Bone marrow-derived cPLA2α contributes to renal fibrosis progression.

The group IVA calcium-dependent cytosolic phospholipase A2 (cPLA2α) enzyme directs a complex "eicosanoid storm" that accompanies the tissue response to injury. cPLA2α and its downstream eicosanoid mediators are also implicated in the pathogenesis of fibrosis in many organs, including the kidney. We aimed to determine the role of cPLA2α in bone marrow-derived cells in a murine model of renal fibrosis, unilateral ureteral obstruction (UUO). WT C57BL/6J mice were irradiated and engrafted with donor bone marrow from either WT mice [WT-bone marrow transplant (BMT)] or mice deficient in cPLA2α (KO-BMT). After full engraftment, mice underwent UUO and kidneys were collected 3, 7, and 14 days after injury. Using picrosirius red, collagen-3, and smooth muscle α actin staining, we determined that renal fibrosis was significantly attenuated in KO-BMT animals as compared with WT-BMT animals. Lipidomic analysis of homogenized kidneys demonstrated a time-dependent upregulation of pro-inflammatory eicosanoids after UUO; KO-BMT animals had lower levels of many of these eicosanoids. KO-BMT animals also had fewer infiltrating pro-inflammatory CD45+CD11b+Ly6Chi macrophages and reduced message levels of pro-inflammatory cytokines. Our results indicate that cPLA2α and/or its downstream mediators, produced by bone marrow-derived cells, play a major role in eicosanoid production after renal injury and in renal fibrinogenesis.

Cytosolic phospholipase A2α increases proliferation and de-differentiation of human renal tubular epithelial cells.

The group IVA calcium-dependent cytosolic phospholipase A2 (cPLA2α) enzyme controls the release of arachidonic acid from membrane bound phospholipids and is the rate-limiting step in production of eicosanoids. A variety of different kidney injuries activate cPLA2α, therefore we hypothesized that cPLA2α activity would regulate pathologic processes in HK-2 cells, a human renal tubular epithelial cell line, by regulating cell phenotype and proliferation. In two lentiviral cPLA2α-silenced knockdowns, we observed decreased proliferation and increased apoptosis compared to control HK-2 cells. cPLA2α-silenced cells also demonstrated an altered morphology, had increased expression E-cadherin, and decreased expression of Ncadherin. Increased levels of E-cadherin were associated with increased promoter activity and decreased levels of SNAIL1, SNAIL2, and ZEB1, transcriptional repressors of E-cadherin expression. Addition of exogenous arachidonic acid, but not PGE2, reversed the phenotypic changes in cPLA2α-silenced cells. These data suggest that cPLA2α may play a key role in renal repair after injury through a PGE2-independent mechanism.

Label-free fluorescence lifetime and second harmonic generation imaging microscopy improves quantification of experimental renal fibrosis.

All forms of progressive renal diseases develop a final pathway of tubulointerstitial fibrosis and glomerulosclerosis. Renal fibrosis is usually quantified using histological staining, a process that is time-consuming and pathologist dependent. Here we develop a fast and operator-independent method to measure fibrosis utilizing the murine unilateral ureteral obstruction model which manifests a time-dependent fibrotic increase in obstructed kidneys while the contralateral kidneys are used as controls. After ureteral obstruction, kidneys were analyzed at 7, 14, and 21 days. Fibrosis was quantified using fluorescence lifetime imaging (FLIM) and second harmonic generation (SHG) in a Deep Imaging via Enhanced photon Recovery deep tissue imaging microscope. This microscope was developed for deep tissue along with second and third harmonic generation imaging and has extraordinary sensitivity toward harmonic generation. SHG data suggest the presence of more fibrillar collagen in the obstructed kidneys. The combination of short-wavelength FLIM and SHG analysis results in a robust assessment procedure independent of observer interpretation and let us create criteria to quantify the extent of fibrosis directly from the image. Thus, the FLIM-SHG technique shows remarkable improvement in quantification of renal fibrosis compared to standard histological techniques.

Activation of the retinoid X receptor modulates angiotensin II-induced smooth muscle gene expression and inflammation in vascular smooth muscle cells.

The retinoid X receptor (RXR) partners with numerous nuclear receptors, such as the peroxisome proliferator activated receptor (PPAR) family, liver X receptors (LXRs), and farnesoid X receptor (FXR). Although each heterodimer can be activated by specific ligands, a subset of these receptors, defined as permissive nuclear receptors, can also be activated by RXR agonists known as rexinoids. Many individual RXR heterodimers have beneficial effects in vascular smooth muscle cells (SMCs). Because rexinoids can potently activate multiple RXR pathways, we hypothesized that treating SMCs with rexinoids would more effectively reverse the pathophysiologic effects of angiotensin II than an individual heterodimer agonist. Cultured rat aortic SMCs were pretreated with either an RXR agonist (bexarotene or 9-cis retinoic acid) or vehicle (dimethylsulfoxide) for 24 hours before stimulation with angiotensin II. Compared with dimethylsulfoxide, bexarotene blocked angiotensin II-induced SM contractile gene induction (calponin and smooth muscle-α-actin) and protein synthesis ([(3)H]leucine incorporation). Bexarotene also decreased angiotensin II-mediated inflammation, as measured by decreased expression of monocyte chemoattractant protein-1 (MCP-1). Activation of p38 mitogen-activated protein (MAP) kinase but not extracellular signal-related kinase (ERK) or protein kinase B (Akt) was also blunted by bexarotene. We compared bexarotene to five agonists of nuclear receptors (PPARα, PPARγ, PPARδ, LXR, and FXR). Bexarotene had a greater effect on calponin reduction, MCP-1 inhibition, and p38 MAP kinase inhibition than any individual agonist. PPARγ knockout cells demonstrated blunted responses to bexarotene, indicating that PPARγ is necessary for the effects of bexarotene. These data demonstrate that RXR is a potent modulator of angiotensin II-mediated responses in the vasculature, partially through inhibition of p38.