12-Lipoxygenase metabolism in mouse distal convoluted tubule cells.
BACKGROUND
Several lines of evidence point to the 12-lipoxygenase (12-LOX) family as important mediators in hypertension, diabetes, and other cardiovascular diseases. The kidney has been a main focus for research of the role of this pathway in several disease models. While most of the studies have focused on mesangial or vascular cells, less is known about 12-LOX regulation at the renal tubular level. The aim of the study was to characterize the expression and regulation by hormones of the family of 12-LOX in mouse distal convoluted tubule at the molecular level.


METHODS
An immortalized mouse distal convoluted tubule (mDCT) cell line was used. mRNA and protein levels were assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively, while 12(S)-HETE production was evaluated by enzyme-linked immunosorbent assay (ELISA). Cells were challenged with aldosterone, angiotensin II, 8Br-cAMP, and vasopressin.


RESULTS
We showed that both platelet (P) and leukocyte (L)-type 12-LOX are expressed in the mDCT cell line, as well as in distal tubules of human kidneys. The production of 12(S)-HETE by mDCT cells was increased in response to cAMP (by two-fold) and by vasopressin (by 1.5-fold). In contrast, neither aldosterone nor angiotensin II exerted appreciable effects on 12(S)-HETE production. The mRNA and protein levels of P-12LOX and L-12LOX were not changed by the different hormones, suggesting that they may act by modulating enzyme activity. We further have demonstrated that this mDCT cell line also expressed the recently cloned 12(R)-LOX.


CONCLUSIONS
mDCT cells show an active 12-LOX metabolism that appears to be modulated by cAMP and vasopressin.
González-Núñez
Daniel
D
Servicio de Nefrologia, Laboratorio de Hormonal, Departamento de Anatomia Patologica, Hospital Clinic, Barcelona, Spain.
Solé
Manel
M
Natarajan
Rama
R
Poch
Esteban
E
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid / Q000096:biosynthesis; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000450:Aldosterone / Q000494:pharmacology; D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001792:Blood Platelets / Q000201:enzymology; D002460:Cell Line; D004247:DNA / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D007686:Kidney Tubules, Distal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007962:Leukocytes / Q000201:enzymology; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism
D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D000804:Angiotensin II; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000450:Aldosterone; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D004247:DNA; D001092:Arachidonate 12-Lipoxygenase