Jerry Morrissey

Osteopontin Expression in the Kidney during Unilateral Ureteral Obstruction

Osteopontin is a bone protein also expressed in other tissues. Increased osteopontin is thought to be associated with tissue inflammation. We used immunocytochemical analyses and polymerase chain reaction amplification of mRNA to examine osteopontin expression and regulation in unilateral ureteral obstruction (UUO) in rats, a model of inflammatory kidney disease. In the obstructed kidney, osteopontin mRNA and protein were significantly increased. The increase reached 4-fold after 1 day of UUO and persisted at this level for the 5-day duration of UUO. Immunocytochemical analyses showed increased osteopontin protein in tubular cells of the obstructed kidney cortex from days 1 through 5 of UUO. No such significant increase was apparent in the glomerulus or interstitium. Increased osteopontin mRNA and protein likewise occurred in the tubular cells of the obstructed kidney of rats that had undergone whole-body irradiation to eliminate macrophage infiltration into the experimental kidney. Purified osteopontin was found to be a chemoattractant for macrophages isolated from the rat peritoneum. Enalapril treatment, which decreases macrophage infiltration of the obstructed kidney, had no effect on the increase in osteopontin mRNA but significantly attenuated the increase in protein in tubular cells. Western blot analysis of whole cortical homogenates revealed that the osteopontin antibody recognized one protein of 67 kD. The amount of this protein was substantially decreased in kidney homogenates obtained from enalapril-treated compared to untreated animals. Increased osteopontin synthesis may, therefore, contribute in part to the inflammatory response that characterizes obstructive nephropathy.

Increases in Glomerular Eicosanoid Production in Rats with Bilateral Ureteral Obstruction Are Mediated by Enhanced Enzyme Activities of Both the Cyclooxygenase and 5-Lipoxygenase Pathways

Abstract Glomeruli isolated from rats with bilateral ureteral obstruction (BUO) of 24 hr duration produced significantly greater amounts of prostaglandin (PG) E2, 6-keto-PGF1α, thromboxane B2, and leukotriene B4 than glomeruli isolated from sham-operated control (SOC) rats. To examine the mechanisms underlying the greater production of eicosa-noids by glomeruli isolated from rats with BUO, we measured the activities of enzymes related to eicosanoid formation such as cyclooxygenase, 5-lipoxygenase, PGE2 isomerase, and PGI2 and thromboxane synthase in glomeruli isolated from SOC rats and rats with BUO. Glomeruli isolated from rats with BUO had a significantly increased activity of cyclooxygenase with de novo synthesis of this enzyme and a markedly augmented activities of PGE2 isomerase and both PGI2 and thromboxane synthases relative to glomeruli isolated from SOC rats. Similarly, the activity of membrane-bound 5-lipoxy-genase, the active location of this enzyme, was significantly greater in glomeruli isolated from rats with BUO than in glomeruli isolated from SOC rats. Thus, BUO of 24 hr duration enhances the glomerular production of eicosanoids via the activation of enzymes in both the cyclooxygenase and 5-lipoxygenase pathways.

Effects of Dietary Protein on Glomerular Eicosanoid Production in Rats with Bilateral Ureteral Obstruction

Abstract Greater protein intake increases glomerular elcosanold production in rats. Bilateral ureteral obstruction (BUO) also enhances glomerular elcosanold production in experimental animals. To examine the effects of dietary protein intake on glomerular elcosanold production in ureteral obstruction, we measured the in vitro production of the vasodilatory prostaglandins, PGE2, and 6-keto PGF1α, and the vasoconstrictor, TxB2, and the mass of cyclooxygenase in glomeruli of sham-operated control (SOC) rats and rats with BUO of 24 hr duration fed a low- (6% casein) or a high- (40% casein) protein diet for approximately 4 weeks. The animals were pretreated or not with the anglotensin converting enzyme inhibitor, enalaprilat, prior to sham-operation or ureteral obstruction. Glomeruli from SOC rats fed a high-protein diet produced significantly greater amounts of PGE2, 6-keto PGF1α, and TxB2, and had substantially increased mass of cyclooxygenase when compared with glomerull from SOC rats fed a low-protein diet. Pretreatment of animals with enalaprilat prior to sham operation prevented the increase in glomerular eicosanoid production and cyclooxygenase content in SOC rats fed a high-protein diet and the levels observed were similar to those in SOC rats fed a low-protein diet. Both elcosanoid production and cyclooxygenase mass were further increased in glomeruli from rats with BUO fed a high-protein diet when compared with glomeruli of SOC rats fed the same diet. The increased levels of these measurements in BUO rats fed a high-protein diet fell markedly when the rats were pretreated with enalaprilat in vivo. The values were essentially comparable to those of SOC rats fed a low-protein diet. By contrast, there was no substantial increase in the production of PGE2, 6-keto PGF1α) and TxB2 and in the mass of cyclooxygenase in glomeruli of BUO versus SOC rats fed a low-protein diet.

Eicosanoid production and levels of newly characterized eicosanoid‐forming enzymes in glomeruli and tubules of rat kidneys

Summary: We examined the production of prostaglandin (P0) E2, 6‐keto PGF1α and thromboxane (Tx) B2, the mass of cyclooxygenase (COX) isoenzymes and the activities of phospholipases A2 and C in glomeruli, cortical tubules and medullary tubules of rat kidneys. Medullary tubules produced significantly greater amounts of PGE2, 6‐keto PGF1α. and TxB2 than glomeruli or cortical tubules. the most abundant eicosanoid in medullary tubules was 6‐keto PGF1α. By contrast, glomeruli and cortical tubules predominantly produced POE2 (glomeruli > cortical tubules). Levels of COX 1 were markedly greater in medullary tubules than in glomeruli or cortical tubules. Glomeruli had significantly greater amounts of COX 1 than cortical tubules. Detectable amounts of COX 2 were not present in the three preparations. the activity of phospholipase (PL) A2 against phosphatidyicholine (PC) was significantly greater in tubules (medullary tubules > cortical tubules) than in glomeruli. By contrast, there was a significant increase in the activity of PLA2 against phosphatidylethanolamine (PE) in glomeruli as compared to tubules (medullary tubules > cortical tubules). the activity of PLC was the Weatest in medullary tubules. Glomeruli had significantly greater activity of PLC than cortical tubules. the order of magnitude for the total activity of the three phospholipases in membranes was medullary tubules> glomeruli> cortical tubules. the total production rate of POE2, 6‐keto PGF1α and TxB2 was in parallel with the amount of COX 1 and the total activity of membranous phospholipases A2 and C in the three preparations. In conclusion, there are differences in the production of PGE2, 6.‐keto PGF1α and TxB2, the ainount of COX 1 and the activities of phospholipases A2 andC among glomeruli, cortical tubules and medullary tubules of rat kidneys; and the different aspects of COX 1 and phospholipases A2 and C have a key role in the control of eicosanoid production in the three preparations.

Mechanism of the decreased eicosanoid production in vitro in response to angiotensin II in glomeruli of rats with bilateral ureteral obstruction

Summary: Glomeruli isolated from rats with bilateral ureteral obstruction (BUO) of 24 h duration synthesized significantly greater amounts of prostaglandin (PG)E2, 6‐keto PGF1a, thromboxane (Tx)B2 and leukotriene (LT)B4 than glomeruli isolated from sham‐operated control (SOC) rats. Glomeruli isolated from SOC rats produced increased amounts of these four eicosanoids compared to basal conditions when 100 nmol/L angiotensin II (AII) was added in vitro to the preparations. However, no significant increases in glomerular eicosanoid production were seen under these conditions in glomeruli of rats with BUO. to examine the mechanims underlying imparied eicosanoid production in glomeruli of rats with BUO exposed to AII in vitro, we measured the activities of phosphatidylethanolamine (PE)‐specific phospholipase A2 (PLA2), 5‐lipoxygenase and the cyclo‐oxygenase pathway enzymes including cyclo‐oxygenase, PGE2 isomerase and PGI2 and Tx synthases under basal conditions and after addition of 100 nmol/L AII in vitro in glomeruli isolated from SOC rats and rats with BUO of 24 h duration. the basal activities of all of these enzymes were significantly greater in glomeruli of rats with BUO compared to SOC rats. In glomeruli of SOC rats, the activities of these enzymes were markedly increased when exposed to 100 nmol/L AII in vitro compared to basal conditions. By contrast, no significant changes in the activities of enzymes involved in eicosanoid formation above baseline were seen in glomeruli of rats with BUO exposed to AII in vitro. the production rates of eicosanoids paralleled the activities of these enzymes under basal and AII‐stimulated conditions in glomeruli obtained from SOC rats and rats with BUO. Thus, the lack of increased levels of PGE2, 6‐keto PGF1a, TxB2 and LTB4, when glomeruli of rats with BUO of 24 h duration are exposed to 100 nmol/L AII in vitro, maybe due mainly to either the action of PE‐specific PLA2 or the combined action of PE‐specific PLA2, cyclo‐oxygenase pathway enzymes and 5‐lipoxygenase set at, or near, maximum levels as a consequence of BUO.