Protein increases glomerular eicosanoid production and activity of related enzymes

Abstract

We examined the in vitro production of PGE2, 6-keto PGF1 alpha and TxB2 by isolated glomeruli from rats fed a low (6% casein) or a high (40% casein) protein diet for approximately eight weeks. Glomeruli from high protein-fed rats produced significantly greater amounts of PGE2, 6-keto PGF1 alpha and TxB2 under basal conditions and in response to the addition of 100 nM angiotensin II (Ang II) than glomeruli from low protein-fed rats. To elucidate the mechanisms by which greater protein intake enhanced the glomerular production of eicosanoids, we explored phospholipase (A2 and C) and cyclooxygenase activity in glomeruli isolated from low- or high-protein fed rats. PE-specific PLA2 activities were significantly increased in glomeruli from rats fed a high protein diet when compared to a low protein diet. On the other hand, PC-specific PLA2 activities were significantly decreased in glomeruli from rats fed a high protein diet. No significant difference in PIP2-PLC activities was detected between glomeruli of the two dietary groups. The cyclooxygenase content and activity was significantly greater in glomeruli from rats fed a high protein diet than in glomeruli from rats fed a low protein diet. Glomeruli of rats fed a 50/50 mixture of the diets (23% casein) had amounts and activity of cyclooxygenase and activities of PE-specific PLA2 intermediate between those of high and low protein-fed animals. In conclusion, increased synthesis of eicosanoids by glomeruli from rats fed a high protein diet may be mediated by increases in the amount and activity of cyclooxygenase coupled with enhanced activity of PE-specific PLA2.

Effects of Dietary Protein on Eicosanoid Production in Rat Renal Tubules

“…The mineral and vitamin contents of the two diets were comparable [1, 7, 8, 11]. The composition of the diets and the method of feeding were previously described in detail [1, 7, 8, 11].…”

“…The production of eicosanoids by renal tubules was determined by the protocol described previously [7, 8, 11, 12, 13, 14, 15, 16]. In brief, cortical or medullary tubules (approximately 200–250 µg of protein) were suspended in warm HBSS and preincubated at 37°C for 5 min.…”

“…Tubular membranes and cytosolic extracts were prepared as reported previously [7, 8, 11, 12, 15, 16]. Cortical or medullary tubules were washed twice with cold Ca 2+ and Mg 2+ -free HBSS and suspended in 250–500 µl of ice-cold homogenate buffer (25 m M Tris-HCl, pH 7.5, containing 1 m M EDTA, 1 m M dithiothreitol, and 100 U/ml trazylol).…”

“…Cortical or medullary tubules were prepared by sieve techniques (mesh sizes 250, 150, and 75 µm) with collagenase digestion as described previously [12]. Briefly, glomeruli were removed from cortices with the consecutive sieves [7, 8, 11, 12, 13, 14, 15, 16]. Cortices without glomeruli remaining on a 150-µm-mesh sieve or medullas were minced and digested enzymatically in Hanks’ balanced salt solution (HBSS) containing 0.1% collagenase type II at 37°C for 30 min under shaking (80 cycles/min).…”

“…We demonstrated previously that the activity of membrane-associated phospholipase (PL) A 2 and cyclooxygenase (COX) and the production of PGE 2 , 6-keto PGF 1α , and TxB 2 were significantly increased in glomeruli of rats fed a high-protein diet relative to those fed a low-protein diet [7, 8]. Treating rats with an angiotensin I converting enzyme inhibitor, however, restored the levels of eicosanoid-forming enzymes and the production of eicosanoids in glomeruli of rats fed a high-protein diet to levels comparable to those observed in glomeruli of rats fed a low-protein diet [8, 9].…”