Renalase peptides reduce pancreatitis severity in mice.

Acute pancreatitis, an acute inflammatory injury of the pancreas, lacks a specific treatment. The circulatory protein renalase is produced by the kidney and other tissues and has potent anti-inflammatory and prosurvival properties. Recombinant renalase can reduce the severity of mild cerulein pancreatitis; the activity is contained in a conserved 20 aa renalase site (RP220). Here, we investigated the therapeutic effects of renalase on pancreatitis using two clinically relevant models of acute pancreatitis. The ability of peptides containing the RP220 site to reduce injury in a 1-day post-endoscopic retrograde cholangiopancreatography (ERCP) and a 2-day severe cerulein induced in mice was examined. The initial dose of renalase peptides was given either prophylactically (before) or therapeutically (after) the initiation of the disease. Samples were collected to determine early pancreatitis responses (tissue edema, plasma amylase, active zymogens) and later histologic tissue injury and inflammatory changes. In both preclinical models, renalase peptides significantly reduced histologic damage associated with pancreatitis, especially inflammation, necrosis, and overall injury. Quantifying inflammation using specific immunohistochemical markers demonstrated that renalase peptides significantly reduced overall bone marrow-derived inflammation and neutrophils and macrophage populations in both models. In the severe cerulein model, administering a renalase peptide with or without pretreatment significantly reduced injury. Pancreatitis and renalase peptide effects appeared to be the same in female and male mice. These studies suggest renalase peptides that retain the anti-inflammatory and prosurvival properties of recombinant renalase can reduce the severity of acute pancreatitis and might be attractive candidates for therapeutic development.NEW & NOTEWORTHY Renalase is a secretory protein. The prosurvival and anti-inflammatory effects of the whole molecule are contained in a 20 aa renalase site (RP220). Systemic treatment with peptides containing this renalase site reduced the severity of post-endoscopic retrograde cholangiopancreatography (ERCP) and severe cerulein pancreatitis in mouse models.

An AI-powered patient triage platform for future viral outbreaks using COVID-19 as a disease model.

Over the last century, outbreaks and pandemics have occurred with disturbing regularity, necessitating advance preparation and large-scale, coordinated response. Here, we developed a machine learning predictive model of disease severity and length of hospitalization for COVID-19, which can be utilized as a platform for future unknown viral outbreaks. We combined untargeted metabolomics on plasma data obtained from COVID-19 patients (n = 111) during hospitalization and healthy controls (n = 342), clinical and comorbidity data (n = 508) to build this patient triage platform, which consists of three parts: (i) the clinical decision tree, which amongst other biomarkers showed that patients with increased eosinophils have worse disease prognosis and can serve as a new potential biomarker with high accuracy (AUC = 0.974), (ii) the estimation of patient hospitalization length with ± 5 days error (R2 = 0.9765) and (iii) the prediction of the disease severity and the need of patient transfer to the intensive care unit. We report a significant decrease in serotonin levels in patients who needed positive airway pressure oxygen and/or were intubated. Furthermore, 5-hydroxy tryptophan, allantoin, and glucuronic acid metabolites were increased in COVID-19 patients and collectively they can serve as biomarkers to predict disease progression. The ability to quickly identify which patients will develop life-threatening illness would allow the efficient allocation of medical resources and implementation of the most effective medical interventions. We would advocate that the same approach could be utilized in future viral outbreaks to help hospitals triage patients more effectively and improve patient outcomes while optimizing healthcare resources.

Challenges in interpreting cytokine data in COVID-19 affect patient care and management.

Challenges in using cytokine data are limiting Coronavirus Disease 2019 (COVID-19) patient management and comparison among different disease contexts. We suggest mitigation strategies to improve the accuracy of cytokine data, as we learn from experience gained during the COVID-19 pandemic.

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.

RENALASE: DISCOVERY, BIOLOGY, AND THERAPEUTIC APPLICATIONS.

While investigating the mechanisms that could mediate the significant burden of cardiovascular complications observed in persons with chronic kidney disease (CKD) and end stage renal disease (ESRD), we identified a previously unknown protein, which we named renalase (RNLS). Over the past 15 years, our understanding of the biology, physiology, and pathophysiology of RNLS has matured. Here we aim to highlight that RNLS is a bifunctional protein. It metabolizes intracellular nicotinamide adenine dinucleotide (NADH), modulates mitochondrial function, and protects energy metabolism. When secreted outside the cell, independent of its enzymatic properties, it functions as a signaling molecule that mediates resistance to stressful stimuli and promotes cell and organ survival. RNLS has been shown to modulate the severity of acute injury to the pancreas, liver, kidney, and heart. It also protects against the development of chronic injury, and here we highlight the potential use of exogenous RNLS peptide agonists to prevent cisplatin-mediated CKD (CP-CKD).

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.

The serum protein renalase reduces injury in experimental pancreatitis.

Acute pancreatitis is a disease associated with inflammation and tissue damage. One protein that protects against acute injury, including ischemic injury to both the kidney and heart, is renalase, which is secreted into the blood by the kidney and other tissues. However, whether renalase reduces acute injury associated with pancreatitis is unknown. Here, we used both in vitro and in vivo murine models of acute pancreatitis to study renalase's effects on this condition. In isolated pancreatic lobules, pretreatment with recombinant human renalase (rRNLS) blocked zymogen activation caused by cerulein, carbachol, and a bile acid. Renalase also blocked cerulein-induced cell injury and histological changes. In the in vivo cerulein model of pancreatitis, genetic deletion of renalase resulted in more severe disease, and administering rRNLS to cerulein-exposed WT mice after pancreatitis onset was protective. Because pathological increases in acinar cell cytosolic calcium levels are central to the initiation of acute pancreatitis, we also investigated whether rRNLS could function through its binding protein, plasma membrane calcium ATPase 4b (PMCA4b), which excretes calcium from cells. We found that PMCA4b is expressed in both murine and human acinar cells and that a PMCA4b-selective inhibitor worsens pancreatitis-induced injury and blocks the protective effects of rRNLS. These findings suggest that renalase is a protective plasma protein that reduces acinar cell injury through a plasma membrane calcium ATPase. Because exogenous rRNLS reduces the severity of acute pancreatitis, it has potential as a therapeutic agent.

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.

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.

Renalase regulates peripheral and central dopaminergic activities.

Renalase is a recently identified FAD/NADH-dependent amine oxidase mainly expressed in kidney that is secreted into blood and urine where it was suggested to metabolize catecholamines. The present study evaluated central and peripheral dopaminergic activities in the renalase knockout (KO) mouse model and examined the changes induced by recombinant renalase (RR) administration on plasma and urine catecholamine levels. Compared with wild-type (WT) mice, KO mice presented increased plasma levels of epinephrine (Epi), norepinephrine (NE), and dopamine (DA) that were accompanied by increases in the urinary excretion of Epi, NE, DA. In addition, the KO mice presented an increase in urinary DA-to-l-3,4-dihydroxyphenylalanine (l-DOPA) ratios without changes in renal tubular aromatic-l-amino acid decarboxylase (AADC) activity. By contrast, the in vivo administration of RR (1.5 mg/kg sc) to KO mice was accompanied by significant decreases in plasma levels of Epi, DA, and l-DOPA as well as in urinary excretion of Epi, DA, and DA-to-l-DOPA ratios notwithstanding the accompanied increase in renal AADC activity. In addition, the increase in renal DA output observed in renalase KO mice was accompanied by an increase in the expression of the L-type amino acid transporter like (LAT) 1 that is reversed by the administration of RR in these animals. These results suggest that the overexpression of LAT1 in the renal cortex of the renalase KO mice might contribute to the enhanced l-DOPA availability/uptake and consequently to the activation of the renal dopaminergic system in the presence of renalase deficiency.

Renalase prevents AKI independent of amine oxidase activity.

AKI is characterized by increased catecholamine levels and hypertension. Renalase, a secretory flavoprotein that oxidizes catecholamines, attenuates ischemic injury and the associated increase in catecholamine levels in mice. However, whether the amine oxidase activity of renalase is involved in preventing ischemic injury is debated. In this study, recombinant renalase protected human proximal tubular (HK-2) cells against cisplatin- and hydrogen peroxide-induced necrosis. Similarly, genetic depletion of renalase in mice (renalase knockout) exacerbated kidney injury in animals subjected to cisplatin-induced AKI. Interestingly, compared with the intact renalase protein, a 20-amino acid peptide (RP-220), which is conserved in all known renalase isoforms, but lacks detectable oxidase activity, was equally effective at protecting HK-2 cells against toxic injury and preventing ischemic injury in wild-type mice. Furthermore, in vitro treatment with RP-220 or recombinant renalase rapidly activated Akt, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinases and downregulated c-Jun N-terminal kinase. In summary, renalase promotes cell survival and protects against renal injury in mice through the activation of intracellular signaling cascades, independent of its ability to metabolize catecholamines, and we have identified the region of renalase required for these effects. Renalase and related peptides show potential as therapeutic agents for the prevention and treatment of AKI.

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.

Renalase in hypertension and kidney disease.

Renalase, a recently discovered flavoprotein, which is strongly expressed in the kidney and heart, effectively metabolizes catecholamines. It was discovered during the search to identify proteins secreted by the kidney that could help explain the high incidence of cardiovascular disease in patients with chronic kidney disease. Recent advances have led to more detailed knowledge of its biology, structure, enzymatic activity, mechanisms of action, associations with human disease states and potential therapeutic value. In this study, we review these advances with a focus on hypertension and kidney disease.

Renalase protects against ischemic AKI.

Elevated levels of plasma catecholamines accompany ischemic AKI, possibly contributing the inflammatory response. Renalase, an amine oxidase secreted by the proximal tubule, degrades circulating catecholamines and reduces myocardial necrosis, suggesting that it may protect against renal ischemia reperfusion injury. Here, mice subjected to renal ischemia reperfusion injury had significantly lower levels of renalase in the plasma and kidney compared with sham-operated mice. Consistent with this, plasma NE levels increased significantly after renal ischemia reperfusion injury. Furthermore, renal tubular inflammation, necrosis, and apoptosis were more severe and plasma catecholamine levels were higher in renalase-deficient mice subjected to renal ischemia reperfusion compared with wild-type mice. Administration of recombinant human renalase reduced plasma catecholamine levels and ameliorated ischemic AKI in wild-type mice. Taken together, these data suggest that renalase protects against ischemic AKI by reducing renal tubular necrosis, apoptosis, and inflammation, and that plasma renalase might be a biomarker for AKI. Recombinant renalase therapy may have potential for the prevention and treatment of AKI.

Renalase Levels are Decreased in Maternal Blood and Placental Tissues in Pregnancies Associated with Preterm Preeclampsia.

Preeclampsia (PEC) is a complication of pregnancy associated with hypertension and the risk of eclampsia. The pathophysiology of PEC is unknown and identifying factors associated with PEC during pregnancy is crucial for placental, fetal, and maternal health. Renalase (RNLS) is an anti-inflammatory secretory flavoprotein associated with hypertension. Recent data demonstrated a correlation between maternal serum RNLS and PEC, and work from our group identified RNLS expression in the placenta. However, it remains unknown whether RNLS levels in placenta are altered by preeclampsia. Additionally, it is unclear if there is a differential effect of preterm and term PEC on RNLS. We demonstrate that serum RNLS was reduced in preterm cases of PEC. Similarly, placental RNLS was diminished in the chorion of preterm cases of PEC. However, a reduction of RNLS in the decidua was observed with all cases of PEC, while the levels of RNLS within the placental villi were similar in all cases. Overall, we demonstrate that RNLS correlates with PEC both systemically in maternal serum and locally within the placenta, with variable effects on the different layers of the placenta and more pronounced in preterm cases.

Chemical complementarity of tumor resident, T-cell receptor CDR3s and renalase-1 correlates with increased melanoma survival.

Overexpression of the secretory protein renalase-1 negatively impacts the survival of melanoma and pancreatic cancer patients, while inhibition of renalase-1 signaling drives tumor rejection by promoting T-cell activation. Thus, we investigated the chemical complementarity between melanoma-resident, T-cell receptor (TCR) complementarity-determining region 3 (CDR3) amino acid sequences (AAs) and the renalase-1 protein. Increasing complementarity of TCR CDR3s to renalase-1 AAs, as assessed by a chemical complementarity scoring algorithm, was associated with improved overall survival (OS) in melanoma patients. The expression levels of several immune signature genes were significantly, positively correlated with increasing TCR CDR3-renalase-1 complementarity scores. Additionally, the survival association observed with high complementarity of TCR CDR3s to renalase-1 AAs was more robust in cases with low renalase-1 gene expression levels. Mapping of TCR CDR3-renalase-1 in silico interaction sites identified major epitope candidates including RP220, the signaling module of the renalase-1 protein, consistent with the fact that a monoclonal antibody to RP220 is a potent inhibitor of melanoma growth. These findings indicate that renalase-1 is a potential antigen for TCR recognition in melanoma and could be considered as a target for immunotherapy.

Renalase regulates renal dopamine and phosphate metabolism.

Renalase is a kidney-secreted catecholamines-degrading enzyme whose expression and activity are downregulated by increased dietary phosphate. A renalase knockout (KO) mouse model was used to explore the mechanisms mediating renalase's effect on phosphate excretion. Compared with wild-type (WT) mice maintained on a regular diet, KO mice show decreased serum PO4(-) (KO = 5.3 ± 0.2 vs. WT = 6.0 ± 0.1, n = 6; P < 0.04) and increased urinary PO4(-) excretion (urine PO4(-)/creatinine: KO = 7.7 ± 0.3 vs. WT = 6.1 ± 0.3, n = 6; P < 0.02). However, both WT and KO mice respond similarly to PO4(-) restriction by increasing renal COMT-1 activity and markedly decreasing PO4(-) excretion, which excludes an intrinsic renal defect in the KO. Renal sodium-phosphate cotransporter Npt2a, sodium proton exchanger NHE3 expression, and MAO-A and B activity did not differ between WT and KO. Only catechol-O-methyl transferase (COMT) expression and activity were significantly increased in KO mice. Despite that, urinary dopamine increased by twofold, whereas urinary l-DOPA excretion decreased by twofold in the KO mouse, indicating an upregulation of renal dopamine (DA) synthesis. These data indicate that renalase deficiency is associated with increased renal DA synthesis, stimulated PO4(-) excretion, and moderately severe hypophosphatemia. The signal to increase renal DA synthesis is strong since it overcomes a compensatory increase in COMT activity.

Does kidney disease cause hypertension?

Hypertension complicates most cases of chronic kidney disease. While the prevalence and severity of hypertension increase as glomerular filtration rate falls, hypertension is often observed in patients with structural kidney disease while renal function is normal, in particular those with polycystic kidney disease or proteinuric glomerular diseases. On the other hand, even severe reductions in renal function may not result in hypertension, especially if there is effective control of extracellular fluid volume. Recent clinical and experimental data indicate that proteinuria may mediate sodium retention and hypertension via plasmin-mediated activation of the epithelial sodium channel. Current evidence supports the notion that chronic kidney disease is a cause of chronic hypertension, even in the absence of detectable changes in glomerular filtration rate.

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.