Fibronectin-Induced Increase in Mesangial Cell Prostaglandin Release: Effect of Hyperglycemia and PKC Inhibition

Abstract

Glomerular accumulation of extracellular matrix in diabetes is a potential regulator of mesangial cell-matrix interactions through transmembrane matrix receptors. We now provide evidence that PG production from rat glomerular mesangial cells is increased by Fn. An increase in PG (measured as PGE) was demonstrated in mesangial cell-enriched glomerular cores after 1-h exposure (149 +/- 8% of timed control) and was sustained over a 24-h period (214 +/- 7%). Increased PG production followed exposure to a chymotryptic fragment (120,000 M(r)) of Fn and occurred concomitant with an increase in particulate PKC activity. A tetrapeptide (Arg-Gly-Asp-Ser) with the Arg-Gly-Asp sequence, contained in Fn and the chymotryptic fragment and recognized by specific membrane receptors (integrin matrix-binding proteins), also raised PG levels. As has been shown previously, exposure to high glucose concentration can increase mesangial cell PGE production (from 677 +/- 61 pg.mg protein-1.2 h-1 at 5.6 mM glucose to 1561 +/- 132 pg.mg protein-1.2 h-1 at 50 mM glucose, P < 0.001). The response to the chymotryptic fragment of Fn also was enhanced by concurrent exposure to high glucose concentration (from 2560 +/- 199 pg.mg protein-1.2 h-1 at 5.6 mM glucose to 4672 +/- 358 pg.mg protein-1.2 h at 50 mM glucose, P < 0.001). Coincubation with H-7, an inhibitor of PKC, abolished the PG response to glucose and the chymotryptic fragment. Involvement of PKC was supported further by abrogation of the effect of chymotryptic fragment in mesangial cells cultured for a prior prolonged period with phorbol ester.(ABSTRACT TRUNCATED AT 250 WORDS)

PPARγ ligands attenuate mesangial contractile dysfunction in high glucose

“…These effects of pioglitazone on cell viability, calcium channel, and angiotensin II receptor may explain the suppression of the angiotensin II-induced cellular contraction at the higher concentration of the drug. The role of prostaglandin E2 on the reduced contractile response of mesangial cells in high glucose has been reported, since prostaglandin E2 decreases mesangial cell tonicity, and an increased production of prostaglandin E2 by mesangial cells in high glucose has also been reported [52,53]. The concentrations of indomethacin and acetyl salicylic acid used in our study were sufficiently high to suppress prostaglandin synthesis [54,55]; however, only indomethacin as a PPARc ligand could attenuate the reduced contractile response.…”

Increased phosphorylation of focal adhesion kinase in diabetic rat kidney glomeruli

“…Our previous data have shown that mesangial cores can respond to soluble fibronectin with an increased production of P G E [8], and in other cultured cell types plated onto fibronectin elevated tyrosine phosphorylation of FAK has been demonstrated [13,14]. We now show that cultured mesangial cells plated for 2 h onto fibronectin-coated plates have increased P G E production (from 12.76+1.83 to 24.52 + 1.10 ng-2 h -1 9 dish -1 p < 0.05, n = 3) and also increased tyrosine phosphorylation of FAK ( Fig.…”

“…The increase in mesangial matrix probably contributes to the structural changes observed in the diabetic kidney, however, the effect of matrix proteins on cellular function may also be important and must be considered. One of the early biochemical changes to occur in the diabetic kidney is the increased production of prostaglandins [5], shown by previous studies to be consequent on hyperglycaemia-induced protein kinase C activation [6][7][8], and we have shown, in vitro, that the addition of exogenous fibronectin to glomerular cores or isolated mesangial cells also results in increased production of prostaglandins [8]. Matrix proteins, including fibronectin, signal to cells via transmembrane cell surface receptors (integrins).…”

“…Mesangial cells were grown out from explanted glomerular cores as described previously [8] and maintained in Dulbecco% modified Eagle's medium (DMEM) containing 20 % (v : v) fetal calf serum (FCS); cells were used between passages one and four. Cells were lysed by direct addition of VO4-containing lysis buffer onto the tissue culture dish, after 5 rain on ice the lysate was centrifuged at 10,000 g for 10 min at 4 ~ In some experiments dishes were coated with 50 ~g/ml plasma fibronectin overnight at 4~ followed by two washes in phosphate buffered saline (PBS) before blocking in 2 mg/ml bovine serum albumin (BSA) for 1 h at 37~ Mesangial cells (5 x 10s), dissociated in trypsin/versene were added to the fibronectin-coated plates in DMEM containing 2 % FCS for 2 h at 37 ~ At the end of the incubation period the medium was removed and stored for PGE measurements and the adherent cells lysed as described above.…”

“…E-series prostaglandins (PGE) are the major eicosanoid metabolites formed by mesangial cells. Therefore, total immunoreactive PGE activity was measured by specific RIA, in the incubation medium of mesangial cells as described previously [8].…”