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Abstract Cardiotrophin-like cytokine factor 1 (CLCF1) is an IL-6 family cytokine with neurotrophic and immuno-modulating functions. CLCF1 mRNA has been detected in primary and secondary lymphoid organs, and up-regulation of CLCF1 mRNA levels has been associated with the T helper (Th) 17 polarization. However, information regarding CLCF1 expression by immune cells at the protein level remains scarce. We have developed a methodology that uses a monoclonal antibody (mAb) directed against CLCF1 for the detection of human and mouse CLCF1 by flow cytometry. We have successfully detected CLCF1 protein expression in cells from the mouse pro-B cell line Ba/F3 that were transduced with CLCF1 cDNA. Interestingly, we found that the anti-CLCF1 mAb inhibits CLCF1 biological activity in vitro by binding to an epitope that encompasses site III of the cytokine. Moreover, we have detected CLCF1 expression in mouse splenic T cells, as well as in vitro differentiated Th1 cells. The specificity of the fluorescence signal was demonstrated using Clcf1-deficient lymphocytes generated using a conditional knock-out mouse model. The detection of CLCF1 protein by flow cytometry will be a valuable tool to study CLCF1 expression during normal and pathological immune responses.

Chronic kidney disease (CKD) is a leading cause of morbidity and mortality globally. Maternal obesity during pregnancy is linked to systemic inflammation and elevated levels of the pro-inflammatory cytokine interleukin-6 (IL-6). In our previous work, we demonstrated that increased maternal IL-6 during gestation impacts intrauterine development in mice. We hypothesized that IL-6-induced inflammation alters gene expression in the developing fetus. To test this, pregnant mice were administered IL-6 or saline during mid-gestation. Newborn mouse kidneys were analyzed using mRNA-seq, miRNA-seq and whole-genome bisulfite-seq (WGBS). A multi-omics approach was employed to quantify mRNA gene expression, miRNA expression and DNA methylation, using advanced bioinformatics and data integration techniques. Our analysis identified 19 key genes present in multiple omics datasets, regulated by epigenetics and miRNAs. We constructed a regulatory network for these genes, revealing disruptions in pathways such as Mannose type O-glycan biosynthesis, the cell cycle, apoptosis and FoxO signaling. Notably, the Atp7b gene was regulated by DNA methylation and miR-223 targeting, whereas the Man2a1 gene was controlled by DNA methylation affecting energy metabolism. These findings suggest that these genes may play a role in fetal programming, potentially leading to CKD later in life due to gestational inflammation.

Epilepsy is one of the most prevalent and serious brain disorders and affects over 70 million people globally. Antiseizure medications (ASMs) relieve symptoms and prevent the occurrence of future seizures in epileptic patients but have a limited effect on epileptogenesis. Addressing the multifaceted nature of epileptogenesis and its association with the Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-mediated neuroinflammation requires a comprehensive understanding of the underlying mechanisms of these medications for the development of targeted therapeutic strategies beyond conventional antiseizure treatments. Several types of NLRP3 inhibitors have been developed and their effect has been validated both in in vitro and in vivo models of epileptogenesis. In this review, we discuss the advances in understanding the regulatory mechanisms of NLRP3 activation as well as progress made, and challenges faced in the development of NLRP3 inhibitors for the treatment of epilepsy.

Mast cells are important components of the immune system, and they perform pro-inflammatory as well as anti-inflammatory roles in the complex process of immune regulation in health and disease. Because of their strategic perivascular localization, sensitivity and adaptability to the microenvironment, and ability to release a variety of preformed and newly synthesized effector molecules, mast cells perform unique functions in almost all organs. Additionally, Mast cells express a wide range of surface and cytoplasmic receptors which enable them to respond to a variety of cytokines, chemicals, and pathogens. The mast cell’s role as a cellular interface between external and internal environments as well as between vasculature and tissues is critical for protection and repair. Mast cell interactions with different immune and nonimmune cells through secreted inflammatory mediators may also turn in favor of disease promoting agents. First and forefront, mast cells are well recognized for their multifaceted functions in allergic diseases. Reciprocal communication between mast cells and endothelial cells in the presence of bacterial toxins in chronic/sub-clinical infections induce persistent vascular inflammation. We have shown that mast cell proteases and histamine induce endothelial inflammatory responses that are synergistically amplified by bacterial toxins. Mast cells have been shown to exacerbate vascular changes in normal states as well as in chronic or subclinical infections, particularly among cigarette smokers. Furthermore, a potential role of mast cells in SARS-CoV-2-induced dysfunction of the capillary-alveolar interface adds to the growing understanding of mast cells in viral infections. The interaction between mast cells and microglial cells in the brain further highlights their significance in neuroinflammation. This review highlights the significant role of mast cells as the interface that acts as sensor and early responder through interactions with cells in systemic organs and the nervous system.

Overexpression of the inducible heme oxygenase (HO-1) isoform in visceral renal glomerular epithelial cells (podocytes) using in vivo transgenesis methods was shown to increase glomerular expression of the complement regulatory protein decay-accelerating factor (DAF, CD55) and reduce complement activation/deposition in a rat model of immune-mediated injury. In this preliminary study, we assessed whether constitutively expressed HO-1 regulates CD55 expression in cultured rat podocytes. We employed methods of flow cytometry, quantitative (q) RT-qPCR and post-transcriptional HO-1 gene silencing (HO-1 interfering RNA, RNAi), to assess changes in constitutive (basal) levels of podocyte HO-1 and CD55 mRNA in cultured rat podocytes. Additionally, the effect of the HO-1 inducer, heme, on HO-1 and CD55 expression was assessed. Results indicate that rat podocytes constitutively express HO-1 and DAF and that the HO-1 inducer, heme, increases both HO-1 and DAF expression. HO-1 gene silencing using RNA interference (RNAi) is feasible but the effect on constitutive CD55 transcription is inconsistent. These observations are relevant to conditions of podocyte exposure to heme that can activate the complementary cascade, as may occur in systemic or intraglomerular hemolysis.

Overactivated NLRP3 inflammasome has been shown to associate with an increasing number of disease conditions. Activation of the NLRP3 inflammasome results in caspase-1-catalyzed formation of active pro-inflammatory cytokines (IL-1β and IL-18) resulting in pyroptosis. The multi-protein composition of the NLRP3 inflammasome and its sensitivity to several damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) make this extensively studied inflammasome an attractive target to treat chronic conditions. However, none of the known NLRP3 inhibitors has been approved for clinical use. Sulfonylurea and covalent inhibitors with electrophilic warhead (Michael acceptor) are among the prominent classes of compounds explored for their NLRP3 inhibitory effects. Chalcone, a small molecule with α, β unsaturated carbonyl group (Michael acceptor), has also been studied as a promising scaffold for the development of NLRP3 inhibitors. Low molecular weight, easy to manipulate lipophilicity and cost-effectiveness have attracted many to use chalcone scaffold for drug development. In this review, we highlight chalcone derivatives with NLRP3 inflammasome inhibitory activities. Recent developments and potential new directions summarized here will, hopefully, serve as valuable perspectives for investigators including medicinal chemists and drug discovery researchers to utilize chalcone as a scaffold for developing novel NLRP3 inflammasome inhibitors.

Depending on their central metal atom, metalloporphyrins (MPs) can attenuate or exacerbate the severity of immune-mediated kidney injury, and this has been attributed to the induction or inhibition of heme oxygenase (HO) activity, particularly the inducible isoform (HO-1) of this enzyme. The role of central metal or porphyrin moieties in determining the efficacy of MPs to attenuate injury, as well as mechanisms underlying this effect, have not been assessed. Using an antibody-mediated complement-dependent model of injury directed against rat visceral glomerular epithelial cells (podocytes) and two MPs (FePPIX, CoPPIX) that induce both HO-1 expression and HO enzymatic activity in vivo but differ in their chelated metal, we assessed their efficacy in reducing albuminuria. Podocyte injury was induced using rabbit immune serum raised against the rat podocyte antigen, Fx1A, and containing an anti-Fx1A antibody that activates complement at sites of binding. FePPIX or CoPPIX were injected intraperitoneally (5 mg/kg) 24 h before administration of the anti-Fx1A serum and on days 1, 3, 6, and 10 thereafter. Upon completion of urine collection on day 14, the kidney cortex was obtained for histopathology and isolation of glomeruli, from which total protein extracts were obtained. Target proteins were analyzed by capillary-based separation and immunodetection (Western blot analysis). Both MPs had comparable efficacy in reducing albuminuria in males, but the efficacy of CoPPIX was superior in female rats. The metal-free protoporphyrin, PPIX, had minimal or no effect on urine albumin excretion. CoPPIX was also the most potent MP in inducing glomerular HO-1, reducing complement deposition, and preserving the expression of the complement regulatory protein (CRP) CD55 but not that of CD59, the expression of which was reduced by both MPs. These observations demonstrate that the metal moiety of HO-1-inducing MPs plays an important role in reducing proteinuria via mechanisms involving reduced complement deposition and independently of an effect on CRPs.

Abstract Systemic inflammation in pregnant obese women is associated with 1.5- to 2-fold increase in serum Interleukin-6 (IL-6) and newborns with lower kidney/body weight ratio but the role of IL-6 in increased susceptibility to chronic kidney (CKD) in adult progeny is not known. Since IL-6 crosses the placental barrier, we administered recombinant IL-6 (10 pg/g) to pregnant mice starting at mid-gestation yielded newborns with lower body (p

Nephron loss initiates compensatory hemodynamic and cellular effects on the remaining nephrons. Increases in single nephron glomerular filtration rate and tubular flow rate exert higher fluid shear stress (FSS) on tubules. In principal cell (PC) culture models FSS induces ERK, and ERK is implicated in the regulation of transepithelial sodium (Na) transport, as well as, proliferation. Thus, we hypothesize that high tubular flow and FSS mediate ERK activation in the cortical collecting duct (CCD) of solitary kidney which regulates amiloride sensitive Na transport and affects CCD cell number. Immunoblotting of whole kidney protein lysate was performed to determine phospho-ERK (pERK) expression. Next, sham and unilateral nephrectomized mice were stained with anti-pERK antibodies, and dolichos biflorus agglutinin (DBA) to identify PCs with pERK. Murine PCs (mpkCCD) were grown on semi-permeable supports under static, FSS, and FSS with U0126 (a MEK1/2 inhibitor) conditions to measure the effects of FSS and ERK inhibition on amiloride sensitive Na short circuit current (Isc). pERK abundance was greater in kidney lysate of unilateral vs. sham nephrectomies. The total number of cells in CCD and pERK positive PCs increased in nephrectomized mice (9.3 ± 0.4 vs. 6.1 ± 0.2 and 5.1 ± 0.5 vs. 3.6 ± 0.3 cell per CCD nephrectomy vs. sham, respectively, n > 6 per group, p < 0.05). However, Ki67, a marker of proliferation, did not differ by immunoblot or immunohistochemistry in nephrectomy samples at 1 month compared to sham. Next, amiloride sensitive Isc in static mpkCCD cells was 25.3 ± 1.7 μA/cm2 (n = 21), but after exposure to 24 h of FSS the Isc increased to 41.4 ± 2.8 μA/cm2 (n = 22; p < 0.01) and returned to 19.1 ± 2.1 μA/cm2 (n = 18, p < 0.01) upon treatment with U0126. Though FSS did not alter α- or γ-ENaC expression in mpkCCD cells, γ-ENaC was reduced in U0126 treated cells. In conclusion, pERK increases in whole kidney and, specifically, CCD cells after nephrectomy, but pERK was not associated with active proliferation at 1-month post-nephrectomy. In vitro studies suggest high tubular flow induces ERK dependent ENaC Na absorption and may play a critical role in Na balance post-nephrectomy.

Hyperfiltration is an important underlying cause of glomerular dysfunction associated with several systemic and intrinsic glomerular conditions leading to chronic kidney disease (CKD). These include obesity, diabetes, hypertension, focal segmental glomerulosclerosis (FSGS), congenital abnormalities and reduced renal mass (low nephron number). Hyperfiltration-associated biomechanical forces directly impact the cell membrane, generating tensile and fluid flow shear stresses in multiple segments of the nephron. Ongoing research suggests these biomechanical forces as the initial mediators of hyperfiltration-induced deterioration of podocyte structure and function leading to their detachment and irreplaceable loss from the glomerular filtration barrier. Membrane lipid-derived polyunsaturated fatty acids (PUFA) and their metabolites are potent transducers of biomechanical stress from the cell surface to intracellular compartments. Omega-6 and ω-3 long-chain PUFA from membrane phospholipids generate many versatile and autacoid oxylipins that modulate pro-inflammatory as well as anti-inflammatory autocrine and paracrine signaling. We advance the idea that lipid signaling molecules, related enzymes, metabolites and receptors are not just mediators of cellular stress but also potential targets for developing novel interventions. With the growing emphasis on lifestyle changes for wellness, dietary fatty acids are potential adjunct-therapeutics to minimize/treat hyperfiltration-induced progressive glomerular damage and CKD.

Background: Suicide rates are on the rise globally. Many suicide completers are previous attempters. Risk identification of attempters will provide an opportunity for prompt and targeted intervention towards suicide prevention. The aim of this study was to determine the incidence of suicide attempt and self-inflicted injury (SASI), and its associations with deployment-related PTSD and/or TBI and attempted suicide among a large national cohort of Veterans deployed pre and post-9/11 2001 who received care from the Veterans Health Administration (VHA) between 1998 - 2014. Methods: Clinical data from over 1,400 VHA establishments provided by the Veterans Administrations Informatics and Computing Infrastructure (VINCI) were used for descriptive and Cox regression analyses. Results: There were 1,327,604 patients in the study - PTSD only (1,121,818), TBI only (100,033), both PTSD and TBI (105,753). The incidence of SASI in the entire cohort was 351 per 100,000 person years; PTSD only (323), TBI only (181), both PTSD and TBI (835). Compared to TBI only group, the adjusted risk for SASI was higher in the PTSD only group (HR=1.129; 95% CI =1.063 - 1.200) and much higher in those with both PTSD and TBI (HR=2.283; 95% CI =2.136 - 2.441). The risk of SASI was higher if they were younger than 50 years, divorced or separated, non-Hispanic females, homeless, suffered adult abuse and neglect, had substance use, generalized anxiety and antisocial personality disorders, and if they were in the Pre-9/11 service period. Proportion of people attempting suicide increased every year post-diagnosis of PTSD and/or TBI. Conclusion: We recommend prioritizing resources to prevent suicide among at risk groups.

Increased fluid flow shear stress (FFSS) in solitary kidney alters podocyte function in vivo. FFSS-treated cultured podocytes show upregulated AKT-GSK3β-β-catenin signaling. The present study was undertaken to confirm (i) the activation of β-catenin signaling in podocytes in vivo using unilaterally nephrectomized (UNX) TOPGAL mice with the β-galactosidase reporter gene for β-catenin activation, (ii) β-catenin translocation in FFSS-treated mouse podocytes, and (iii) β-catenin signaling using publicly available data from UNX mice. The UNX of TOPGAL mice resulted in glomerular hypertrophy and increased the mesangial matrix consistent with hemodynamic adaptation. Uninephrectomized TOPGAL mice showed an increased β-galactosidase expression at 4 weeks but not at 12 weeks, as assessed using immunofluorescence microscopy (p < 0.001 at 4 weeks; p = 0.16 at 12 weeks) and X-gal staining (p = 0.008 at 4 weeks; p = 0.65 at 12 weeks). Immunofluorescence microscopy showed a significant increase in phospho-β-catenin (Ser552, p = 0.005) at 4 weeks but not at 12 weeks (p = 0.935) following UNX, and the levels of phospho-β-catenin (Ser675) did not change. In vitro FFSS caused a sustained increase in the nuclear translocation of phospho-β-catenin (Ser552) but not phospho-β-catenin (Ser675) in podocytes. The bioinformatic analysis of the GEO dataset, #GSE53996, also identified β-catenin as a key upstream regulator. We conclude that transcription factor β-catenin mediates FFSS-induced podocyte (glomerular) injury in solitary kidney.

Objective: To examine the effect of cigarette smoking (CS) status and total testosterone (TT) levels after testosterone replacement therapy (TRT) on all-cause mortality, myocardial infarction (MI), and stroke in male smokers and nonsmokers without history of MI and stroke. Participants and Methods: Data from 18,055 males with known CS status and low TT levels who received TRT at the Veterans Health Administration between December 1, 1999, and May 31, 2014, were grouped into (1) current smokers with normalized TT, (2) current smokers with nonnormalized TT, (3) nonsmokers with normalized TT, and (4) nonsmokers with nonnormalized TT. Combined effect of CS status and TT level normalization after TRT on all-cause mortality, MI, and stroke was compared using propensity score–weighted Cox proportional hazard models. Results: Normalization of serum TT levels in nonsmokers was associated with a significant decrease in all-cause mortality (hazard ratio [HR]=0.526; 95% CI, 0.477-0.581; P

Cardiotrophin-like cytokine factor 1 (CLCF1) is secreted as a complex with the cytokine receptor-like factor 1 (CRLF1). Syndromes caused by mutations in the genes encoding CLCF1 or CRLF1 suggest an important role for CLCF1 in the development and regulation of the immune system. In mice, CLCF1 induces B-cell expansion, enhances humoral responses and triggers autoimmunity. Interestingly, inactivation of CRLF1, which impedes CLCF1 secretion, leads to a marked reduction in the number of bone marrow (BM) progenitor cells, while mice heterozygous for CLCF1 display a significant decrease in their circulating leukocytes. We therefore hypothesized that CLCF1 might be implicated in the regulation of hematopoiesis. To test this hypothesis, murine hematopoietic progenitor cells defined as Lin−Sca1+c-kit+ (LSK) were treated in vitro with ascending doses of CLCF1. The frequency and counts of LSK cells were significantly increased in the presence of CLCF1, which may be mediated by several CLCF1-induced soluble factors including IL-6, G-CSF, IL-1β, IL-10, and VEGF. CLCF1 administration to non-diseased C57BL/6 mice resulted in a pronounced increase in circulating myeloid cells, which was concomitant with augmented LSK and myeloid cell counts in the BM. Likewise, CLCF1 administration to mice following sub-lethal irradiation or congeneic BM transplantation (BMT) resulted in accelerated LSK recovery along with a sustained increase in BM-derived CD11b+ cells. Altogether, our observations establish an important and unforeseen role for CLCF1 in regulating hematopoiesis with a bias toward myeloid cell differentiation.

Non-small-cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutation eventually develop resistance to EGFR-targeted tyrosine kinase inhibitors (TKIs). Treatment resistance remains the primary obstacle to the successful treatment of NSCLC. Although drug resistance mechanisms have been studied extensively in NSCLC, the regulation of these mechanisms has not been completely understood. Recently, increasing numbers of microRNAs (miRNAs) are implicated in EGFR-TKI resistance, indicating that miRNAs may serve as novel targets and may hold promise as predictive biomarkers for anti-EGFR therapy. MicroRNA-506 (miR-506) has been identified as a tumor suppressor in many cancers, including lung cancer; however, the role of miR-506 in lung cancer chemoresistance has not yet been addressed. Here we report that miR-506-3p expression was markedly reduced in erlotinib-resistant (ER) cells. We identified Sonic Hedgehog (SHH) as a novel target of miR-506-3p, aberrantly activated in ER cells. The ectopic overexpression of miR-506-3p in ER cells downregulates SHH signaling, increases E-cadherin expression, and inhibits the expression of vimentin, thus counteracting the epithelial–mesenchymal transition (EMT)-mediated chemoresistance. Our results advanced our understanding of the molecular mechanisms underlying EGFR-TKI resistance and indicated that the miR-506/SHH axis might represent a novel therapeutic target for future EGFR mutated lung cancer treatment.

BackgroundAtrial fibrillation (AF) is the most common cardiac dysrhythmia associated with significant morbidity and mortality. Several small studies have reported that low serum total testosterone (TT) levels were associated with a higher incidence of AF. In contrast, it is also reported that anabolic steroid use is associated with an increase in the risk of AF. To date, no study has explored the effect of testosterone normalization on new incidence of AF after testosterone replacement therapy (TRT) in patients with low testosterone. Methods and ResultsUsing data from the Veterans Administrations Corporate Data Warehouse, we identified a national cohort of 76 639 veterans with low TT levels and divided them into 3 groups. Group 1 had TRT resulting in normalization of TT levels (normalized TRT), group 2 had TRT without normalization of TT levels (nonnormalized TRT), and group 3 did not receive TRT (no TRT). Propensity score–weighted stabilized inverse probability of treatment weighting Cox proportional hazard methods were used for analysis of the data from these groups to determine the association between post‐TRT levels of TT and the incidence of AF. Group 1 (40 856 patients, median age 66 years) had significantly lower risk of AF than group 2 (23 939 patients, median age 65 years; hazard ratio 0.90, 95% CI 0.81–0.99, P=0.0255) and group 3 (11 853 patients, median age 67 years; hazard ratio 0.79, 95% CI 0.70–0.89, P=0.0001). There was no statistical difference between groups 2 and 3 (hazard ratio 0.89, 95% CI 0.78– 1.0009, P=0.0675) in incidence of AF. ConclusionsThese novel results suggest that normalization of TT levels after TRT is associated with a significant decrease in the incidence of AF.

Circulating permeability factors have been identified in the plasma of patients with focal segmental glomerulosclerosis (FSGS). Post-transplant recurrence of proteinuria, improvement of proteinuria after treatment with plasmapheresis, and induction of proteinuria in experimental animals by plasma fractions each provide evidence for such plasma factors. Advanced proteomic methods have identified candidate molecules in recurrent FSGS. We have proposed cardiotrophin-like cytokine-1 as an active factor in FSGS. Another potential permeability factor in FSGS is soluble urokinase receptor. In our studies, in vitro plasma permeability activity is blocked by substances that may decrease active molecules or block their effects. We have shown that the simple sugar galactose blocks the effect of FSGS serum in vitro and decreases permeability activity when administered to patients. Since the identities of permeability factors and their mechanisms of action are not well defined, treatment of FSGS is empiric. Corticosteroids are the most common agents for initial treatment. Calcineurin inhibitors, such as cyclosporine A, and tacrolimus and immunosuppressive medications, including mycophenylate, induce remission is some patients with steroid-resistant or -dependent nephrotic syndrome. Therapies that diminish proteinuria and slow progression in FSGS as well as other conditions include renin-angiotensin blockade, blood pressure lowering and plasma lipid control. Use of findings from in vitro studies, coupled with definitive identification of pathogenic molecules, may lead to new treatments to arrest FSGS progression and prevent recurrence after transplantation.

CLCF-1 is a cytokine known for B-cell stimulation and for neurotrophic properties. We have identified CLCF-1 as a potential injurious factor in the human renal disease focal segmental glomerulosclerosis (FSGS). We investigated its effects on renal cells and renal function in in vitro and in vivo studies. Methods include measurement of the effect of CLCF-1 on phosphorylation of target molecules of the JAK/STAT pathway, on cytoskeleton and cell morphology in cultured podocytes, on albumin permeability of isolated rat glomeruli, and on tissue phosphorylation and urine albumin after acute or chronic CLCF-1 injection. In addition, cell sorting was performed to determine the presence of cells expressing CLCF-1 in spleen and bone marrow of normal mice and the effect of CLCF-1 infusion on splenic B-cell populations. CLCF-1 increased phosphorylation of STAT3 in multiple cell types, activated podocytes leading to formation of lamellipodia and decrease in basal stress fibers, increased glomerular albumin permeability, and increased STAT3 phosphorylation of peripheral blood cells and renal cortex. CLCF-1 increased urine albumin/creatinine ratio in mice and increased B-cell expression of IgG in mouse spleen. We conclude that CLCF-1 has potentially important systemic effects, alters podocyte function, and may contribute to renal dysfunction and albuminuria.

We examined the effect of intact human mast cells (HMC-1 5C6) and their selected mediators on interleukin-6 (IL-6) production and bone morphogenetic protein-2 (BMP-2) expression in human coronary artery endothelial cells (HCAEC) in the presence and absence of lipopolysaccharide (LPS). Scanning electron microscopy showed that HMC-1 5C6 cells adhere to HCAEC in cocultures. Addition of HMC-1 5C6 cells markedly enhanced the IL-6 production by quiescent and LPS-activated HCAEC even at the maximal concentration of LPS. Furthermore, mast cell-derived histamine and proteases accounted for the direct and synergistic effect of mast cells on IL-6 production that was completely blocked by the combination of histamine receptor-1 antagonist and protease inhibitors. Another novel finding is that histamine was able to induce BMP-2 expression in HCAEC. Collectively, our results suggest that endotoxin and mast cell products synergistically amplify vascular inflammation and that histamine participates in the early events of vascular calcification.

Abstract??Increased infiltration of the kidney by mast cells is associated with proteinuria, and interstitial fibrosis in various renal diseases. Mast cells produce serine proteases including tryptase and chymase (MCC) that act via protease-activated receptors (PARs) to induce synthesis of fibrogenic cytokines by renal cells. In the present study, we investigated direct effect of MCC and role of PARs on glomerular albumin permeability (Palb). Isolated rat glomeruli were incubated with MCC (0.1, 1, 10, and 100?ng/ml) for 5?30?min in presence or absence of PAR-1 and PAR-2 blocking antibodies. Palbwas determined from the change in glomerular volume in response to an albumin oncotic gradient. The effect of direct activation of PARs on Palbwas verified by incubating glomeruli with synthetic hexapeptide known to activate PAR-1 and PAR-2. MCC increased Palbof isolated rat glomeruli in a dose- and time-dependent manner. Blocking PAR-2 prevented MCC-mediated increase in Palb. RT-PCR analysis of glomerular RNA demonstrated the presence of constitutively expressed PAR-1, -2, and -3 and low levels of PAR-4. In addition, direct activation of PAR-2 by hexapeptide SLIGKV increased Palbcomparable to MCC, whereas PAR-1 activation by TFLLRN had no effect on Palb. Our results document that MCC induces increase in Palband that this effect is mediated through PAR-2. MCC may also play a role in renal scarring. We propose that inhibiting MCC activity or blocking the activation of PAR-2 may provide new targets for therapy in renal diseases. [ABSTRACT FROM AUTHOR]