An ultrastructural study of proliferative nephritis induced experimentally by a monoclonal antibody against mesangial cells: replacement of mesangial cells by cells of the monocyte-macrophage system

An experimental nephritis accompanied by transient proteinuria can be produced by an intravenous injection of the monoclonal antibody, 1-22-3, raised against isolated rat glomeruli. The present study deals with the ultrastructural changes in the glomeruli in rats after the injection of this antibody. At 2 h after injection, all the mesangial cells had completely degenerated and neutrophils invaded most mesangial areas. Monocytes occupied the vacant mesangial areas at 24 h and gradually increased in number over the next 4 days. At 4 and 6 days, macrophage-like cells, possibly derived from monocytes, underwent frequent mitosis, resulting in a remarkable proliferation of these cells. The interpretation of these cells as macrophages was strongly supported by the fact that they contained previously injected latex particles in large numbers. From 2 to 4 weeks after injection, the macrophage-like cells gradually transformed into cells indistinguishable from normal mesangial cells. In the present experimental nephritis where all mesangial cells were initially destroyed, cells of the monocyte-macrophage system appear to play a leading role in the pathogenesis of the ensuing proliferative glomerulonephritis, and represent the source of the replacing mesangial cells.     

Interleukin 1 and tumor necrosis factor synergistically stimulate prostaglandin synthesis and phospholipase A2 release from rat renal mesangial cells

Treatment of rat glomerular mesangial cells with recombinant human interleukin 1 alpha (rIL-1 alpha), recombinant human interleukin 1 beta (rIL-1 beta) or recombinant human tumor necrosis factor (rTNF) induces prostaglandin E2 (PGE2) synthesis and the release of a phospholipase A2 (PLA2) activity. rIL-1 beta is significantly more potent than rIL-1 alpha or rTNF in stimulating PGE2 as well as PLA2 release from mesangial cells. When given together, rTNF interacts in a synergistic fashion with rIL-1 alpha and rIL-1 beta to enhance both, PGE2 synthesis and PLA2 release. The released PLA2 has a neutral pH optimum and is calcium-dependent. Pretreatment of cells with actinomycin D or cycloheximide inhibits basal and cytokine-stimulated PGE2 and PLA2 release.     

Protocadherin 12 (VE-cadherin 2) is expressed in endothelial, trophoblast, and mesangial cells

Protocadherin 12 protein (PCDH12, VE-cadherin 2) is a cell adhesion molecule that has been isolated from endothelial cells. Here, we have used Northern and Western blots, immunohistology, and flow cytometry to examine the distribution of PCDH12 in mouse tissues. It is an N-glycosylated protein of 150-kDa mass. In the endothelium, PCDH12 immunoreactivity was variable and dependent upon the vascular bed. In both the embryo and embryonic stem cell differentiation system, signals were localized in vasculogenic rather than angiogenic endothelium. In addition, the protein was strongly expressed in a subset of invasive cells of the placenta, which were identified as glycogen-rich trophoblasts. In adult mice, strong PCDH12 signals were observed in mesangial cells of kidney glomeruli whereas expression was not detected in other types of perivascular cells. As opposed to most protocadherins, PCDH12 is not expressed in early embryonic (day 12.5) and adult brains. As a first approach to obtain insight into PCDH12 function, we produced transgenic mice deficient in PCDH12, which were viable and fertile. They did not display any obvious histomorphological defects. We conclude that PCDH12 has a unique expression pattern and that its deficiency does not lead to conspicuous abnormalities. Moreover, PCDH12 is the first specific marker for both glycogen-rich trophoblasts and mesangial cells.     

Purification and characterization of angiotensin converting enzyme 2 (ACE2) from murine model of mesangial cell in culture

Angiotensin converting enzyme 2 (ACE2) is a component of the renin-angiotensin system (RAS) which converts Ang II, a potent vasoconstrictor peptide into Ang 1-7, a vasodilator peptide which may act as a negative feedback hormone to the actions of Ang II. The discovery of this enzyme added a new level of complexity to this system. The mesangial cells (MC) have multiple functions in glomerular physiology and pathophysiology and are able to express all components of the RAS. Despite of being localized in these cells, ACE2 has not yet been purified or characterized. In this study ACE2 from mice immortalized MC (IMC) was purified by ion-exchange chromatography. The purified enzyme was identified as a single band around 60-70 kDa on SDS-polyacrylamide gel and by Western blotting using a specific antibody. The optima pH and chloride concentrations were 7.5 and 200 mM, respectively. The N-terminal sequence was homologous with many species ACE2 N-terminal sequences as described in the literature. ACE2 purified from IMC was able to hydrolyze Ang II into Ang 1-7 and the K_m value for Ang II was determined to be 2.87 ± 0.76 μM. In conclusion, we purified and localized, for the first time, ACE2 in MC, which was able to generate Ang 1-7 from Ang II. Ang 1-7 production associated to Ang II degradation by ACE2 may exert a protective effect in the renal hemodynamic.     

Identification and characterization of a hormonally regulated form of phospholipase A2 in rat renal mesangial cells

The activity of phospholipase A2 (PLA2), the regulatory enzyme in arachidonic acid release and prostaglandin synthesis, was measured in cell-free extracts of rat renal mesangial cells. Arginine vasopressin (AVP) and phorbol myristate acetate (PMA) both stimulated PLA2 activity as assayed by the release of free arachidonic acid from exogenously added [14C]arachidonyl-phosphatidylcholine. This represents a direct in vitro demonstration of hormone-induced changes in PLA2 activity. The stimulated activity was recovered following fractionation by DEAE-cellulose anion exchange and FPLC Superose 12 gel filtration. Stimulated activity from AVP- and PMA-treated cells comigrated as a single peak, suggesting that these agents are stimulating a single form of the enzyme. The molecular weight of this hormonally regulated form of PLA2 is approximately 60,000. The enzyme has an obligate requirement for Ca2+, having no activity in the presence of EGTA, and has a pH optimum in the alkaline range. Following cell disruption in the presence of chelators, the enzyme is recovered in a high speed supernatant. However, it appears that it can bind to a crude membrane fraction in a Ca2+-dependent manner, similar to other Ca2+-phospholipid binding proteins. The observed stable modification in PLA2 activity by AVP and PMA suggests a phosphorylation of PLA2 or PLA2 modulators by protein kinase C.     

Development of a potent 2-oxoamide inhibitor of secreted phospholipase A2 guided by molecular docking calculations and molecular dynamics simulations

Inhibition of group IIA secreted phospholipase A₂ (GIIA sPLA₂) has been an important objective for medicinal chemists. We have previously shown that inhibitors incorporating the 2-oxoamide functionality may inhibit human and mouse GIIA sPLA₂s. Herein, the development of new potent inhibitors by molecular docking calculations using the structure of the known inhibitor 7 as scaffold, are described. Synthesis and biological evaluation of the new compounds revealed that the long chain 2-oxoamide based on (S)-valine GK241 led to improved activity (IC₅₀ = 143 nM and 68 nM against human and mouse GIIA sPLA₂, respectively). In addition, molecular dynamics simulations were employed to shed light on GK241 potent and selective inhibitory activity.     

A role for G-proteins in the epidermal growth factor stimulation of phospholipase A2 in rat kidney mesangial cells

We have previously demonstrated phospholipase C (PLC) independent activation of phospholipase A₂(PLA₂) by epidermal growth factor (EGF) in glomerular mesangial cells in culture. In the current study using glass beads to permeabilize [³H]- or [¹⁴C]-arachidonate labelled mesangial cells we demonstrate that guanine nucleotides modulate the EGF-mediated stimulation of arachidonic acid release (75% inhibition with 100 M GDPS and 108% augmentation with 100 M GTPS). GTPS alone stimulated both the release of free arachidonic acid and production of diacylglycerol (DAG), while EGF itself neither stimulated DAG nor augmented the DAG response to GTPS. These findings suggest the intermediacy of a G-protein in PLC-independent stimulation of PLA₂ by a growth factor, and provide a model system for determining the relationship between G-protein intermediacy and the intrinsic tyrosine kinase activity of the growth factor receptor.Abbreviations EGF Epidermal Growth Factor - PLC phospholipase C - PLA₂ phospholipase A₂ - DAG Diacylglycerol - NEFA non-esterified fatty acid - GTPS guanosine-5-0-[3-thio]triphosphate - GDP\S guanosine-5-0-[2-thio]diphosphate     

The antioxidative effect of bone morphogenetic protein-7 against high glucose-induced oxidative stress in mesangial cells

Bone morphogenetic protein-7 (BMP-7) protects kidneys from diabetic nephropathy (DN), and high glucose (HG)-induced oxidative stress is involved in DN. We investigated the antioxidative ability of BMP-7 using HG-treated mesangial cells. We treated rat mesangial cells (RMCs) with recombinant human BMP-7 (rhBMP-7) and examined changes in reactive oxygen species (ROS) levels and intracellular signals in response to HG-induced oxidative stress. rhBMP-7 decreased the level of ROS in HG-treated RMCs. In contrast, lowering endogenous BMP-7 by siRNA or BMP receptor II (BMP-RII) by anti-BMP-RII antibodies increased the level of ROS in HG-treated RMCs. rhBMP-7 increased Smad-1,5,8 phosphorylation, decreased PKCζ and c-Jun N-terminal kinase (JNK) phosphorylation, and decreased fibronectin and collagen IV synthesis in HG-treated RMCs. In conclusion, we found that BMP-7 could protect mesangial cells from HG-induced oxidative stress by activating BMP-RII. The antioxidative activity of BMP-7 was primarily due to inhibition of PKCζ, JNK phosphorylation, and c-jun activation.     

Tryptase activates calcium-independent phospholipase A2 and releases PGE2 in airway epithelial cells

Human small airway epithelial cells (HSAEC) form the boundary between the external environmental allergens and the internal lung milieu. Mast cells are present in human lung tissue interspersed within the pulmonary epithelium and can secrete a host of pre- and newly formed mediators from their granules, which may propagate small airway inflammation. In this study, tryptase stimulation of HSAEC increased membrane-associated, calcium-independent phospholipase A(2)gamma (iPLA(2)gamma) activity, resulting in increased arachidonic acid and PGE(2) release. These responses were inhibited by pretreating HSAEC with the iPLA(2)-selective inhibitor bromoenol lactone. The tryptase-stimulated PGE(2) production was inhibited by treating HSAEC with the cyclooxygenase (COX)-1-selective inhibitor SC-560 and the nonselective COX inhibitor aspirin but not by the COX-2-selective inhibitor CAY10404, indicating that the early release of arachidonic acid is metabolized by constitutive COX-1 to form PGE(2) in tryptase-stimulated HSAEC. Additionally, platelet-activating factor production and neutrophil adherence to tryptase-stimulated HSAEC was also increased. This complex response can set up a cascade of inflammatory mediator production in small airways. We speculate that selective inhibition of iPLA(2)gamma-mediated phospholipid hydrolysis may prove beneficial in inflammatory airway diseases.     

The novel gene AngRem104 downregulates glucocorticoid receptor expression and activates NF-kappaB in human mesangial cells

AngRem104 [angiotensin II (Ang II)-related genes in human mesangial cells (MCs), clone104], a novel gene in human MCs induced by Ang II, was previously identified in human MCs and found to interact with several proteins. The current study used a yeast two-hybrid system and co-immunoprecipitation to investigate the interaction between AngRem104 and glucocorticoid receptor (GR) AF-1-specific elongation factor (GR-EF). GR expression was downregulated and the number of MCs positive for activated nuclear factor κB (NF-κB) was increased when AngRem104 was overexpressed. Transfection with antisense AngRem104 vector resulted in the upregulation of GR protein and reduced numbers of MCs with activated NF-κB. These results indicate that the novel gene AngRem104 is involved in the in vivo regulation of GR expression and the activation of NF-κB through interaction with GR-EF in human MCs.     

The lack of PDGE-stimulated PGE2 release from ras-transformed NIH-3T3 cells results from reduced phospholipase C but not phospholipase A2 activity

Our previous work demonstrated that NIH-3T3 cells expressing high levels of the mutated cellular ras oncogene (EJ-ras gene) exhibited reduced hormone-sensitive adenylate cyclase and platelet-derived growth factor-stimulated (PDGF) phospholipase A2/C activities. We now report that although the ras-transformed cells display markedly reduced phospholipase C activity, as measured by the levels of inositol 1,4,5-trisphosphate synthesized after PDGF-stimulation, normal levels of phospholipase A2 activity can be uncovered; thus, similar levels of prostaglandin E2 were synthesized in EJ-ras transformed and control cells after stimulation with phorbol myristate acetate (PMA) and/or the calcium ionophore A-23187, agents which stimulate protein kinase C and intracellular Ca2+ levels, respectively. These data suggest that the EJ-ras gene product uncouples the PDGF receptor from the phospholipase C, resulting in reduced PDGF-stimulated Ca2+ mobilization, protein kinase C stimulation and an apparent decrease in Ca2+-dependent phospholipase A2.     

Evidence that PAR2-triggered prostaglandin E2 (PGE2) formation involves the ERK-cytosolic phospholipase A2-COX-1-microsomal PGE synthase-1 cascade in human lung epithelial cells

We investigated possible involvement of three isozymes of prostaglandin E synthase (PGES), microsomal PGES-1 (mPGES-1), mPGES-2 and cytosolic PGES (cPGES) in COX-2-dependent prostaglandin E(2) (PGE(2)) formation following proteinase-activated receptor-2 (PAR2) stimulation in human lung epithelial cells. PAR2 stimulation up-regulated mPGES-1 as well as COX-2, but not mPGES-2 or cPGES, leading to PGE(2) formation. The PAR2-triggered up-regulation of mPGES-1 was suppressed by inhibitors of COX-1, cytosolic phospholipase A(2) (cPLA(2)) and MEK, but not COX-2. Finally, a selective inhibitor of mPGES-1 strongly suppressed the PAR2-evoked PGE(2) formation. PAR2 thus appears to trigger specific up-regulation of mPGES-1 that is dependent on prostanoids formed via the MEK/ERK/cPLA(2)/COX-1 pathway, being critical for PGE(2) formation.     

NG2 proteoglycan increases mesangial cell proliferation and extracellular matrix production

As a membrane-spanning protein, NG2 chondroitin sulfate proteoglycan interacts with molecules on both sides of plasma membrane. The present study explored the role of NG2 in the pathogenesis of diabetic nephropathy. In the normal kidneys, NG2 was observed predominantly in glomerular mesangium, Bowman's capsule and interstitial vessels. Both mRNA and protein expression in kidneys was significantly higher in strepozotocin-induced diabetic rats than that in normal rats. In the cultured rat mesangial cell line HBZY-1, overexpression of NG2 promoted mesangial cell proliferation and extracellular matrix (ECM) production, such as type VI collagen and laminin. Furthermore, target knockdown of NG2 resulted in decreased cell proliferation and ECM formation. The observations suggest that NG2 is up-regulated in diabetic nephropathy. It actively participates in the development and progression of glomerulosclerosis by stimulating proliferation of mesangial cells and deposition of ECM.     

Inhibition of endogenous BMP in the glomerulus leads to mesangial matrix expansion

Bone morphogenetic protein7 (BMP7) attenuates renal tubular and interstitial damage in a variety of experimental models. The function of BMP in the glomerulus is, however, not well understood. In the present study, we generated transgenic mice carrying cDNA for noggin, an endogenous inhibitor of BMPs, driven by the podocyte-specific promoter nephrin. Transgenic founder mice could be divided into two groups based on gross histological analyses at 2 months of age. One group was characterized by the presence of cystic glomeruli with collapsed capillary tufts and a decrease in mesangial cell number, representing a developmental defect during glomerular morphogenesis ("cystic" Tg mice). In contrast, the kidneys appeared to be normal in the other group ("non-cystic" Tg mice). In both groups, however, massive mesangial expansion developed at 10 months of age. The lesion was characterized by the accumulation of fibronectin, but not type I collagen, type IV collagen or laminin. This phenotype is similar to the fibronectin nephropathy. These results suggest that endogenous BMP can have an important role in regulating glomerular structural homeostasis.     

Emodin suppresses interleukin-1beta induced mesangial cells proliferation and extracellular matrix production via inhibiting P38 MAPK

Previous findings indicate that emodin has anti-proliferation and anti-fibrosis effects on several cell lines. In this study, we investigated the effects of emodin on IL-1β induced proliferation of mesangial cells (MCs) and on their production of extracellular matrix (ECM), and explored the possible mechanisms. To test the therapeutic effect of emodin on progressive renal disease, we administered emodin to rats in renal failure models induced by subtotal nephrectomy, the renal function was analyzed. Our results showed emodin significantly suppressed IL-1β induced MC proliferation and arrested the cell-cycle progress in vitro. Fibronectin and collagen IV production by MC were significantly reduced after emodin treatment. P38 mRNA, protein levels of P-P38, P-MKK3/6 and P-MKK4 were quantified. We observed no alterations of P38 expression and P-MKK4 protein content; however, protein levels of P-P38 and P-MKK3/6 significantly decreased after emodin treatment. In the renal failure models, after administration of emodin for eight weeks, the rat renal lesions were significantly ameliorated, as evidenced by the decreased blood creatinine, urea, and the 24-hour urine protein. In conclusion, emodin suppresses IL-1β induced MC proliferation and ECM production in vitro. We hypothesize that this is achieved by inactivating MKK3/6 and P38. Emodin ameliorates renal failure in subtotal nephrectomized rats, which suggests a potential role of emodin in the treatment of progressive renal diseases.