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Published on: September 9, 2021
12-Lipoxygenase metabolism in mouse distal convoluted tubule cells
Daniel González-Núñez1, Manel Solé, Rama Natarajan
1Servicio de Nefrologia, Laboratorio de Hormonal, Departamento de Anatomia Patologica, Hospital Clinic, Barcelona, Spain.
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.
Conclusion:
mDCT cells show an active 12-LOX metabolism that appears to be modulated by cAMP and vasopressin.
Insights
The 12-lipoxygenase (12-LOX) pathway is active in mouse kidney tubules and regulated by cAMP and vasopressin, but not aldosterone or angiotensin II. This highlights a novel mechanism in cardiovascular disease research.
Area of Science:
- Renal Physiology
- Molecular Biology
- Cardiovascular Research
Background:
- The 12-lipoxygenase (12-LOX) pathway is implicated in cardiovascular diseases like hypertension and diabetes.
- Research has primarily focused on 12-LOX in kidney mesangial and vascular cells, with limited understanding of its role in renal tubules.
Purpose of the Study:
- To investigate the expression and hormonal regulation of the 12-LOX family in mouse distal convoluted tubule (mDCT) cells.
- To elucidate the molecular mechanisms of 12-LOX activity in renal tubular cells.
Main Methods:
- Utilized an immortalized mDCT cell line for experiments.
- Assessed gene and protein expression via RT-PCR and Western blot.
- Quantified 12(S)-HETE production using ELISA after stimulation with hormones (aldosterone, angiotensin II, cAMP, vasopressin).
Main Results:
- Both platelet (P)-12-LOX and leukocyte (L)-12-LOX are expressed in mDCT cells and human kidney distal tubules.
- cAMP and vasopressin significantly increased 12(S)-HETE production, while aldosterone and angiotensin II had no significant effect.
- Hormonal stimulation did not alter P-12LOX or L-12LOX mRNA or protein levels, suggesting post-translational regulation of enzyme activity.
- The mDCT cell line also expresses 12(R)-LOX.
Conclusions:
- Mouse distal convoluted tubule cells exhibit active 12-LOX metabolism.
- This activity is modulated by cAMP and vasopressin, indicating a potential role in renal tubular function and disease.
