APOBEC-1 deletion enhances cisplatin-induced acute kidney injury.

Cisplatin (CP) induces acute kidney injury (AKI) whereby proximal tubules undergo regulated necrosis. Repair is almost complete after a single dose. We now demonstrate a role for Apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1 (Apobec-1) that is prominently expressed at the interface between acute and chronic kidney injury (CKD), in the recovery from AKI. Apobec-1 knockout (KO) mice exhibited greater mortality than in wild type (WT) and more severe AKI in both CP- and unilateral ischemia reperfusion (IR) with nephrectomy. Specifically, plasma creatinine (pCr) 2.6 ± 0.70 mg/dL for KO, n = 10 and 0.16 ± 0.02 for WT, n = 6, p < 0.0001 in CP model and 1.34 ± 0.22 mg/dL vs 0.75 ± 0.06, n = 5, p < 0.05 in IR model. The kidneys of Apobec-1 KO mice showed increased necrosis, increased expression of KIM-1, NGAL, RIPK1, ASCL4 and increased lipid accumulation compared to WT kidneys (p < 0.01). Neutrophils and activated T cells were both increased, while macrophages were reduced in kidneys of Apobec-1 KO animals. Overexpression of Apobec-1 in mouse proximal tubule cells protected against CP-induced cytotoxicity. These findings suggest that Apobec-1 mediates critical pro-survival responses to renal injury and increasing Apobec-1 expression could be an effective strategy to mitigate AKI.

Kidney-Targeted Renalase Agonist Prevents Cisplatin-Induced Chronic Kidney Disease by Inhibiting Regulated Necrosis and Inflammation.

BACKGROUND: Repeated administration of cisplatin causes CKD. In previous studies, we reported that the kidney-secreted survival protein renalase (RNLS) and an agonist peptide protected mice from cisplatin-induced AKI. METHODS: To investigate whether kidney-targeted delivery of RNLS might prevent cisplatin-induced CKD in a mouse model, we achieved specific delivery of a RNLS agonist peptide (RP81) to the renal proximal tubule by encapsulating the peptide in mesoscale nanoparticles (MNPs). We used genetic deletion of RNLS, single-cell RNA sequencing analysis, and Western blotting to determine efficacy and to explore underlying mechanisms. We also measured plasma RNLS in patients with advanced head and neck squamous cell carcinoma receiving their first dose of cisplatin chemotherapy. RESULTS: In mice with CKD induced by cisplatin, we observed an approximate 60% reduction of kidney RNLS; genetic deletion of RNLS was associated with significantly more severe cisplatin-induced CKD. In this severe model of cisplatin-induced CKD, systemic administration of MNP-encapsulated RP81 (RP81-MNP) significantly reduced CKD as assessed by plasma creatinine and histology. It also decreased inflammatory cytokines in plasma and inhibited regulated necrosis in kidney. Single-cell RNA sequencing analyses revealed that RP81-MNP preserved epithelial components of the nephron and the vasculature and suppressed inflammatory macrophages and myofibroblasts. In patients receiving their first dose of cisplatin chemotherapy, plasma RNLS levels trended lower at day 14 post-treatment. CONCLUSIONS: Kidney-targeted delivery of RNLS agonist RP81-MNP protects against cisplatin-induced CKD by decreasing cell death and improving the viability of the renal proximal tubule. These findings suggest that such an approach might mitigate the development of CKD in patients receiving cisplatin cancer chemotherapy.

Inhibition of Endocytosis of Clathrin-Mediated Angiotensin II Receptor Type 1 in Podocytes Augments Glomerular Injury.

BACKGROUND: Inhibition of the renin-angiotensin system remains a cornerstone in reducing proteinuria and progression of kidney failure, effects believed to be the result of reduction in BP and glomerular hyperfiltration. However, studies have yielded conflicting results on whether podocyte-specific angiotensin II (AngII) signaling directly induces podocyte injury. Previous research has found that after AngII stimulation, ß-arrestin-bound angiotensin II receptor type 1 (AT1R) is internalized in a clathrin- and dynamin-dependent manner, and that Dynamin1 and Dynamin2 double-knockout mice exhibit impaired clathrin-mediated endocytosis. METHODS: We used podocyte-specific Dyn double-knockout mice to examine AngII-stimulated AT1R internalization and signaling in primary podocytes and controls. We also examined the in vivo effect of AngII in these double-knockout mice through renin-angiotensin system blockers and through deletion of Agtr1a (which encodes the predominant AT1R isoform expressed in kidney, AT1aR). We tested calcium influx, Rac1 activation, and lamellipodial extension in control and primary podocytes of Dnm double-knockout mice treated with AngII. RESULTS: We confirmed augmented AngII-stimulated AT1R signaling in primary Dnm double-knockout podocytes resulting from arrest of clathrin-coated pit turnover. Genetic ablation of podocyte Agtr1a in Dnm double-knockout mice demonstrated improved albuminuria and kidney function compared with the double-knockout mice. Isolation of podocytes from Dnm double-knockout mice revealed abnormal membrane dynamics, with increased Rac1 activation and lamellipodial extension, which was attenuated in Dnm double-knockout podocytes lacking AT1aR. CONCLUSIONS: Our results indicate that inhibiting aberrant podocyte-associated AT1aR signaling pathways has a protective effect in maintaining the integrity of the glomerular filtration barrier.

Early B Cell Factor 1 (EBF1) Regulates Glomerular Development by Controlling Mesangial Maturation and Consequently COX-2 Expression.

BACKGROUND: We recently showed the transcription factor Early B cell factor 1 (EBF1) is essential for the last stages of metanephric development, and that mice globally deficient in EBF1 display impaired maturation of peripheral glomeruli. EBF1 is present within multiple glomerular cell types, including the glomerular mesangium and podocytes. METHODS: To identify which cell type is driving the glomerular developmental defects in the global EBF1 knockout mice, we deleted EBF1 from the mesangium/pericytes (Foxd1-cre) or podocytes (Podocin-cre) in mice. RESULTS: Deletion of EBF1 from Foxd1 lineage cells resulted in hypoplastic kidneys, poorly differentiated peripheral glomeruli, and decreased proximal tubular mass in the outer cortex. Renal insufficiency was apparent at P21 when proteinuria presents, fibrosis of both the glomeruli and interstitium rapidly progresses, microthrombi appear, and hematuria develops. Approximately half of the Foxd1+, Ebf1fl/fl mice die before they are 3 months old. Mice with podocyte-targeted deletion of EBF1 exhibited no developmental abnormalities. Mice with Ebf1 deficiency in Foxd1 lineage cells shared characteristics with Ptgs2/COX-2-insufficient models, and mechanistic investigation revealed impaired calcineurin/NFATc1 activation and decreased COX-2 expression. Deletion of COX-2 from the interstitial/mesangial lineage displayed a less severe phenotype than EBF1 deficiency in mice. Overexpressing COX-2 in the EBF1-deficient mice, however, partially restored glomerular development. CONCLUSIONS: The results suggest that EBF1 regulates metanephric development at the last stages of glomerular maturation through its actions in the stromal progenitor (Foxd1+) lineage where it mediates proper regulation of calcineurin/NFAT signaling and COX-2 expression.

Caveolin-1 Regulates Atherogenesis by Attenuating Low-Density Lipoprotein Transcytosis and Vascular Inflammation Independently of Endothelial Nitric Oxide Synthase Activation.

BACKGROUND: Atherosclerosis is driven by synergistic interactions between pathological, biomechanical, inflammatory, and lipid metabolic factors. Our previous studies demonstrated that absence of caveolin-1 (Cav1)/caveolae in hyperlipidemic mice strongly inhibits atherosclerosis, which was attributed to activation of endothelial nitric oxide (NO) synthase (eNOS) and increased production of NO and reduced inflammation and low-density lipoprotein trafficking. However, the contribution of eNOS activation and NO production in the athero-protection of Cav1 and the exact mechanisms by which Cav1/caveolae control the pathogenesis of diet-induced atherosclerosis are still not clear. METHODS: Triple-knockout mouse lacking expression of eNOS, Cav1, and Ldlr were generated to explore the role of NO production in Cav1-dependent athero-protective function. The effects of Cav1 on lipid trafficking, extracellular matrix remodeling, and vascular inflammation were studied both in vitro and in vivo with a mouse model of diet-induced atherosclerosis. The expression of Cav1 and distribution of caveolae regulated by flow were analyzed by immunofluorescence staining and transmission electron microscopy. RESULTS: We found that absence of Cav1 significantly suppressed atherogenesis in Ldlr-/-eNOS-/- mice, demonstrating that athero-suppression is independent of increased NO production. Instead, we find that the absence of Cav1/caveolae inhibited low-density lipoprotein transport across the endothelium and proatherogenic fibronectin deposition and disturbed flow-mediated endothelial cell inflammation. Consistent with the idea that Cav1/caveolae may play a role in early flow-dependent inflammatory priming, distinct patterns of Cav1 expression and caveolae distribution were observed in athero-prone and athero-resistant areas of the aortic arch even in wild-type mice. CONCLUSIONS: These findings support a role for Cav1/caveolae as a central regulator of atherosclerosis that links biomechanical, metabolic, and inflammatory pathways independently of endothelial eNOS activation and NO production.

Endothelial ERK1/2 signaling maintains integrity of the quiescent endothelium.

To define the role of ERK1/2 signaling in the quiescent endothelium, we induced endothelial Erk2 knockout in adult Erk1-/- mice. This resulted in a rapid onset of hypertension, a decrease in eNOS expression, and an increase in endothelin-1 plasma levels, with all mice dying within 5 wk. Immunostaining and endothelial fate mapping showed a robust increase in TGFß signaling leading to widespread endothelial-to-mesenchymal transition (EndMT). Fibrosis affecting the cardiac conduction system was responsible for the universal lethality in these mice. Other findings included renal endotheliosis, loss of fenestrated endothelia in endocrine organs, and hemorrhages. An ensemble computational intelligence strategy, comprising deep learning and probabilistic programing of RNA-seq data, causally linked the loss of ERK1/2 in HUVECs in vitro to activation of TGFß signaling, EndMT, suppression of eNOS, and induction of endothelin-1 expression. All in silico predictions were verified in vitro and in vivo. In summary, these data establish the key role played by ERK1/2 signaling in the maintenance of vascular normalcy.

Regulated necrosis and failed repair in cisplatin-induced chronic kidney disease.

Cisplatin is an effective chemotherapeutic agent, but significant nephrotoxicity limits its clinical use. Despite extensive investigation of the acute cellular and molecular responses to cisplatin, the mechanisms of progression from acute to chronic kidney injury have not been explored. We used functional and morphological metrics to establish a time-point when the transition from acute and reversible kidney injury to chronic and irreparable kidney disease is clearly established. In mice administered 1 or 2 doses of intraperitoneal cisplatin separated by 2 weeks, kidney function returned toward baseline two weeks after the first dose, but failed to return to normal two weeks following a second dose. Multiphoton microscopy revealed increased glomerular epithelial and proximal tubular damage in kidneys exposed to two doses of cisplatin compared with those exposed to a single dose. In contrast, there was no evidence of fibrosis, macrophage invasion, or decrease in endothelial cell mass in chronically diseased kidneys. Pathway analysis of microarray data revealed regulated necrosis as a key determinant in the development of chronic kidney disease after cisplatin administration. Western blot analysis demonstrated activation of proteins involved in necroptosis and increased expression of kidney injury markers, cellular stress response regulators, and upstream activators of regulated necrosis, including Toll-like receptors 2 and 4. These data suggest that unresolved injury and sustained activation of regulated necrosis pathways, rather than fibrosis, promote the progression of cisplatin-induced acute kidney injury to chronic kidney disease.

Characterization of renal NaCl and oxalate transport in Slc26a6-/- mice.

The apical membrane Cl-/oxalate exchanger SLC26A6 has been demonstrated to play a role in proximal tubule NaCl transport based on studies in microperfused tubules. The present study is directed at characterizing the role of SLC26A6 in NaCl homeostasis in vivo under physiological conditions. Free-flow micropuncture studies revealed that volume and Cl- absorption were similar in surface proximal tubules of wild-type and Slc26a6-/- mice. Moreover, the increments in urine flow rate and sodium excretion following thiazide and furosemide infusion were identical in wild-type and Slc26a6-/- mice, indicating no difference in NaCl delivery out of the proximal tubule. The absence of an effect of deletion of SLC26A6 on NaCl homeostasis was further supported by the absence of lower blood pressure in Slc26a6-/- compared with wild-type mice on normal or low-salt diets. Moreover, raising plasma and urine oxalate by feeding mice a diet enriched in soluble oxalate did not affect mean blood pressure. In contrast to the lack of effect of SLC26A6 deletion on NaCl homeostasis, fractional excretion of oxalate was reduced from 1.6 in wild-type mice to 0.7 in Slc26a6-/- mice. We conclude that, although SLC26A6 is dispensable for renal NaCl homeostasis, it is required for net renal secretion of oxalate.

CLOCK phosphorylation by AKT regulates its nuclear accumulation and circadian gene expression in peripheral tissues.

Circadian locomotor output cycles kaput (CLOCK) is a transcription factor that activates transcription of clock-controlled genes by heterodimerizing with BMAL1 and binding to E-box elements on DNA. Although several phosphorylation sites on CLOCK have already been identified, this study characterizes a novel phosphorylation site at serine 845 (Ser-836 in humans). Here, we show that CLOCK is a novel AKT substrate in vitro and in cells, and this phosphorylation site is a negative regulator of CLOCK nuclear localization by acting as a binding site for 14-3-3 proteins. To examine the role of CLOCK phosphorylation in vivo, ClockS845A knockin mice were generated using CRISPR/Cas9 technology. ClockS845A mice are essentially normal with normal central circadian rhythms and hemodynamics. However, examination of core circadian gene expression from peripheral tissues demonstrated that ClockS845A mice have diminished expression of Per2, Reverba, Dbp, and Npas2 in skeletal muscle and Per2, Reverba, Dbp, Per1, Rora, and Npas2 in the liver during the circadian cycle. The reduction in Dbp levels is associated with reduced H3K9ac at E-boxes where CLOCK binds despite no change in total CLOCK levels. Thus, CLOCK phosphorylation by AKT on Ser-845 regulates its nuclear translocation and the expression levels of certain core circadian genes in insulin-sensitive tissues.

Endothelial Cell Autonomous Role of Akt1: Regulation of Vascular Tone and Ischemia-Induced Arteriogenesis.

OBJECTIVE: The importance of PI3K/Akt signaling in the vasculature has been demonstrated in several models, as global loss of Akt1 results in impaired postnatal ischemia- and VEGF-induced angiogenesis. The ubiquitous expression of Akt1, however, raises the possibility of cell-type-dependent Akt1-driven actions, thereby necessitating tissue-specific characterization. APPROACH AND RESULTS: Herein, we used an inducible, endothelial-specific Akt1-deleted adult mouse model (Akt1iECKO) to characterize the endothelial cell autonomous functions of Akt1 in the vascular system. Endothelial-targeted ablation of Akt1 reduces eNOS (endothelial nitric oxide synthase) phosphorylation and promotes both increased vascular contractility in isolated vessels and elevated diastolic blood pressures throughout the diurnal cycle in vivo. Furthermore, Akt1iECKO mice subject to the hindlimb ischemia model display impaired blood flow and decreased arteriogenesis. CONCLUSIONS: Endothelial Akt1 signaling is necessary for ischemic resolution post-injury and likely reflects the consequence of NO insufficiency critical for vascular repair.

Akt1 Controls the Timing and Amplitude of Vascular Circadian Gene Expression.

The AKT signaling pathway is important for circadian rhythms in mammals and flies ( Drosophila). However, AKT signaling in mammals is more complicated since there are 3 isoforms of AKT, each performing slightly different functions. Here we study the most ubiquitous AKT isoform, Akt1, and its role at the organismal level in the central and vascular peripheral clocks. Akt1-/- mice exhibit relatively normal behavioral rhythms with only minor differences in circadian gene expression in the liver and heart. However, circadian gene expression in the Akt1-/- aorta, compared with control aorta, follows a distinct pattern. In the Akt1-/- aorta, positive regulators of circadian transcription have lower amplitude rhythms and peak earlier in the day, and negative circadian regulators are expressed at higher amplitudes and peak later in the day. In endothelial cells, negative circadian regulators exhibit an increased amplitude of expression, while the positive circadian regulators are arrhythmic with a decreased amplitude of expression. This indicates that Akt1 conditions the normal circadian rhythm in the vasculature more so than in other peripheral tissues where other AKT isoforms or kinases might be important for daily rhythms.

Extracellular renalase protects cells and organs by outside-in signalling.

Renalase was discovered as a protein synthesized by the kidney and secreted in blood where it circulates at a concentration of approximately 3-5 µg/ml. Initial reports suggested that it functioned as an NAD(P)H oxidase and could oxidize catecholamines. Administration of renalase lowers blood pressure and heart rate and also protects cells and organs against ischaemic and toxic injury. Although renalase's protective effect was initially ascribed to its oxidase properties, a paradigm shift in our understanding of the cellular actions of renalase is underway. We now understand that, independent of its enzymatic properties, renalase functions as a cytokine that provides protection to cells, tissues and organs by interacting with its receptor to activate protein kinase B, JAK/STAT, and the mitogen-activated protein kinase pathways. In addition, recent studies suggest that dysregulated renalase signalling may promote survival of several tumour cells due to its capacity to augment expression of growth-related genes. In this review, we focus on the cytoprotective actions of renalase and its capacity to sustain cancer cell growth and also the translational opportunities these findings represent for the development of novel therapeutic strategies for organ injury and cancer.

Renalase Expression by Melanoma and Tumor-Associated Macrophages Promotes Tumor Growth through a STAT3-Mediated Mechanism.

To sustain their proliferation, cancer cells overcome negative-acting signals that restrain their growth and promote senescence and cell death. Renalase (RNLS) is a secreted flavoprotein that functions as a survival factor after ischemic and toxic injury, signaling through the plasma calcium channel PMCA4b to activate the PI3K/AKT and MAPK pathways. We show that RNLS expression is increased markedly in primary melanomas and CD163(+) tumor-associated macrophages (TAM). In clinical specimens, RNLS expression in the tumor correlated inversely with disease-specific survival, suggesting a pathogenic role for RNLS. Attenuation of RNLS by RNAi, blocking antibodies, or an RNLS-derived inhibitory peptide decreased melanoma cell survival, and anti-RNLS therapy blocked tumor growth in vivo in murine xenograft assays. Mechanistic investigations showed that increased apoptosis in tumor cells was temporally related to p38 MAPK-mediated Bax activation and that increased cell growth arrest was associated with elevated expression of the cell-cycle inhibitor p21. Overall, our results established a role for the secreted flavoprotein RNLS in promoting melanoma cell growth and CD163(+) TAM in the tumor microenvironment, with potential therapeutic implications for the management of melanoma. Cancer Res; 76(13); 3884-94. ©2016 AACR.

Inhibition of renalase expression and signaling has antitumor activity in pancreatic cancer.

An essential feature of cancer is dysregulation of cell senescence and death. Renalase, a recently discovered secreted flavoprotein, provides cytoprotection against ischemic and toxic cellular injury by signaling through the PI3K-AKT and MAPK pathways. Here we show that renalase expression is increased in pancreatic cancer tissue and that it functions as a growth factor. In a cohort of patients with pancreatic ductal adenocarcinoma, overall survival was inversely correlated with renalase expression in the tumor mass, suggesting a pathogenic role for renalase. Inhibition of renalase signaling using siRNA or inhibitory anti-renalase antibodies decreased the viability of cultured pancreatic ductal adenocarcinoma cells. In two xenograft mouse models, either the renalase monoclonal antibody m28-RNLS or shRNA knockdown of renalase inhibited pancreatic ductal adenocarcinoma growth. Inhibition of renalase caused tumor cell apoptosis and cell cycle arrest. These results reveal a previously unrecognized role for the renalase in cancer: its expression may serve as a prognostic maker and its inhibition may provide an attractive therapeutic target in pancreatic cancer.

Uncoupling Caveolae From Intracellular Signaling In Vivo.

RATIONALE: Caveolin-1 (Cav-1) negatively regulates endothelial nitric oxide (NO) synthase-derived NO production, and this has been mapped to several residues on Cav-1, including F92. Herein, we reasoned that endothelial expression of an F92ACav-1 transgene would let us decipher the mechanisms and relationships between caveolae structure and intracellular signaling. OBJECTIVE: This study was designed to separate caveolae formation from its downstream signaling effects. METHODS AND RESULTS: An endothelial-specific doxycycline-regulated mouse model for the expression of Cav-1-F92A was developed. Blood pressure by telemetry and nitric oxide bioavailability by electron paramagnetic resonance and phosphorylation of vasodilator-stimulated phosphoprotein were determined. Caveolae integrity in the presence of Cav-1-F92A was measured by stabilization of caveolin-2, sucrose gradient, and electron microscopy. Histological analysis of heart and lung, echocardiography, and signaling were performed. CONCLUSIONS: This study shows that mutant Cav-1-F92A forms caveolae structures similar to WT but leads to increases in NO bioavailability in vivo, thereby demonstrating that caveolae formation and downstream signaling events occur through independent mechanisms.

Three-Dimensional Morphology by Multiphoton Microscopy with Clearing in a Model of Cisplatin-Induced CKD.

Traditional histologic methods are limited in their ability to detect pathologic changes of CKD, of which cisplatin therapy is an important cause. In addition, poor reproducibility of available methods has limited analysis of the role of fibrosis in CKD. Highly labor-intensive serial sectioning studies have demonstrated that three-dimensional perspective can reveal useful morphologic information on cisplatin-induced CKD. By applying the new technique of multiphoton microscopy (MPM) with clearing to a new mouse model of cisplatin-induced CKD, we obtained detailed morphologic and collagen reconstructions of millimeter-thick renal sections that provided new insights into pathophysiology. Quantitative analysis revealed that a major long-term cisplatin effect is reduction in the number of cuboidal cells of the glomerular capsule, a change we term the "uncapped glomerulus lesion." Glomerulotubular disconnection was confirmed, but connection remnants between damaged tubules and atubular glomeruli were observed. Reductions in normal glomerular capsules corresponded to reductions in GFR. Mild increases in collagen were noted, but the fibrosis was not spatially correlated with atubular glomeruli. Glomerular volume and number remained unaltered with cisplatin exposure, but cortical tubulointerstitial mass decreased. In conclusion, new observations were made possible by using clearing MPM, demonstrating the utility of this technique for studies of renal disease. This technique should prove valuable for further characterizing the evolution of CKD with cisplatin therapy and of other conditions.

The Protein Acyl Transferase ZDHHC21 Modulates α1 Adrenergic Receptor Function and Regulates Hemodynamics.

OBJECTIVE: Palmitoylation, the reversible addition of the lipid palmitate to a cysteine, can alter protein localization, stability, and function. The ZDHHC family of protein acyl transferases catalyzes palmitoylation of numerous proteins. The role of ZDHHC enzymes in intact tissue and in vivo is largely unknown. Herein, we characterize vascular functions in a mouse that expresses a nonfunctional ZDHHC21 (F233Δ). APPROACH AND RESULTS: Physiological studies of isolated aortae and mesenteric arteries from F233Δ mice revealed an unexpected defect in responsiveness to phenylephrine, an α1 adrenergic receptor agonist. In vivo, F233Δ mice displayed a blunted response to infusion of phenylephrine, and they were found to have elevated catecholamine levels and elevated vascular α1 adrenergic receptor gene expression. Telemetry studies showed that the F233Δ mice were tachycardic and hypotensive at baseline, consistent with diminished vascular tone. In biochemical studies, ZDHHC21 was shown to palmitoylate the α1D adrenoceptor and to interact with it in a molecular complex, thus suggesting a possible molecular mechanism by which the receptor can be regulated by ZDHHC21. CONCLUSIONS: Together, the data support a model in which ZDHHC21 F233Δ diminishes the function of vascular α1 adrenergic receptors, leading to reduced vascular tone, which manifests in vivo as hypotension and tachycardia. This is to our knowledge the first demonstration of a ZDHHC isoform affecting vascular function in vivo and identifies a novel molecular mode of regulation of vascular tone and blood pressure.

Identification of a receptor for extracellular renalase.

BACKGROUND: An increased risk for developing essential hypertension, stroke and diabetes is associated with single nucleotide gene polymorphisms in renalase, a newly described secreted flavoprotein with oxidoreductase activity. Gene deletion causes hypertension, and aggravates acute ischemic kidney (AKI) and cardiac injury. Independent of its intrinsic enzymatic activities, extracellular renalase activates MAPK signaling and prevents acute kidney injury (AKI) in wild type (WT) mice. Therefore, we sought to identity the receptor for extracellular renalase. METHODS AND RESULTS: RP-220 is a previously identified, 20 amino acids long renalase peptide that is devoid of any intrinsic enzymatic activity, but it is equally effective as full-length recombinant renalase at protecting against toxic and ischemic injury. Using biotin transfer studies with RP-220 in the human proximal tubular cell line HK-2 and protein identification by mass spectrometry, we identified PMCA4b as a renalase binding protein. This previously characterized plasma membrane ATPase is involved in cell signaling and cardiac hypertrophy. Co-immunoprecipitation and co-immunolocalization confirmed protein-protein interaction between endogenous renalase and PMCA4b. Down-regulation of endogenous PMCA4b expression by siRNA transfection, or inhibition of its enzymatic activity by the specific peptide inhibitor caloxin1b each abrogated RP-220 dependent MAPK signaling and cytoprotection. In control studies, these maneuvers had no effect on epidermal growth factor mediated signaling, confirming specificity of the interaction between PMCA4b and renalase. CONCLUSIONS: PMCA4b functions as a renalase receptor, and a key mediator of renalase dependent MAPK signaling.

Loss of the endothelial glucocorticoid receptor prevents the therapeutic protection afforded by dexamethasone after LPS.

Glucocorticoids are normally regarded as anti-inflammatory therapy for a wide variety of conditions and have been used with some success in treating sepsis and sepsis-like syndromes. We previously demonstrated that mice lacking the glucocorticoid receptor in the endothelium (GR EC KO mice) are extremely sensitive to low-dose LPS and demonstrate prolonged activation and up regulation of NF-κB. In this study we pre-treated these GR EC KO mice with dexamethasone and assessed their response to an identical dose of LPS. Surprisingly, the GR EC KO mice fared even worse than when given LPS alone demonstrating increased mortality, increased levels of the inflammatory cytokines TNF-α and IL-6 and increased nitric oxide release after the dexamethasone pre-treatment. As expected, control animals pre-treated with dexamethasone showed improvement in all parameters assayed. Mechanistically we demonstrate that GR EC KO mice show increased iNOS production and NF-κB activation despite treatment with dexamethasone.

Renalase: its role as a cytokine, and an update on its association with type 1 diabetes and ischemic stroke.

PURPOSE OF REVIEW: Remarkable progress has been achieved over the past 2 years in understanding the cellular actions of renalase, its pathophysiology and potential therapeutic utility. RECENT FINDINGS: There has been a paradigm shift in our thinking about the mechanisms underlying the cellular actions of renalase. We now understand that, independent of its enzymatic properties, renalase functions as a signaling molecule, a cytokine that interacts with a yet-to-be identified plasma membrane receptor(s) to activate protein kinase B and the mitogen-activated protein kinase pathway. These signaling properties are critical to its cytoprotective effects. New information regarding renalase's enzymatic function as an α-nicotinamide adenine dinucleotide oxidase/anomerase will be reviewed. Lastly, we will discuss the association of certain single nucleotide polymorphisms in the renalase gene with type 1 diabetes and with ischemic stroke, and the clinical implications of these findings. SUMMARY: The consistent association of renalase single nucleotide polymorphisms and the development of type 1 diabetes is a great interest particularly because we now understand that renalase functions as a cytokine. Future work on renalase should focus on exploring the identity of its receptor(s), and its potential role as an immune modulator.