Inner Medullary Collecting Duct Cells of the Kidney Synthesize Cholesterol
Poster #: 133
Session/Time: B
Author:
Rachel Marie Lisner, BA
Mentor:
Eman Gohar, PhD
Research Type: Basic Science
Abstract
INTRODUCTION:
Cholesterol biosynthesis occurs through a multistep pathway that converts acetyl coenzyme A into cholesterol, with 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) catalyzing the rate-limiting step. One function of cholesterol is to serve as a precursor for estrogen biosynthesis. Activation of the renal G protein-coupled estrogen receptor 1 (GPER1) by estrogen promotes sodium excretion and lowers blood pressure. It has not yet been tested whether the kidney can locally biosynthesize cholesterol, the precursor of estrogen. Given the vital role of the kidney's collecting duct (CD) in fine tuning sodium reabsorption and regulating blood pressure, we hypothesize that the CD cells, which are actively involved in the GPER1 pathway, exhibit an intrinsic capacity for local cholesterol biosynthesis.
METHODS:
To test our hypothesis, mouse inner medullary CD cells (mIMCD3) were cultured and allowed to reach confluency. Then, cells were starved for 3 hours and subsequently treated with vehicle, or the HMGCR inhibitor, rosuvastatin (1, 5 or 10 µM), or the precursor substrate for cholesterol biosynthesis, acetate (10 mM). Western blotting was used to assess HMGCR enzyme abundance in cell extracts. Amplex Red cholesterol assay was conducted on media samples to determine cholesterol levels. Serum-containing and serum-starved media samples were assayed to determine whether the detected cholesterol values were dependent on pre-existing serum in the media.
RESULTS:
Western blotting of mIMCD3 cell extracts revealed the presence of HMGCR. Media conditioned with mIMCD3 cells exhibited a statistically significant increase in cholesterol levels after 48 hours of culture compared to unconditioned media (67.33 ±1.73 vs. 23.73 ± 0.54 µg/ml, respectively; n = 3 per group; P < 0.0001). Treatment of mIMCD3 cells with 1 µM rosuvastatin significantly reduced the cholesterol levels in the conditioned media compared to vehicle-treated cells. Assessment of cholesterol levels for cells cultured in serum-starved media revealed that cholesterol was significantly higher in serum-starved media conditioned by mIMCD3 cells compared to serum-starved unconditioned media (3.97 ± 0.08 vs. 0.59 ± 0.041 µg/ml, respectively; n = 3 per group; P < 0.0001). Additionally, treatment of mIMCD3 cells with rosuvastatin (0, 1, 5, and 10 µM) resulted in dose-dependent decreases in the cholesterol levels in the media (33.46 ± 0.03; 32.70 ± 0.19; 32.53 ± 0.28 µg/ml; 31.39 ± 0.37 µg/ml, respectively; n = 3 per group), with a statistically significant difference between the 1 µM and 10 µM concentrations (P = 0.025). Lastly, western blotting of mIMCD3 cells treated with 10mM acetate displayed a greater HMGCR abundance than vehicle-treated cells.
CONCLUSION:
Our data illustrated the presence of key cholesterol synthesis enzyme HMGCR in mIMCD3 cells. It also demonstrated increased cholesterol levels in both serum-containing and serum-starved media when conditioned with mIMCD3 cells compared to unconditioned media. Inhibition of HMGCR by rosuvastatin resulted in a dose-dependent decrease in cholesterol levels in cell media. Treatment of cells with acetate increased HMGCR abundance, demonstrating the ability of mIMCD3 cells to upregulate their cholesterol synthesizing machinery, and potentially their ability to synthesize cholesterol. Altogether, our results provide novel evidence that mIMCD3 cells have an endogenous capacity to biosynthesize cholesterol.
Cholesterol biosynthesis occurs through a multistep pathway that converts acetyl coenzyme A into cholesterol, with 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) catalyzing the rate-limiting step. One function of cholesterol is to serve as a precursor for estrogen biosynthesis. Activation of the renal G protein-coupled estrogen receptor 1 (GPER1) by estrogen promotes sodium excretion and lowers blood pressure. It has not yet been tested whether the kidney can locally biosynthesize cholesterol, the precursor of estrogen. Given the vital role of the kidney's collecting duct (CD) in fine tuning sodium reabsorption and regulating blood pressure, we hypothesize that the CD cells, which are actively involved in the GPER1 pathway, exhibit an intrinsic capacity for local cholesterol biosynthesis.
METHODS:
To test our hypothesis, mouse inner medullary CD cells (mIMCD3) were cultured and allowed to reach confluency. Then, cells were starved for 3 hours and subsequently treated with vehicle, or the HMGCR inhibitor, rosuvastatin (1, 5 or 10 µM), or the precursor substrate for cholesterol biosynthesis, acetate (10 mM). Western blotting was used to assess HMGCR enzyme abundance in cell extracts. Amplex Red cholesterol assay was conducted on media samples to determine cholesterol levels. Serum-containing and serum-starved media samples were assayed to determine whether the detected cholesterol values were dependent on pre-existing serum in the media.
RESULTS:
Western blotting of mIMCD3 cell extracts revealed the presence of HMGCR. Media conditioned with mIMCD3 cells exhibited a statistically significant increase in cholesterol levels after 48 hours of culture compared to unconditioned media (67.33 ±1.73 vs. 23.73 ± 0.54 µg/ml, respectively; n = 3 per group; P < 0.0001). Treatment of mIMCD3 cells with 1 µM rosuvastatin significantly reduced the cholesterol levels in the conditioned media compared to vehicle-treated cells. Assessment of cholesterol levels for cells cultured in serum-starved media revealed that cholesterol was significantly higher in serum-starved media conditioned by mIMCD3 cells compared to serum-starved unconditioned media (3.97 ± 0.08 vs. 0.59 ± 0.041 µg/ml, respectively; n = 3 per group; P < 0.0001). Additionally, treatment of mIMCD3 cells with rosuvastatin (0, 1, 5, and 10 µM) resulted in dose-dependent decreases in the cholesterol levels in the media (33.46 ± 0.03; 32.70 ± 0.19; 32.53 ± 0.28 µg/ml; 31.39 ± 0.37 µg/ml, respectively; n = 3 per group), with a statistically significant difference between the 1 µM and 10 µM concentrations (P = 0.025). Lastly, western blotting of mIMCD3 cells treated with 10mM acetate displayed a greater HMGCR abundance than vehicle-treated cells.
CONCLUSION:
Our data illustrated the presence of key cholesterol synthesis enzyme HMGCR in mIMCD3 cells. It also demonstrated increased cholesterol levels in both serum-containing and serum-starved media when conditioned with mIMCD3 cells compared to unconditioned media. Inhibition of HMGCR by rosuvastatin resulted in a dose-dependent decrease in cholesterol levels in cell media. Treatment of cells with acetate increased HMGCR abundance, demonstrating the ability of mIMCD3 cells to upregulate their cholesterol synthesizing machinery, and potentially their ability to synthesize cholesterol. Altogether, our results provide novel evidence that mIMCD3 cells have an endogenous capacity to biosynthesize cholesterol.