Endoplasmic reticulum (ER) stress is a prominent mechanism of macrophage apoptosis in advanced atherosclerotic lesions. Recent studies from our laboratory showed that advanced atherosclerotic plaques in Apoe(-/-) mice with bone marrow deficiency of the calcium-permeable channel Transient Receptor Potential Canonical 3 (TRPC3) are characterized by reduced areas of necrosis and fewer apoptotic macrophages than animals transplanted with Trpc3(+/+) bone marrow. In vitro, proinflammatory M1 but not anti-inflammatory M2 macrophages derived from Trpc3(-/-)Apoe(-/-) animals exhibited reduced ER stress-induced apoptosis. However, whether this was due to a specific effect of TRPC3 deficiency on macrophage ER stress signaling remained to be determined. In the present work we used polarized macrophages derived from mice with macrophage-specific deficiency of TRPC3 to examine the expression level of ER stress markers and the activation status of some typical mediators of macrophage apoptosis. We found that the reduced susceptibility of TRPC3-deficient M1 macrophages to ER stress-induced apoptosis correlates with an impaired unfolded protein response (UPR), reduced mitochondrion-dependent apoptosis, and reduced activation of the proapoptotic molecules calmodulin-dependent protein kinase II and signal transducer and activator of transcription 1. Notably, none of these pathways was altered in TRPC3-deficient M2 macrophages. These findings show for the first time an obligatory requirement for a member of the TRPC family of cation channels in ER stress-induced apoptosis in macrophages, underscoring a rather selective role of the TRPC3 channel on mechanisms related to the UPR signaling in M1 macrophages.

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.

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 μg/kg, 100 μg/kg MC-LR or vehicle every 48 h for 4 weeks (n = 15–17 mice/group). Early mortality was observed in both the 50 μg/kg (1/17, 6%), and 100 μg/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.

Vascular calcification (VC) is one of the major causes of cardiovascular morbidity and mortality in patients with chronic kidney disease (CKD). VC is a complex process expressing similarity to bone metabolism in onset and progression. VC in CKD is promoted by various factors not limited to hyperphosphatemia, Ca/Pi imbalance, uremic toxins, chronic inflammation, oxidative stress, and activation of multiple signaling pathways in different cell types, including vascular smooth muscle cells (VSMCs), macrophages, and endothelial cells. In the current review, we provide an in-depth analysis of the various kinds of VC, the clinical significance and available therapies, significant contributions from multiple cell types, and the associated cellular and molecular mechanisms for the VC process in the setting of CKD. Thus, we seek to highlight the key factors and cell types driving the pathology of VC in CKD in order to assist in the identification of preventative, diagnostic, and therapeutic strategies for patients burdened with this disease.

Inflammatory Bowel Disease (IBD) represents a collection of gastrointestinal disorders resulting from genetic and environmental factors. Microcystin-leucine arginine (MC-LR) is a toxin produced by cyanobacteria during algal blooms and demonstrates bioaccumulation in the intestinal tract following ingestion. Little is known about the impact of MC-LR ingestion in individuals with IBD. In this study, we sought to investigate MC-LR’s effects in a dextran sulfate sodium (DSS)-induced colitis model. Mice were separated into four groups: (a) water only (control), (b) DSS followed by water (DSS), (c) water followed by MC-LR (MC-LR), and (d) DSS followed by MC-LR (DSS + MC-LR). DSS resulted in weight loss, splenomegaly, and severe colitis marked by transmural acute inflammation, ulceration, shortened colon length, and bloody stools. DSS + MC-LR mice experienced prolonged weight loss and bloody stools, increased ulceration of colonic mucosa, and shorter colon length as compared with DSS mice. DSS + MC-LR also resulted in greater increases in pro-inflammatory transcripts within colonic tissue (TNF-α, IL-1β, CD40, MCP-1) and the pro-fibrotic marker, PAI-1, as compared to DSS-only ingestion. These findings demonstrate that MC-LR exposure not only prolongs, but also worsens the severity of pre-existing colitis, strengthening evidence of MC-LR as an under-recognized environmental toxin in vulnerable populations, such as those with IBD.

In previous work we reported that ApoeKO mice transplanted with bone marrow cells deficient in the Transient Receptor Potential Canonical 3 (TRPC3) channel have reduced necrosis and number of apoptotic macrophages in advanced atherosclerotic plaques. Also, in vitro studies with polarized macrophages derived from mice with macrophage-specific loss of TRPC3 showed that M1, but not M2 macrophages, deficient in Trpc3 are less susceptible to ER stress-induced apoptosis than Trpc3 expressing cells. The questions remained (a) whether the plaque phenotype in transplanted mice resulted from a genuine effect of Trpc3 on macrophages, and (b) whether the reduced necrosis and macrophage apoptosis in plaques of these mice was a manifestation of the selective effect of TRPC3 on apoptosis of M1 macrophages previously observed in vitro. Here, we addressed these questions using Ldlr knockout (Ldlr−/−) mice with macrophage-specific loss of Trpc3 (MacTrpc3−/−/Ldlr−/− → Ldlr−/−). Compared to controls, we observed decreased plaque necrosis and number of apoptotic macrophages in MacTrpc3−/−/Ldlr−/− → Ldlr−/− mice. Immunohistochemical analysis revealed a reduction in apoptotic M1, but not apoptotic M2 macrophages. These findings confirm an effect of TRPC3 on plaque necrosis and support the notion that this is likely a reflection of the reduced susceptibility of Trpc3-deficient M1 macrophages to apoptosis.

Insulin resistance and obesity are associated with reduced gonadotropin-releasing hormone (GnRH) release and infertility. Mice that lack insulin receptors (IRs) throughout development in both neuronal and non-neuronal brain cells are known to exhibit subfertility due to hypogonadotropic hypogonadism. However, attempts to recapitulate this phenotype by targeting specific neurons have failed. To determine whether astrocytic insulin sensing plays a role in the regulation of fertility, we generated mice lacking IRs in astrocytes (astrocyte-specific insulin receptor deletion [IRKO GFAP ] mice). IRKO GFAP males and females showed a delay in balanopreputial separation or vaginal opening and first estrous, respectively. In adulthood, IRKO GFAP female mice also exhibited longer, irregular estrus cycles, decreased pregnancy rates, and reduced litter sizes. IRKO GFAP mice show normal sexual behavior but hypothalamic-pituitary-gonadotropin (HPG) axis dysregulation, likely explaining their low fecundity. Histological examination of testes and ovaries showed impaired spermatogenesis and ovarian follicle maturation. Finally, reduced prostaglandin E synthase 2 (PGES2) levels were found in astrocytes isolated from these mice, suggesting a mechanism for low GnRH/luteinizing hormone (LH) secretion. These findings demonstrate that insulin sensing by astrocytes is indispensable for the function of the reproductive axis. Additional work is needed to elucidate the role of astrocytes in the maturation of hypothalamic reproductive circuits.

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.