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Background: Understanding how sex influences the pathophysiology of cardiovascular and renal disease may enhance precision medicine approaches that account for sex specific biologic differences in these diseases. Nevertheless, characterization of preclinical models often fail to account for the role of sex on important pathophysiologic mechanisms. The renin-angiotensin-aldosterone system (RAAS) plays a central role in the development and progression of cardiovascular and renal disease. In addition to the classic RAAS pathway, an alternative RAAS pathway produces angiotensins (e.g. Ang [1-7]) that can counteract classical RAAS hormones such as Angiotensin (Ang) II. We hypothesized that RAAS profiling in a high-salt fed Dahl-S rat model would reveal significant sex differences in both classic and alternative RAAS pathways. Methods/Results: Ten week-old male and female Dahl-S rats were fed either 0.2% salt normal chow (SS-NC), or 8% high-salt diet (SS-HS) for eight weeks (n=6-8/group). Equilibrium RAAS profiling via LC-MS/MS was performed in lithium heparin plasma samples. In response to high-salt diet, both male and female SS-HS rats had significant (p Conclusion: High-salt diet induces significant sex-dependent differences in both classic and alternative RAAS pathways of Dahl-S rats and may provide important insight into the sex-dependent differences of cardiovascular and renal injury seen in this model.

Objective: Cardiotonic steroids (CTS) are known ligands of the Na+/K+-ATPase (NKA) and chronic elevations in volume expanded conditions such as hypertension and chronic kidney disease (CKD) can mediate a pro-inflammatory and pro-fibrotic phenotype. Paraoxonase1 (PON1) is a lactonase enzyme that circulates bound to HDL and possesses antioxidant, anti-inflammatory and anti-atherogenic properties. PON1 can hydrolyze CTS to inactive open-ring forms making them incapable of stimulating NKA and initiating pro-inflammatory signaling cascades. Diminished PON1 activity results in detrimental clinical outcomes in the context of CKD but whether reduced PON1 activity is mechanistically linked to adverse cardiovascular events in CKD remains unknown. We hypothesized that PON-1 can attenuate progression of cardiac inflammation in CKD via modulating the pathogenic pathways induced by CTS signaling using a well characterized Dahl salt-sensitive rat model of hypertensive renal disease and elevated CTS. Methods: Dahl salt-sensitive wild type ( SS-WT ), PON1 knockout ( SS-PON1KO ), and PON1 knockout rats treated with 3E9 anti-CTS antibody (3E9 mAb,50μg/kg i.p. every week for a total of 4 injections) were fed a high salt (8%) diet for five weeks to induce hypertensive renal disease and elevate CTS levels. H&E staining was performed on hearts to analyze immune cell infiltration. Real-time PCR (RT-PCR) analysis was performed for markers of inflammation (IL6, IL1β, and CCL2), hypertrophy (Myh7, NPPA, and Slc8a), and fibrosis (Timp1). Results: RT-PCR analysis revealed significantly increased (≥1.5 fold) expression of cardiac inflammatory (pSS-PON1 KO compared to SS-WT rats after high salt feeding. Treatment of SS-PON1 KO rats with 3E9 mAb significantly decreased (≥ 2.5 fold) expression of Timp1, IL6, Ccl2, IL1β, NPPA, Myh7 and Slc8a (all pSS-PON1KO rats treated with 3E9 mAb compared to SS-PON-1 KO rats (3.5±0.5 VS1.813±0.4094,p Conclusion: Our findings suggest that PON-1 via its counter-regulatory mechanism of the CTS signaling axis exhibits a cardioprotective role in chronic kidney disease.

Background: Paraoxonase 3 (Pon3), is one of the three isoforms of the paraoxonase gene family. While Pon1 and Pon2 are widely studied, there is a paucity of knowledge regarding Pon3. Pon3 is synthesized in the liver and can circulate bound to high-density lipoproteins. There is significant expression in the kidney also. Pon3 has the ability to metabolize eicosanoids, which can act as signaling molecules and have known roles in the pathophysiology of some renal diseases. Decreased Pon activity is associated with elevated levels of eicosanoid metabolites and adverse clinical outcomes. We tested the hypothesis that targeted disruption of Pon3 results in elevated levels of pro-inflammatory eicosanoids and progression of renal injury. Methods/ Results: Ten week old male Dahl salt-sensitive (SS rats) and Pon3 mutant rats (SS Pon3 KO) were maintained on 8% high salt diet for eight weeks, to initiate salt-sensitive hypertensive renal disease. Previously we observed that SS Pon3 KO rats on eight weeks high salt diet demonstrated significantly increased phenotypic renal injury and mortality. In the current study, we noted that SS Pon3 KO had significantly decreased (pPon3 KO and SS wild type rats at the end of eight weeks of high salt diet. We found that hydroxyl fatty acids 5-HEPE and 5-HETE (5-lipoxygenase dependent arachidonic acid metabolites) were significantly (pPon3 KO compared to SS wild type rats. In addition to being mediators of inflammation, these metabolites are associated with renal cell injury and death. Furthermore, prostaglandin 6-keto-PGF 1α , which has known links to renal inflammation, was significantly (pPon3 KO compared to SS wild type rats. Conclusion: These findings suggest that targeted deletion of Pon3 increases pro-inflammatory eicosanoids (5-HETE and 5-HEPE) and prostaglandins (6-keto-PGF 1α ), as well as increases renal damage independent of blood pressure.

Background Renal artery stenosis is a common cause of renal ischemia, contributing to the development of chronic kidney disease. To investigate the role of local CD 40 expression in renal artery stenosis, Goldblatt 2‐kidney 1‐clip surgery was performed on hypertensive Dahl salt‐sensitive rats (S rats) and genetically modified S rats in which CD 40 function is abolished ( Cd40 mutant ). Methods and Results Four weeks following the 2‐kidney 1‐clip procedure, Cd40 mutant rats demonstrated significantly reduced blood pressure and renal fibrosis in the ischemic kidneys compared with S rat controls. Similarly, disruption of Cd40 resulted in reduced 24‐hour urinary protein excretion in Cd40 mutant rats versus S rat controls (46.2±1.9 versus 118.4±5.3 mg/24 h; P Cd40 mutant rats versus S rat controls ( P Cd40 mutant rats demonstrated a significant decrease in gene expression of the profibrotic mediator, plasminogen activator inhibitor‐1 ( P P P P PCR assay. Reciprocal renal transplantation documented that CD 40 exclusively expressed in the kidney contributes to ischemia‐induced renal fibrosis. Furthermore, human CD 40‐knockout proximal tubule epithelial cells suggested that suppression of CD 40 signaling significantly inhibited expression of proinflammatory and ‐fibrotic genes. Conclusions Taken together, our data suggest that activation of CD 40 induces a significant proinflammatory and ‐fibrotic response and represents an attractive therapeutic target for treatment of ischemic renal disease.

The burden of cardiovascular disease and death in chronic kidney disease (CKD) outpaces that of the other diseases and is not adequately described by traditional risk factors alone. Diminished activity of paraoxonase (PON)-1 is associated with increased oxidant stress, a common feature underlying the pathogenesis of CKD. We aimed to assess the prognostic value of circulating PON-1 protein and PON lactonase activity on adverse clinical outcomes across various stages and etiologies of CKD. Circulating PON-1 protein levels and PON lactonase activity were measured simultaneously in patients with CKD as well as a cohort of apparently healthy non-CKD subjects. Both circulating PON-1 protein levels and PON lactonase activity were significantly lower in CKD patients compared to the non-CKD subjects. Similarly, across all stages of CKD, circulating PON-1 protein and PON lactonase activity were significantly lower in patients with CKD compared to the non-CKD controls. Circulating PON lactonase activity, but not protein levels, predicted future adverse clinical outcomes, even after adjustment for traditional risk factors. The combination of lower circulating protein levels and higher activity within the CKD subjects were associated with the best survival outcomes. These findings demonstrate that diminished circulating PON lactonase activity, but not protein levels, predicts higher risk of future adverse clinical outcomes in patients with CKD.

Microcystins are potent hepatotoxins that have become a global health concern in recent years. Their actions in at-risk populations with pre-existing liver disease is unknown. We tested the hypothesis that the No Observed Adverse Effect Level (NOAEL) of Microcystin-LR (MC-LR) established in healthy mice would cause exacerbation of hepatic injury in a murine model (Leprdb/J) of Non-alcoholic Fatty Liver Disease (NAFLD). Ten-week-old male Leprdb/J mice were gavaged with 50 &mu, g/kg, 100 &mu, g/kg MC-LR or vehicle every 48 h for 4 weeks (n = 15&ndash, 17 mice/group). Early mortality was observed in both the 50 &mu, g/kg (1/17, 6%), and 100 &mu, g/kg (3/17, 18%) MC-LR exposed mice. MC-LR exposure resulted in significant increases in circulating alkaline phosphatase levels, and histopathological markers of hepatic injury as well as significant upregulation of genes associated with hepatotoxicity, necrosis, nongenotoxic hepatocarcinogenicity and oxidative stress response. In addition, we observed exposure dependent changes in protein phosphorylation sites in pathways involved in inflammation, immune function, and response to oxidative stress. These results demonstrate that exposure to MC-LR at levels that are below the NOAEL established in healthy animals results in significant exacerbation of hepatic injury that is accompanied by genetic and phosphoproteomic dysregulation in key signaling pathways in the livers of NAFLD mice.