From Inflammation to the Onset of Fibrosis through A2A Receptors in Kidneys from Deceased Donors.

Pretransplant graft inflammation could be involved in the worse prognosis of deceased donor (DD) kidney transplants. A2A adenosine receptor (A2AR) can stimulate anti-inflammatory M2 macrophages, leading to fibrosis if injury and inflammation persist. Pre-implantation biopsies of kidney donors (47 DD and 21 living donors (LD)) were used to analyze expression levels and activated intracellular pathways related to inflammatory and pro-fibrotic processes. A2AR expression and PKA pathway were enhanced in DD kidneys. A2AR gene expression correlated with TGF-ß1 and other profibrotic markers, as well as CD163, C/EBPß, and Col1A1, which are highly expressed in DD kidneys. TNF-α mRNA levels correlated with profibrotic and anti-inflammatory factors such as TGF-ß1 and A2AR. Experiments with THP-1 cells point to the involvement of the TNF-α/NF-κB pathway in the up-regulation of A2AR, which induces the M2 phenotype increasing CD163 and TGF-ß1 expression. In DD kidneys, the TNF-α/NF-κB pathway could be involved in the increase of A2AR expression, which would activate the PKA-CREB axis, inducing the macrophage M2 phenotype, TGF-ß1 production, and ultimately, fibrosis. Thus, in inflamed DD kidneys, an increase in A2AR expression is associated with the onset of fibrosis, which may contribute to graft dysfunction and prognostic differences between DD and LD transplants.

Mycophenolate Mofetil in C3 Glomerulopathy and Pathogenic Drivers of the Disease.

BACKGROUND AND OBJECTIVES: C3 glomerulopathy is a complement-mediated disease arising from abnormalities in complement genes and/or antibodies against complement components. Previous studies showed that treatment with corticosteroids plus mycophenolate mofetil (MMF) was associated with improved outcomes, although the genetic profile of these patients was not systematically analyzed. This study aims to analyze the main determinants of disease progression and response to this therapeutic regimen. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We conducted a retrospective, multicenter, observational cohort study in 35 nephrology departments belonging to the Spanish Group for the Study of Glomerular Diseases. Patients diagnosed with C3 glomerulopathy (n=81) or dense deposit disease (n=16) between January 1995 and March 2018 were enrolled. Multivariable and propensity score matching analyses were used to evaluate the association of clinical and genetic factors with response to treatment with corticosteroids and MMF as measured by proportion of patients with disease remission and kidney survival (status free of kidney failure). RESULTS: The study group comprised 97 patients (84% C3 glomerulopathy, 16% dense deposit disease). Forty-two patients were treated with corticosteroids plus MMF, and this treatment was associated with a higher rate of remission and lower probability of kidney failure (79% and 14%, respectively) compared with patients treated with other immunosuppressives (24% and 59%, respectively), or ecluzimab (33% and 67%, respectively), or conservative management (18% and 65%, respectively). The therapeutic superiority of corticosteroids plus MMF was observed both in patients with complement abnormalities and with autoantibodies. However, patients with pathogenic variants in complement genes only achieved partial remission, whereas complete remissions were common among patients with autoantibody-mediated forms. The main determinant of no remission was baseline proteinuria. Relapses occurred after treatment discontinuation in 33% of the patients who had achieved remission with corticosteroids plus MMF, and a longer treatment length of MMF was associated with a lower risk of relapse. CONCLUSIONS: The beneficial response to corticosteroids plus MMF treatment in C3 glomerulopathy appears independent of the pathogenic drivers analyzed in this study.

Reduced Adenosine Uptake and Its Contribution to Signaling that Mediates Profibrotic Activation in Renal Tubular Epithelial Cells: Implication in Diabetic Nephropathy.

Altered nucleoside levels may be linked to pathogenic signaling through adenosine receptors. We hypothesized that adenosine dysregulation contributes to fibrosis in diabetic kidney disease. Our findings indicate that high glucose levels and experimental diabetes decreased uptake activity through the equilibrative nucleoside transporter 1 (ENT1) in proximal tubule cells. In addition, a correlation between increased plasma content of adenosine and a marker of renal fibrosis in diabetic rats was evidenced. At the cellular level, exposure of HK2 cells to high glucose, TGF-ß and the general adenosine receptor agonist NECA, induced the expression of profibrotic cell activation markers α-SMA and fibronectin. These effects can be avoided by using a selective antagonist of the adenosine A3 receptor subtype in vitro. Furthermore, induction of fibrosis marker α-SMA was prevented by the A3 receptor antagonist in diabetic rat kidneys. In conclusion, we evidenced the contribution of purinergic signaling to renal fibrosis in experimental diabetic nephropathy.

HLA-DQA1 and PLA2R1 polymorphisms and risk of idiopathic membranous nephropathy.

BACKGROUND AND OBJECTIVES: Single nucleotide polymorphisms (SNPs) within HLA complex class II HLA-DQ α-chain 1 (HLA-DQA1) and M-type phospholipase A2 receptor (PLA2R1) genes were identified as strong risk factors for idiopathic membranous nephropathy (IMN) development in a recent genome-wide association study. Copy number variants (CNVs) within the Fc gamma receptor III (FCGR3) locus have been associated with several autoimmune diseases, but their role in IMN has not been studied. This study aimed to validate the association of HLA-DQA1 and PLA2R1 risk alleles with IMN in a Spanish cohort, test the putative association of FCGR3A and FCGR3B CNVs with IMN, and assess the use of these genetic factors to predict the clinical outcome of the disease. DESIGN, SETTINGS, PARTICIPANTS, & MEASUREMENTS: A Spanish cohort of 89 IMN patients and 286 matched controls without nephropathy was recruited between October of 2009 and July of 2012. Case-control studies for SNPs within HLA-DQA1 (rs2187668) and PLA2R1 (rs4664308) genes and CNVs for FCGR3A and FCGR3B genes were performed. The contribution of these polymorphisms to predict clinical outcome and renal function decline was analyzed. RESULTS: This study validated the association of these HLA-DQA1 and PLA2R1 SNPs with IMN in a Spanish cohort and its increased risk when combining both risk genotypes. No significant association was found between FCGR3 CNVs and IMN. These results revealed that HLA-DQA1 and PLA2R1 genotype combination adjusted for baseline proteinuria strongly predicted response to immunosuppressive therapy. HLA-DQA1 genotype adjusted for proteinuria was also linked with renal function decline. CONCLUSION: This study confirms that HLA-DQA1 and PLA2R1 genotypes are risk factors for IMN, whereas no association was identified for FCGR3 CNVs. This study provides, for the first time, evidence of the contribution of these HLA-DQA1 and PLA2R1 polymorphisms in predicting IMN response to immunosuppressors and disease progression. Future studies are needed to validate and identify prognostic markers.

Adenosine A(2B) receptor-mediated VEGF induction promotes diabetic glomerulopathy.

Diabetic nephropathy ranks as the most devastating kidney disease worldwide. It characterizes in the early onset by glomerular hypertrophy, hyperfiltration and mesangial expansion. Experimental models show that overproduction of vascular endothelial growth factor (VEGF) is a pathogenic condition for podocytopathy; however the mechanisms that regulate this growth factor induction are not clearly identified. We determined that the adenosine A(2B) receptor (A(2B)AR) mediates VEGF overproduction in ex vivo glomeruli exposed to high glucose concentration, requiring PKCα and Erk1/2 activation. The glomerular content of A(2B)AR was concomitantly increased with VEGF at early stages of renal disease in streptozotocin-induced diabetic rats. Further, in vivo administration of an antagonist of A(2B)AR in diabetic rats blocked the glomerular overexpression of VEGF, mesangial cells activation and proteinuria. In addition, we also determined that the accumulation of extracellular adenosine occurs in glomeruli of diabetic rats. Correspondingly, raised urinary adenosine levels were found in diabetic rats. In conclusion, we evidenced that adenosine signaling at the onset of diabetic kidney disease is a pathogenic event that promotes VEGF induction.

New role of the human equilibrative nucleoside transporter 1 (hENT1) in epithelial-to-mesenchymal transition in renal tubular cells.

Epithelial-to-mesenchymal transition (EMT) is an important pro-fibrotic event in which tubular epithelial cells are transformed into myofibroblasts. Nucleoside transporters (NT) are regulated by many factors and processes, some of which are involved in fibrosis, such as cytokines, inflammation, and proliferation. Equilibrative nucleoside transporter 1 (ENT1) has been proved to be the most widely expressed adenosine transporter. In that sense, ENT1 may be a key player in cell damage signaling. Here we analyze the role of human ENT1 (hENT1) in the EMT process in proximal tubular cells. Addition of the main inducer of EMT, the transforming growth factor-ß1, to HK-2 cells increased hENT1 mRNA and protein level expression. ENT1-mediated adenosine uptake was also enhanced. When cells were incubated with dipyridamole to evaluate the potential contribution of ENT1 to EMT by blocking its transport activity, EMT was induced. Moreover, the knock down of hENT1 with siRNA induced EMT and collagen production in HK-2 cells. Kidneys isolated from ENT1 knockout mice showed higher levels of interstitial collagen and α-SMA positive cells than wild-type mice. Our results point to a new potential role of hENT1 as a modulator of EMT in proximal tubular cells. In this sense, hENT1 could be involved in renal protection processes, and the loss or reduced expression of hENT1 would lead to an increased vulnerability of cells to the onset and/or progression of renal fibrosis.

n-3 Fatty acids block TNF-α-stimulated MCP-1 expression in rat mesangial cells.

Monocyte chemoattractant protein 1 (MCP-1) is a CC cytokine that fundamentally contributes to the pathogenesis of inflammatory renal disease. MCP-1 is highly expressed in cytokine-stimulated mesangial cells in vitro and following glomerular injury in vivo. Interventions to limit MCP-1 expression are commonly effective in assorted experimental models. Fish oil, an abundant source of n-3 fatty acids, has anti-inflammatory properties, the basis of which remains incompletely defined. We examined potential mechanisms whereby fish oil reduces MCP-1 expression and thereby suppresses inflammatory responses to tissue injury. Cultured mesangial cells were treated with TNF-α in the presence of the n-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA); equimolar concentrations of the n-6 fatty acids LA and OA served as controls. MCP-1 mRNA expression was assessed by Northern blotting, and transcriptional activity of the MCP-1 promoter was assessed by transient transfection. The involvement of the ERK and NF-κB pathways was evaluated through transfection analysis and the use of the MEK inhibitor U0126. DHA and EPA decreased TNF-α-stimulated MCP-1 mRNA expression by decreasing transcription of the MCP-1 gene. DHA and EPA decreased p-ERK expression and nuclear translocation of NF-κB, both of which are necessary for TNF-α-stimulated MCP-1 expression. Both NF-κB and AP-1 sites were involved in transcriptional regulation of the MCP-1 gene by DHA and EPA. We conclude that DHA and EPA inhibit TNF-α-stimulated transcription of the MCP-1 gene through interaction of signaling pathways involving ERK and NF-κB. We speculate that such effects may contribute to the salutary effect of fish oil in renal and vascular disease.

Signaling pathways modulated by fish oil in salt-sensitive hypertension.

Although many studies have indicated that fish oil (FO) improves cardiovascular risk factors and reduces histopathological manifestations of injury in experimental renal injury models, potential mechanisms underlying this protective effect have not been adequately defined. The objective of this study was to identify potential signaling pathways that confer protection in the Dahl rat model of salt-sensitive hypertension. Male Dahl salt-sensitive rats (n = 10/group) were provided with formulated diets containing 8% NaCl, 20% protein, and 25% FO or 25% corn oil (CO) for 28 days. FO reduced blood pressure (-11% at 4 wk; P < 0.05), urine protein excretion (-45% at 4 wk; P < 0.05), plasma cholesterol and triglyceride levels (-54%, P < 0.001; and -58%, P < 0.05), and histopathological manifestations of renal injury, including vascular hypertrophy, segmental and global glomerular sclerosis, interstitial fibrosis, and tubular atrophy. Interstitial inflammation was significantly reduced by FO (-32%; P < 0.001), as assessed by quantitative analysis of ED1-positive cells in sections of the renal cortex. FO reduced tubulointerstitial proliferative activity, as assessed by Western blot analysis of cortical homogenates for PCNA (-51%; P < 0.01) and quantitative analysis of Mib-1-stained sections of the renal cortex (-42%; P < 0.001). Decreased proliferative activity was associated with reduced phospho-ERK expression (-37%; P < 0.005) and NF-kappaB activation (-42%; P < 0.05). FO reduced cyclooxygenase (COX)-2 expression (-63%; P < 0.01) and membrane translocation of the NADPH oxidase subunits p47(phox) and p67(phox) (-26 and -34%; P < 0.05). We propose that FO ameliorates renal injury in Dahl salt-sensitive rats through the inhibition of ERK, decreased NF-kappaB activation, inhibition of COX-2 expression, and decreased NADPH oxidase activation.

TGF-beta1 stimulates monocyte chemoattractant protein-1 expression in mesangial cells through a phosphodiesterase isoenzyme 4-dependent process.

Monocyte chemoattractant protein-1 (MCP-1) and transforming growth factor (TGF)-beta1 are critical mediators of renal injury by promoting excessive inflammation and extracellular matrix deposition, thereby contributing to progressive renal disease. In renal disease models, MCP-1 stimulates the production of TGF-beta1. However, a potential role for TGF-beta1 in the regulation of MCP-1 production by mesangial cells (MCs) has not previously been evaluated. The objectives of this study were to define the role of TGF-beta1 in regulation of MCP-1 expression in cultured MCs and to define mechanisms through which rolipram (Rp), a phosphodiesterase isoenzyme 4 (PDE4) inhibitor with anti-inflammatory properties, alters MCP-1 expression. TGF-beta1 induced MCP-1 in a time- and dose-dependent manner without increasing transcription of the MCP-1 gene. TGF-beta1-mediated induction of MCP-1 occurred without activation of the NF-kappaB pathway. Rp blocked TGF-beta1-stimulated MCP-1 expression via a protein kinase A-dependent process, at least in part, by decreasing MCP-1 message stability. Rp exerted no effect on activation of the Smad pathway by TGF-beta1. TGF-beta1-mediated induction of MCP-1 required activation of ERK and p38, both of which were suppressed by a PDE4 inhibitor. TGF-beta1-stimulated reactive oxygen species (ROS) generation by MCs, and Rp inhibited ROS generation in TGF-beta1-stimulated MCs; in addition, both Rp and ROS scavengers blocked TGF-beta1-stimulated MCP-1 expression. We conclude that TGF-beta1 stimulates MCP-1 expression through pathways involving activation of ERK, p38, and ROS generation. Positive cross-talk between TGF-beta1 and MCP-1 signaling in MCs may underlie the development of progressive renal disease. Rp, by preventing TGF-beta1-stimulated MCP-1 production, may offer a therapeutic approach in retarding the progression of renal disease.

Differential regulation of mesangial cell mitogenesis by cAMP phosphodiesterase isozymes 3 and 4.

Mesangial cell (MC) mitogenesis is regulated through "negative cross talk" between cAMP-PKA and ERK signaling. Although it is widely accepted that cAMP inhibits mitogenesis through PKA-mediated phosphorylation of Raf-1, recent studies have indicated that cAMP-mediated inhibition of mitogenesis may occur independently of Raf-1 phosphorylation or without inhibiting ERK activity. We previously showed that MCs possess functionally compartmentalized intracellular pools of cAMP that are differentially regulated by cAMP phosphodiesterases (PDE); an intracellular pool directed by PDE3 but not by PDE4 suppresses mitogenesis. We therefore sought to determine whether there was a differential effect of PDE3 vs. PDE4 inhibitors on the Ras-Raf-MEK-ERK pathway in cultured MC. Although PDE3 and PDE4 inhibitors activated PKA and modestly elevated cAMP levels to a similar extent, only PDE3 inhibitors suppressed MC mitogenesis (-57%) and suppressed Raf-1 kinase and ERK activity (-33 and -68%, respectively). Both PDE3 and PDE4 inhibitors suppressed B-Raf kinase activity. PDE3 inhibitors increased phosphorylation of Raf-1 on serine 43 and serine 259 and decreased phosphorylation on serine 338; PDE4 inhibitors were without effect. Overexpression of a constitutively active MEK-1 construct reversed the antiproliferative effect of PDE3 inhibitors. PDE3 inhibitors also reduced cyclin A levels (-27%), cyclin D and cyclin E kinase activity (-30 and -50%, respectively), and induced expression of the cell cycle inhibitor p21 (+90%). We conclude that the antiproliferative effects of PDE3 inhibitors are mechanistically related to inhibition of the Ras-Raf-MEK-ERK pathway. Additional cell cycle targets of PDE3 inhibitors include cyclin A, cyclin D, cyclin E, and p21.

Correlation of quantitative digital image analysis with the glomerular filtration rate in chronic allograft nephropathy.

Chronic allograft nephropathy (CAN) is characterized by progressive renal functional loss and histologic abnormalities of one or more tissue compartments. In this study, correlations between histologic abnormalities and graft function [glomerular filtration rate (GFR, measured by iothalamate clearance), serum creatinine (SCr) and urinary protein (UPr)] were investigated using biopsies from 49 patients with newly diagnosed CAN. Extent of tubulointerstitial fibrosis (%TIF), as assessed by a semi-quantitative score, correlated significantly with GFR, SCr and UPr. The close correlation between %TIF and GFR suggested that quantitative measurement of %TIF may predict functional consequences of CAN. Calculation of %TIF by computerized digital analysis was performed using four strategies: (a) quantitation of blue material in Masson's trichrome (MT)-stained sections, (b) quantitation of red material in Sirius Red-stained sections (SR-nonpolarized), (c) quantitation of birefringent material in Sirius Red stained-sections examined under polarized light (SR-polarized) and (d) quantification of brown material in sections stained by immunoperoxidase for alpha-smooth muscle actin. Only the SR-nonpolarized score correlated significantly with GFR at the time of biopsy-diagnosis of CAN. We conclude that digital analysis strategies demonstrate variable accuracy in quantifying %TIF. Validation of the SR-nonpolarized strategy against histologic scoring and GFR supports the application of this technique to longitudinal studies of CAN.