Interaction of nitric oxide and transforming growth factor-beta1 induced by angiotensin II and mechanical stretch in rat renal tubular epithelial cells.
OBJECTIVE
Changes in intrarenal pressure accompanying unilateral ureteral obstruction can result in tubular mechanical stretch and mediator release from renal tubules. Therefore, we examined the synthesis of nitric oxide and transforming growth factor-beta (TGF-beta), and their interaction in rat renal epithelial cells (NRK-52E) exposed to either angiotensin II or mechanical stretch.


METHODS
NRK-52E were exposed to either angiotensin II or mechanical stretch. Nitrite and TGF-beta in the supernatant were assessed by the Greiss reaction and bioassay, respectively. The level of cell hypertrophy and intracellular TGF-beta protein was determined by flow cytometry. TGF-beta messenger RNA and inducible nitric oxide synthase protein were detected by reverse transcriptase polymerase chain reaction and Western blot, respectively.


RESULTS
Angiotensin II stimulated TGF-beta1 and nitric oxide. The nitric oxide synthase inhibitor, N-nitro-L-arginine (L-NAME) or angiotensin II type I receptor blocker, losartan, inhibited nitric oxide and TGF-beta1 induced by angiotensin II. Flow cytometry showed that either L-NAME or losartan inhibited angiotensin II induced cell hypertrophy. TGF-beta1 inhibited iNOS protein and nitric oxide, whereas an anti-TGF-beta antibody enhanced iNOS. Mechanical stretch induced TGF-beta, inducible NOS protein and nitric oxide. However, TGF-beta expression was not affected by L-arginine or L-NAME when cells were exposed to mechanical stretch.


CONCLUSIONS
These results demonstrate that nitric oxide is an intermediate in angiotensin II stimulated TGF-beta1 expression but not in stretch induced TGF-beta expression, and that TGF-beta1 is a negative regulator of nitric oxide in rat renal epithelial cells. The complex interaction of these cytokines may be a target for intervention in the fibrotic and apoptotic processes in the obstructed kidney.
Miyajima
A
A
Department of Urology, Center for Pediatric Urology and Laboratory for Minimally Invasive Urologic Surgery, New York Presbyterian Hospital, Weill Medical College of Cornell University, New York, New York, USA.nt
Chen
J
J
Kirman
I
I
Poppas
D P
DP
Darracott Vaughan E
J R
JR
Felsen
D
D
D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D017077:Culture Media, Conditioned; D004847:Epithelial Cells; D005434:Flow Cytometry; D007684:Kidney Tubules / Q000378:metabolism*; D008907:Mink; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013314:Stress, Mechanical; D016212:Transforming Growth Factor beta / Q000096:biosynthesis*
D017077:Culture Media, Conditioned; D016212:Transforming Growth Factor beta; D000804:Angiotensin II; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat