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The quest for Mineralocorticoid Receptors. Sodium. MR. Yutong Liu Gomez-Sanchez Lab. Aldosterone. The Focus: Unraveling causes of hypertension. The Players:. Mineralocorticoid receptor (MR) Activation of MR in the amygdala increases salt appetite

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the quest for mineralocorticoid receptors

The quest for Mineralocorticoid Receptors



Yutong Liu

Gomez-Sanchez Lab



The Focus: Unraveling causes of hypertension

The Players:

  • Mineralocorticoid receptor (MR)
  • Activation of MR in the amygdala increases salt appetite
  • 2. MR in circumventricular organs are involved in cardiovascular homeostasis & inflammatory process
  • 3. MR in hippocampal neurons are involved in learning and memory
  • Aldosterone (aldo)
  • Aldo is a steroid hormone of the mineralocorticoid family, which maintains fluid, electrolyte, and blood pressure homeostasis
  • 2. Synthesis-
  • a. Adrenal glands
  • b. synthesis tightly regulated by Na, K, & angiotensin II

The Focus: Unraveling causes of hypertension

  • Chronic excessive activation of MR
  • Hypertension
  • 2. Heart, vessel, and kidney inflammation and scarring
  • 3. Heart and Vessel hypertrophy

The Connections


Mineralocorticoid Receptor



In kidney, heart, brain,

& many other organs

  • Effects 65 million Americans according to the American Heart Association
  • 2. Primary risk factor for heart attack, stroke, and kidney failure
  • 3. 7-12% hypertension is due to hyperaldosteronism



Developing tools to study MR & Aldo action in the brain

Molecular Biology






… so many to tell, which one?

  • Small interfering (si) mRNA carried by a lentivirus vector can be used to eliminate MR expression

2. An ELISA can be used for a very sensitive MR detection

3. A transgenic rat over-expressing aldosynthase in neurons will have an increased appetite for sodium


The purpose of the Western Blot

In general:

To confirm the existence/absence of a protein of the same molecular weight & charge as MR in cells transfected with si mRNA for MR or scrambled si mRNA.

In this case:

To demonstrate a decrease in the expression of MR by the si mRNA method.


1. Sample Preparation

  • *apparatus: a) water bath, b) centrifuge
  • a. boil
  • b. centrifuge
  • 2. Electrophoresis
  • *apparatus: Hoefer Mighty Small II
  • a. load sample
  • b. load marker
  • c. run @ 150v for 1 hour
  • 3. Transfer
  • *apparatus: BioRad MiniProtean II
  • a. soak filter pads in transfer buffer
  • b. soak PVDF membrane in methanol (1min), then transfer buffer (30min)
  • c. soak gel in transfer buffer (5min)
  • d. Assemble transfer sandwich
  • e. fill apparatus w/ transfer buffer
  • f. run for 1.5 hours at 350 mA
  • 4. Immuno Detection
  • *apparatus: Belly Dancer
  • a. block w/ milk solution for 1 hr
  • b. incubate w/ primary antibody overnight in cold room
  • c. wash membrane w/ 1x PBS + .05% Tween
  • d. incubate w/ secondary antibody for 1 hr
  • e. wash membrane in 1x PBSTween
  • 5. Developing
  • ~location: a) 4° C cold room, b) dark room
  • make a 1:1 working solution of West Pico reagents
  • incubate membrane in West Pico solution in cold room (5 min)
  • in dark room, dab excess WP
  • wrap membrane in plastic wrap
  • expose film to membrane

Western Blot



Developing an Enzyme Linked ImmunoSorbent Assay (ELISA) Protocol for MR

What the MR ELISA reveals:

How much MR protein is in a particular solution

More protocols in planning

  • Chicken anti-sheep
  • Sheep anti-mr
  • Mouse anti-mr
  • Primary AB (MR 365 & MRN2 2B7)
  • Donkey anti-mouse HRP

Protocols we have tried

  • anti-sheep IgG
  • Sheep anti-mr
  • Mouse anti-mr
  • Primary AB
  • Goat anti-mouse

Developing an Enzyme Linked ImmunoSorbent Assay (ELISA) Protocol for MR

Sandwich ELISA for MR: side view of one well in a 96 well plate

Rabbit x-mouse Horseradish Peroxidase

Primary Antibody: 365 and 2B7

Samples: Variable & Control

Sheep x-MR

Coating antibody: chicken x-sheep; donkey x-sheep


Molecular weight combined w/ antibody is more accurate

The Good

Antibody may not be completely specific

The Bad

Western Blot


Can’t do many samples simultaneously; not very quantitative

1.Quantitative (can make a standard curve)

2. More sensitive: measure smaller amounts

The Bad

The Good


Biologists had a dogma that what you observe in one organism, if it’s significant to life itself, will likely be 

appropriate to other organisms.

Gina Kolata


F3; AS4433 +/+ male, normal salt diet,

13.5 mo old,

Congestive Heart Failure & glomerulosclerosis

Sprague Dawley male

normal salt diet

12 mo old



Experimental Setup of Behavioral Study:

Do Aldosynthase-over-expressing rats prefer saline over water?

Aldosynthase rats

(7 male, 7 female)

Normal Sprague-Dawley rats

(7 male, 7 female)







The siRNA and the ELISA for MR are still works in progress.

The salt appetite study of transgenic rats

revealed that both type of rats preferred saline over water. As to which type of rat preferred saline more, over the length of 3 exposures, a guesstimated conclusion would be the same.



Dr. Ian Paul

Anne Dautenhahn

Catherine Kaime

Neuroscience Summer Scholars


Dr. Elise P. Gomez-Sanchez

Dr. Celso E. Gomez-Sanchez

Angela F. de Rodriguez

Gomez-Sanchez lab