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We were the first to previously report that microcystin-LR (MC-LR) has limited effects within the colons of healthy mice but has toxic effects within colons of mice with pre-existing inflammatory bowel disease. In the current investigation, we aimed to elucidate the mechanism by which MC-LR exacerbates colitis and to identify effective therapeutic targets. Through our current investigation, we report that there is a significantly greater recruitment of macrophages into colonic tissue with pre-existing colitis in the presence of MC-LR than in the absence of MC-LR. This is seen quantitatively through IHC staining and the enumeration of F4/80-positive macrophages and through gene expression analysis for Cd68, Cd11b, and Cd163. Exposure of isolated macrophages to MC-LR was found to directly upregulate macrophage activation markers Tnf and Il1b. Through a high-throughput, unbiased kinase activity profiling strategy, MC-LR-induced phosphorylation events were compared with potential inhibitors, and doramapimod was found to effectively prevent MC-LR-induced inflammatory responses in macrophages.

Cardiotonic steroids (CTSs) are NKA α-1 (Na + /K + -ATPase α-1) ligands that are increased in volume expanded states and associated with cardiac and renal diseases. Although initiation and resolution of inflammation is an important component of cellular injury and repair in renal disease, it is unknown whether CTS activation of NKA α-1 signaling in this setting regulates this inflammatory response. On this background, we hypothesized that CTS signaling through the NKA α-1–Src kinase complex promotes a proinflammatory response in renal epithelial and immune cells. First, we observed that the CTS telocinobufagin activated multiple proinflammatory cytokines/chemokines in renal epithelial cells, and these effects were attenuated after either NKA α-1 knockdown or with a specific inhibitor of the NKA α-1–Src kinase complex (pNaKtide). Similar findings were observed in immune cells, where we demonstrated that while telocinobufagin induced both oxidative burst and enhanced Nuclear factor kappa-light-chain-enhancer of activated B cells activation in macrophages ( P +/− macrophages or by pretreatment with pNaKtide or the Src inhibitor PP2 ( P +/− versus wild-type mice. Similarly, 5/6th partial nephrectomy yielded decreased levels of the urinary oxidative stress marker 8-Oxo-2′-deoxyguanosine in NKA α-1 +/− versus wild-type mice. Finally, we found that in vivo inhibition of the NKA α-1–Src kinase complex with pNaKtide significantly inhibited renal proinflammatory gene expression after 5/6th partial nephrectomy. These findings suggest that the NKA α-1–Src kinase complex plays a central role in regulating the renal inflammatory response induced by elevated CTS both in vitro and in vivo.

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 Recent studies have highlighted a critical role for a group of natriuretic hormones, cardiotonic steroid (CTS), in mediating renal inflammation and fibrosis associated with volume expanded settings, such as chronic kidney disease. Immune cell adhesion is a critical step in the inflammatory response; however, little is currently understood about the potential regulatory role of CTS signaling in this setting. Herein, we tested the hypothesis that CTS signaling through Na+/K+‐ATPase α‐1 (NKA α‐1) enhances immune cell recruitment and adhesion to renal epithelium that ultimately advance renal inflammation. Methods and Results We demonstrate that knockdown of the α‐1 isoform of Na/K‐ATPase causes a reduction in CTS‐induced macrophage infiltration in renal tissue as well reduces the accumulation of immune cells in the peritoneal cavity in vivo. Next, using functional adhesion assay, we demonstrate that CTS‐induced increases in the adhesion of macrophages to renal epithelial cells were significantly diminished after reduction of NKA α‐1 in either macrophages or renal epithelial cells as well after inhibition of NKA α‐1‐Src signaling cascade with a specific peptide inhibitor, pNaKtide in vitro. Finally, CTS‐induced expression of adhesion markers in both endothelial and immune cells was significantly inhibited in an NKA α‐1‐Src signaling dependent manner in vitro. Conclusions These findings suggest that CTS potentiates immune cell migration and adhesion to renal epithelium through an NKA α‐1–dependent mechanism; our new findings suggest that pharmacological inhibition of this feed‐forward loop may be useful in the treatment of renal inflammation associated with renal disease.

In 1972 Neal Bricker presented the “trade-off” hypothesis in which he detailed the role of physiological adaptation processes in mediating some of the pathophysiology associated with declines in renal function. In the late 1990’s Xie and Askari published seminal studies indicating that the Na+/K+-ATPase (NKA) was not only an ion pump, but also a signal transducer that interacts with several signaling partners. Since this discovery, numerous studies from multiple laboratories have shown that the NKA is a central player in mediating some of these long-term “trade-offs” of the physiological adaptation processes which Bricker originally proposed in the 1970’s. In fact, NKA ligands such as cardiotonic steroids (CTS), have been shown to signal through NKA, and consequently been implicated in mediating both adaptive and maladaptive responses to volume overload such as fibrosis and oxidative stress. In this review we will emphasize the role the NKA plays in this “trade-off” with respect to CTS signaling and its implication in inflammation and fibrosis in target organs including the heart, kidney, and vasculature. As inflammation and fibrosis exhibit key roles in the pathogenesis of a number of clinical disorders such as chronic kidney disease, heart failure, atherosclerosis, obesity, preeclampsia, and aging, this review will also highlight the role of newly discovered NKA signaling partners in mediating some of these conditions.