ABSTRACT
AIM: The anti-inflammatory protein annexin A1 (AnxA1) and its formyl peptide receptor 2 (FPR2) have protective effects in organ fibrosis. Their role in chronic kidney disease (CKD) has not yet been elucidated. Our aim was to characterize the AnxA1/FPR2 system in models of renal fibrosis. METHODS: Rats were treated with angiotensin receptor antagonist during the nephrogenic period (ARAnp) to induce late-onset hypertensive nephropathy and fibrosis. Localization and regulation of AnxA1 and FPR2 were studied by quantitative real-time PCR and double labelling immunofluorescence. Biological effects of AnxA1 were studied in cultured renal fibroblasts from AnxA1(-/-) and wild-type mice. RESULTS: Angiotensin receptor antagonist during the nephrogenic period kidneys displayed matrix foci containing CD73(+) fibroblasts, alpha-smooth muscle actin (a-SMA)(+) myofibroblasts and CD68(+) macrophages. TGF-ß and AnxA1 mRNAs were ~threefold higher than in controls. AnxA1 was localized to macrophages and fibroblasts; myofibroblasts were negative. FPR2 was localized to fibroblasts, myofibroblasts, macrophages and endothelial cells. AnxA1 and FPR2 immunoreactive signals were increased in the foci, with fibroblasts and macrophages expressing both proteins. AnxA1(-/-) fibroblasts revealed higher α-SMA (sevenfold) and collagen 1A1 (Col1A1; 144-fold) mRNA levels than controls. Treatment of murine WT fibroblasts with TGF-ß (22.5 ng mL 24 h(-1)) increased mRNA levels of α-SMA (9.3-fold) and Col1A1 (fourfold). These increases were greatly attenuated upon overexpression of AnxA1 (1.5- and 1.7-fold, respectively; P < 0.05). Human fibroblasts reacted similarly when receiving the FPR2 inhibitor WRW4. CONCLUSION: Our results demonstrate that AnxA1 and FPR2 are abundantly expressed in the renal interstitium and modulate fibroblast phenotype and extracellular matrix synthesis activity.
Subject(s)
Annexin A1/metabolism , Fibroblasts/metabolism , Fibrosis/metabolism , Kidney Diseases/metabolism , Kidney/metabolism , Signal Transduction/physiology , Animals , Blotting, Western , Disease Models, Animal , Fibrosis/pathology , Fluorescent Antibody Technique , Humans , Kidney/pathology , Kidney Diseases/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Rats , Real-Time Polymerase Chain Reaction , Receptors, Lipoxin/metabolismABSTRACT
Annexin A1 (ANXA1) exerts anti-inflammatory effects through multiple mechanisms including inhibition of prostaglandin synthesis. Once secreted, ANXA1 can bind to G protein-coupled formyl peptide receptors (Fpr) and activate diverse cellular signaling pathways. ANXA1 is known to be expressed in cells of the juxtaglomerular apparatus, but its relation to the expression of cyclooxygenase 2 (COX-2) in thick ascending limb and macula densa cells has not been elucidated. We hypothesized that ANXA1 regulates the biosynthesis of COX-2. ANXA1 abundance in rat kidney macula densa was extensively colocalized with COX-2 (95%). Furosemide, an established stimulus for COX-2 induction, caused enhanced expression of both ANXA1 and COX-2 with maintained colocalization (99%). In ANXA1-deficient mice, COX-2-positive cells were more numerous than in control mice (+107%; normalized to glomerular number; P < 0.05) and renin expression was increased (+566%; normalized to glomerular number; P < 0.05). Cultured macula densa cells transfected with full-length rat ANXA1 revealed downregulation of COX-2 mRNA (-59%; P < 0.05). Similarly, treatment with dexamethasone suppressed COX-2 mRNA in the cells (-49%; P < 0.05), while inducing ANXA1 mRNA (+56%; P < 0.05) and ANXA1 protein secretion. Inhibition of the ANXA-1 receptor Fpr1 with cyclosporin H blunted the effect of dexamethasone on COX-2 expression. These data show that ANXA1 exerts an inhibitory effect on COX-2 expression in the macula densa. ANXA1 may be a novel intrinsic modulator of renal juxtaglomerular regulation by inhibition of PGE(2) synthesis.
Subject(s)
Annexin A1/metabolism , Cyclooxygenase 2/metabolism , Kidney/metabolism , Animals , Cells, Cultured , Dexamethasone/pharmacology , Diuretics/pharmacology , Furosemide/pharmacology , Glucocorticoids/pharmacology , Kidney/cytology , Kidney/drug effects , Male , Mice , Mice, Inbred C57BL , Rats , Rats, Sprague-Dawley , Renin/biosynthesisABSTRACT
Na(+)-K(+)-2Cl(-) cotransporter (NKCC2)-mediated NaCl reabsorption in the thick ascending limb (TAL) is stimulated by AVP via V2 receptor/PKA/cAMP signaling. This process is antagonized by locally produced eicosanoids such as 20-HETE or prostaglandin E(2), which are synthesized in a phospholipase A(2)-dependent reaction cascade. Using microarray-based gene expression analysis, we found evidence for an AVP-dependent downregulation of the calcium-independent isoform of PLA(2), iPLA(2)ß, in the outer medulla of rats. In the present study, we therefore examined the contribution of iPLA(2)ß to NKCC2 regulation. Immunoreactive iPLA(2)ß protein was detected in cultured mTAL cells as well as in the entire TAL of rodents and humans with the exception of the macula densa. Administration of the V2 receptor-selective agonist desmopressin (5 ng/h; 3 days) to AVP-deficient diabetes insipidus rats increased outer medullary phosphorylated NKCC2 (pNKCC2) levels more than twofold in association with a marked reduction in iPLA(2)ß abundance (-65%; P < 0.05), thus confirming microarray results. Inhibition of iPLA(2)ß in Sprague-Dawley rats with FKGK 11 (0.5 µM) or in mTAL cells with FKGK 11 (10 µM) or (S)-bromoenol lactone (5 µM) for 1 h markedly increased pNKCC2 levels without affecting total NKCC2 expression. Collectively, these data indicate that iPLA(2)ß acts as an inhibitory modulator of NKCC2 activity and suggest that downregulation of iPLA(2)ß may be a relevant step in AVP-mediated urine concentration.
