diabetic nephropathy. We also demonstrate that PTUPB has renal actions comparable to enalapril which is currently the standard of care to treat diabetic nephropathy.Support or Funding InformationNational Institute of Health (NIH) grant (DK103616) and Dr. Ralph and Marian Falk Medical Research Trust Bank of America, N.A., Trustee grant to JDI; NIEHS/R01 ES002710 (HAM1400) and NIEHS/Superfund Research Program P42 ES004699 (SUPR?30) to BDHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.