Novel Model of Oxalate Diet-Induced Chronic Kidney Disease in Dahl-Salt-Sensitive Rats.

Diet-induced models of chronic kidney disease (CKD) offer several advantages, including clinical relevance and animal welfare, compared with surgical models. Oxalate is a plant-based, terminal toxic metabolite that is eliminated by the kidneys through glomerular filtration and tubular secretion. An increased load of dietary oxalate leads to supersaturation, calcium oxalate crystal formation, renal tubular obstruction, and eventually CKD. Dahl-Salt-Sensitive (SS) rats are a common strain used to study hypertensive renal disease; however, the characterization of other diet-induced models on this background would allow for comparative studies of CKD within the same strain. In the present study, we hypothesized that SS rats on a low-salt, oxalate rich diet would have increased renal injury and serve as novel, clinically relevant and reproducible CKD rat models. Ten-week-old male SS rats were fed either 0.2% salt normal chow (SS-NC) or a 0.2% salt diet containing 0.67% sodium oxalate (SS-OX) for five weeks.Real-time PCR demonstrated an increased expression of inflammatory marker interleukin-6 (IL-6) (p < 0.0001) and fibrotic marker Timp-1 metalloproteinase (p < 0.0001) in the renal cortex of SS-OX rat kidneys compared with SS-NC. The immunohistochemistry of kidney tissue demonstrated an increase in CD-68 levels, a marker of macrophage infiltration in SS-OX rats (p < 0.001). In addition, SS-OX rats displayed increased 24 h urinary protein excretion (UPE) (p < 0.01) as well as significant elevations in plasma Cystatin C (p < 0.01). Furthermore, the oxalate diet induced hypertension (p < 0.05). A renin-angiotensin-aldosterone system (RAAS) profiling (via liquid chromatography-mass spectrometry; LC-MS) in the SS-OX plasma showed significant (p < 0.05) increases in multiple RAAS metabolites including angiotensin (1-5), angiotensin (1-7), and aldosterone. The oxalate diet induces significant renal inflammation, fibrosis, and renal dysfunction as well as RAAS activation and hypertension in SS rats compared with a normal chow diet. This study introduces a novel diet-induced model to study hypertension and CKD that is more clinically translatable and reproducible than the currently available models.

Paraoxonases at the Heart of Neurological Disorders.

Paraoxonase enzymes serve as an important physiological redox system that participates in the protection against cellular injury caused by oxidative stress. The PON enzymes family consists of three members (PON-1, PON-2, and PON-3) that share a similar structure and location as a cluster on human chromosome 7. These enzymes exhibit anti-inflammatory and antioxidant properties with well-described roles in preventing cardiovascular disease. Perturbations in PON enzyme levels and their activity have also been linked with the development and progression of many neurological disorders and neurodegenerative diseases. The current review summarizes the available evidence on the role of PONs in these diseases and their ability to modify risk factors for neurological disorders. We present the current findings on the role of PONs in Alzheimer's disease, Parkinson's disease, and other neurodegenerative and neurological diseases.

Cardioprotective Role for Paraoxonase-1 in Chronic Kidney Disease.

Paraoxonase-1 (PON-1) is a hydrolytic enzyme associated with HDL, contributing to its anti-inflammatory, antioxidant, and anti-atherogenic properties. Deficiencies in PON-1 activity result in oxidative stress and detrimental clinical outcomes in the context of chronic kidney disease (CKD). However, it is unclear if a decrease in PON-1 activity is mechanistically linked to adverse cardiovascular events in CKD. We investigated the hypothesis that PON-1 is cardioprotective in a Dahl salt-sensitive model of hypertensive renal disease. Experiments were performed on control Dahl salt-sensitive rats (SSMcwi, hereafter designated SS-WT rats) and mutant PON-1 rats (SS-Pon1em1Mcwi, hereafter designated SS-PON-1 KO rats) generated using CRISPR gene editing technology. Age-matched 10-week-old SS and SS-PON-1 KO male rats were maintained on high-salt diets (8% NaCl) for five weeks to induce hypertensive renal disease. Echocardiography showed that SS-PON-1 KO rats but not SS-WT rats developed compensated left ventricular hypertrophy after only 4 weeks on the high-salt diet. RT-PCR analysis demonstrated a significant increase in the expression of genes linked to cardiac hypertrophy, inflammation, and fibrosis, as well as a significant decrease in genes essential to left ventricular function in SS-PON-1 KO rats compared to SS-WT rats. A histological examination also revealed a significant increase in cardiac fibrosis and immune cell infiltration in SS-PON-1 KO rats, consistent with their cardiac hypertrophy phenotype. Our data suggest that a loss of PON-1 in the salt-sensitive hypertensive model of CKD leads to increased cardiac inflammation and fibrosis as well as a molecular and functional cardiac phenotype consistent with compensated left ventricular hypertrophy.

Microcystin-LR aerosol induces inflammatory responses in healthy human primary airway epithelium.

Harmful algal blooms plague bodies of freshwater globally. These blooms are often composed of outgrowths of cyanobacteria capable of producing the heptapeptide Microcystin-LR (MC-LR) which is a well-known hepatotoxin. Recently, MC-LR has been detected in aerosols generated from lake water. However, the risk for human health effects due to MC-LR inhalation exposure have not been extensively investigated. In this study, we exposed a fully differentiated 3D human airway epithelium derived from 14 healthy donors to MC-LR-containing aerosol once a day for 3 days. Concentrations of MC-LR ranged from 100 pM to 1 µM. Although there were little to no detrimental alterations in measures of the airway epithelial function (i.e. cell survival, tissue integrity, mucociliary clearance, or cilia beating frequency), a distinct shift in the transcriptional activity was found. Genes related to inflammation were found to be upregulated such as C-C motif chemokine 5 (CCL5; log2FC = 0.57, p = 0.03) and C-C chemokine receptor type 7 (CCR7; log2FC = 0.84, p = 0.03). Functionally, conditioned media from MC-LR exposed airway epithelium was also found to have significant chemo-attractive properties for primary human neutrophils. Additionally, increases were found in the concentration of secreted chemokine proteins in the conditioned media such as CCL1 (log2FC = 5.07, p = 0.0001) and CCL5 (log2FC = 1.02, p = 0.046). These results suggest that MC-LR exposure to the human airway epithelium is capable of inducing an inflammatory response that may potentiate acute or chronic disease.

Antioxidant Therapy Significantly Attenuates Hepatotoxicity following Low Dose Exposure to Microcystin-LR in a Murine Model of Diet-Induced Non-Alcoholic Fatty Liver Disease.

We have previously shown in a murine model of Non-alcoholic Fatty Liver Disease (NAFLD) that chronic, low-dose exposure to the Harmful Algal Bloom cyanotoxin microcystin-LR (MC-LR), resulted in significant hepatotoxicity including micro-vesicular lipid accumulation, impaired toxin metabolism as well as dysregulation of the key signaling pathways involved in inflammation, immune response and oxidative stress. On this background we hypothesized that augmentation of hepatic drug metabolism pathways with targeted antioxidant therapies would improve MC-LR metabolism and reduce hepatic injury in NAFLD mice exposed to MC-LR. We chose N-acetylcysteine (NAC, 40 mM), a known antioxidant that augments the glutathione detoxification pathway and a novel peptide (pNaKtide, 25 mg/kg) which is targeted to interrupting a specific Src-kinase mediated pro-oxidant amplification mechanism. Histological analysis showed significant increase in hepatic inflammation in NAFLD mice exposed to MC-LR which was attenuated on treatment with both NAC and pNaKtide (both p ≤ 0.05). Oxidative stress, as measured by 8-OHDG levels in urine and protein carbonylation in liver sections, was also significantly downregulated upon treatment with both antioxidants after MC-LR exposure. Genetic analysis of key drug transporters including Abcb1a, Phase I enzyme-Cyp3a11 and Phase II metabolic enzymes-Pkm (Pyruvate kinase, muscle), Pklr (Pyruvate kinase, liver, and red blood cell) and Gad1 (Glutamic acid decarboxylase) was significantly altered by MC-LR exposure as compared to the non-exposed control group (all p ≤ 0.05). These changes were significantly attenuated with both pNaKtide and NAC treatment. These results suggest that MC-LR metabolism and detoxification is significantly impaired in the setting of NAFLD, and that these pathways can potentially be reversed with targeted antioxidant treatment.

Dirty Jobs: Macrophages at the Heart of Cardiovascular Disease.

Cardiovascular disease (CVD) is one of the greatest public health concerns and is the leading cause of morbidity and mortality in the United States and worldwide. CVD is a broad yet complex term referring to numerous heart and vascular conditions, all with varying pathologies. Macrophages are one of the key factors in the development of these conditions. Macrophages play diverse roles in the maintenance of cardiovascular homeostasis, and an imbalance of these mechanisms contributes to the development of CVD. In the current review, we provide an in-depth analysis of the diversity of macrophages, their roles in maintaining tissue homeostasis within the heart and vasculature, and the mechanisms through which imbalances in homeostasis may lead to CVD. Through this review, we aim to highlight the potential importance of macrophages in the identification of preventative, diagnostic, and therapeutic strategies for patients with CVD.

Evaluating the Roles of Different Types of Laser Therapy in Becker's Nevus Treatment.

Becker's nevus (BN) is a cutaneous hamartoma of benign nature that develops through adolescence and affects mostly young men. The nevus is usually located unilaterally and is characterized by hypertrichosis and hyperpigmentation. Despite recent advances in treatment modalities, no effective treatment has been established for BN hyperpigmentation. We sought to assess the efficacy and safety of fractional Erbium: YAG 2940 nm and Q-switched Nd: YAG 1064 nm lasers in the treatment of BN hyperpigmentation. Twenty-three patients with BN were included in a prospective, randomized-controlled, observer-blinded, split-lesion comparative technique trial. In each patient, two similar square test regions were randomized to either be treated with a fractional Erbium: YAG 2940 nm laser or with a Q-switched Nd: YAG 1064 nm laser. Each patient was treated with three sessions at six-week intervals. At the follow-up, clearance of hyperpigmentation was assessed by physician global assessment, visual analogue scale, grade of improvement, patient global assessment, and patient satisfaction. Regions treated with the fractional Erbium: YAG 2940 nm laser demonstrated significantly better improvement compared to ones treated with the Q-switched Nd: YAG 1064 nm (p-value = 0.001) laser. Adverse effects such as repigmentation and hypertrophic scarring were not reported during the follow-up period. The outcomes were cosmetically acceptable with overall high satisfaction among the included patients. Our data suggest a superior role for the fractional Erbium: YAG (2940 nm) laser in the treatment of BN hyperpigmentation compared to the Q-switched Nd: YAG (1064 nm) laser, along with being a safer method and having no reported side effects.

Paraoxonase-1 Regulation of Renal Inflammation and Fibrosis in Chronic Kidney Disease.

Papraoxonase-1 (PON1) is a hydrolytic lactonase enzyme that is synthesized in the liver and circulates attached to high-density lipoproteins (HDL). Clinical studies have demonstrated an association between diminished PON-1 and the progression of chronic kidney disease (CKD). However, whether decreased PON-1 is mechanistically linked to renal injury is unknown. We tested the hypothesis that the absence of PON-1 is mechanistically linked to the progression of renal inflammation and injury in CKD. Experiments were performed on control Dahl salt-sensitive rats (SSMcwi, hereafter designated SS rats) and Pon1 knock-out rats (designated SS-Pon1em1Mcwi, hereafter designated SS-PON-1 KO rats) generated by injecting a CRISPR targeting the sequence into SSMcwi rat embryos. The resulting mutation is a 7 bp frameshift insertion in exon 4 of the PON-1 gene. First, to examine the renal protective role of PON-1 in settings of CKD, ten-week-old, age-matched male rats were maintained on a high-salt diet (8% NaCl) for up to 5 weeks to initiate the salt-sensitive hypertensive renal disease characteristic of this model. We found that SS-PON-1 KO rats demonstrated several hallmarks of increased renal injury vs. SS rats including increased renal fibrosis, sclerosis, and tubular injury. SS-PON-1 KO also demonstrated increased recruitment of immune cells in the renal interstitium, as well as increased expression of inflammatory genes compared to SS rats (all p < 0.05). SS-PON-1 KO rats also showed a significant (p < 0.05) decline in renal function and increased renal oxidative stress compared to SS rats, despite no differences in blood pressure between the two groups. These findings suggest a new role for PON-1 in regulating renal inflammation and fibrosis in the setting of chronic renal disease independent of blood pressure.

A PON for All Seasons: Comparing Paraoxonase Enzyme Substrates, Activity and Action including the Role of PON3 in Health and Disease.

Paraoxonases (PONs) are a family of hydrolytic enzymes consisting of three members, PON1, PON2, and PON3, located on human chromosome 7. Identifying the physiological substrates of these enzymes is necessary for the elucidation of their biological roles and to establish their applications in the biomedical field. PON substrates are classified as organophosphates, aryl esters, and lactones based on their structure. While the established native physiological activity of PONs is its lactonase activity, the enzymes' exact physiological substrates continue to be elucidated. All three PONs have antioxidant potential and play an important anti-atherosclerotic role in several diseases including cardiovascular diseases. PON3 is the last member of the family to be discovered and is also the least studied of the three genes. Unlike the other isoforms that have been reviewed extensively, there is a paucity of knowledge regarding PON3. Thus, the current review focuses on PON3 and summarizes the PON substrates, specific activities, kinetic parameters, and their association with cardiovascular as well as other diseases such as HIV and cancer.

Microcystin-LR (MC-LR) Triggers Inflammatory Responses in Macrophages.

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.

Renal Fibrosis Is Significantly Attenuated Following Targeted Disruption of Cd40 in Experimental Renal Ischemia.

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 CD40 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 CD40 function is abolished (Cd40mutant). Methods and Results Four weeks following the 2-kidney 1-clip procedure, Cd40mutant 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 Cd40mutant rats versus S rat controls (46.2±1.9 versus 118.4±5.3 mg/24 h; P<0.01), as well as protection from oxidative stress, as indicated by increased paraoxonase activity in Cd40mutant rats versus S rat controls (P<0.01). Ischemic kidneys from Cd40mutant rats demonstrated a significant decrease in gene expression of the profibrotic mediator, plasminogen activator inhibitor-1 (P<0.05), and the proinflammatory mediators, C-C motif chemokine ligand 19 (P<0.01), C-X-C Motif Chemokine Ligand 9 (P<0.01), and interleukin-6 receptor (P<0.001), compared with S rat ischemic kidneys, as assessed by quantitative PCR assay. Reciprocal renal transplantation documented that CD40 exclusively expressed in the kidney contributes to ischemia-induced renal fibrosis. Furthermore, human CD40-knockout proximal tubule epithelial cells suggested that suppression of CD40 signaling significantly inhibited expression of proinflammatory and -fibrotic genes. Conclusions Taken together, our data suggest that activation of CD40 induces a significant proinflammatory and -fibrotic response and represents an attractive therapeutic target for treatment of ischemic renal disease.

Epithelial and Endothelial Adhesion of Immune Cells Is Enhanced by Cardiotonic Steroid Signaling Through Na+/K+-ATPase-α-1.

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.

Chronic Low Dose Oral Exposure to Microcystin-LR Exacerbates Hepatic Injury in a Murine Model of Non-Alcoholic Fatty Liver Disease.

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.

Exposure to the Harmful Algal Bloom (HAB) Toxin Microcystin-LR (MC-LR) Prolongs and Increases Severity of Dextran Sulfate Sodium (DSS)-Induced Colitis.

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-1ß, 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.

Proinflammatory Effects of Cardiotonic Steroids Mediated by NKA α-1 (Na+/K+-ATPase α-1)/Src Complex in Renal Epithelial Cells and Immune Cells.

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<0.05), the effects were abolished in NKA α-1+/- macrophages or by pretreatment with pNaKtide or the Src inhibitor PP2 ( P<0.01). In a series of in vivo studies, we found that 5/6th partial nephrectomy induced significantly less oxidative stress in the remnant kidney of NKA α-1+/- 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.

Cardiotonic Steroids and the Sodium Trade Balance: New Insights into Trade-Off Mechanisms Mediated by the Na⁺/K⁺-ATPase.

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.

Telocinobufagin, a Novel Cardiotonic Steroid, Promotes Renal Fibrosis via Na⁺/K⁺-ATPase Profibrotic Signaling Pathways.

Cardiotonic steroids (CTS) are Na⁺/K⁺-ATPase (NKA) ligands that are elevated in volume-expanded states and associated with cardiac and renal dysfunction in both clinical and experimental settings. We test the hypothesis that the CTS telocinobufagin (TCB) promotes renal dysfunction in a process involving signaling through the NKA α-1 in the following studies. First, we infuse TCB (4 weeks at 0.1 µg/g/day) or a vehicle into mice expressing wild-type (WT) NKA α-1, as well as mice with a genetic reduction (~40%) of NKA α-1 (NKA α-1+/-). Continuous TCB infusion results in increased proteinuria and cystatin C in WT mice which are significantly attenuated in NKA α-1+/- mice (all p < 0.05), despite similar increases in blood pressure. In a series of in vitro experiments, 24-h treatment of HK2 renal proximal tubular cells with TCB results in significant dose-dependent increases in both Collagens 1 and 3 mRNA (2-fold increases at 10 nM, 5-fold increases at 100 nM, p < 0.05). Similar effects are seen in primary human renal mesangial cells. TCB treatment (100 nM) of SYF fibroblasts reconstituted with cSrc results in a 1.5-fold increase in Collagens 1 and 3 mRNA (p < 0.05), as well as increases in both Transforming Growth factor beta (TGFb, 1.5 fold, p < 0.05) and Connective Tissue Growth Factor (CTGF, 2 fold, p < 0.05), while these effects are absent in SYF cells without Src kinase. In a patient study of subjects with chronic kidney disease, TCB is elevated compared to healthy volunteers. These studies suggest that the pro-fibrotic effects of TCB in the kidney are mediated though the NKA-Src kinase signaling pathway and may have relevance to volume-overloaded conditions, such as chronic kidney disease where TCB is elevated.

Na/K-ATPase/src complex mediates regulation of CD40 in renal parenchyma.

Background: Recent studies have highlighted a critical role for CD40 in the pathogenesis of renal injury and fibrosis. However, little is currently understood about the regulation of CD40 in this setting. Methods: We use novel Na/K-ATPase cell lines and inhibitors in order to demonstrate the regulatory function of Na/K-ATPase with regards to CD40 expression and function. We utilize 5/6 partial nephrectomy as well as direct infusion of a Na/K-ATPase ligand to demonstrate this mechanism exists in vivo. Results: We demonstrate that knockdown of the α1 isoform of Na/K-ATPase causes a reduction in CD40 while rescue of the α1 but not the α2 isoform restores CD40 expression in renal epithelial cells. Second, because the major functional difference between α1 and α2 is the ability of α1 to form a functional signaling complex with Src, we examined whether the Na/K-ATPase/Src complex is important for CD40 expression. We show that a gain-of-Src binding α2 mutant restores CD40 expression while loss-of-Src binding α1 reduces CD40 expression. Furthermore, loss of a functional Na/K-ATPase/Src complex also disrupts CD40 signaling. Importantly, we show that use of a specific Na/K-ATPase/Src complex antagonist, pNaKtide, can attenuate cardiotonic steroid (CTS)-induced induction of CD40 expression in vitro. Conclusions: Because the Na/K-ATPase/Src complex is also a key player in the pathogenesis of renal injury and fibrosis, our new findings suggest that Na/K-ATPase and CD40 may comprise a pro-fibrotic feed-forward loop in the kidney and that pharmacological inhibition of this loop may be useful in the treatment of renal fibrosis.