Effects of Renal N-methyl-D-aspartate Receptors in Regulating Renal Blood Flow and Hypertension Restricted; Files Only

Abstract

N-methyl-D-aspartate receptors (NMDAr) are glutamate receptors that act as calcium channels. Previous research demonstrates that NMDAr in the kidney contribute to renal hemodynamics, potentially via interaction with epithelial sodium channel (ENaC) through a physiological mechanism not yet known. This thesis investigates how the NMDAr profile in the kidney contributes to blood pressure phenotype, specifically through scrutinization of the NMDAr subtypes. We evaluated change in blood pressure in response to NMDAr inhibition in Liddle Syndrome (LS)—a genetic disorder with overactivation of ENaC—mice in under normal (0.4% NaCl) and high salt (HS, 4% NaCl) diet. We repeated the experiment with GluN2C—a specific NMDA receptor subunit—antagonist to evaluate subtypical implications. GluN2C expression was confirmed and quantified via RNAscope and immunofluorescence. We also traced renal blood flow (RBF) in response to   NMDAr inhibitor injection to gain mechanistic insight on the results. LS mice on normal salt diet exhibited acute increase in blood pressure upon NMDAr inhibition (SBP 130.2 ±14 vs. 104.3±17 mmHg, β=1.33 vs 1.116, p<0.01), followed by subacute normalization. Male LS mice on HS diet displayed elevated BP in response to increase in dietary sodium intake, and they exhibited acute decrease in BP subsequent to infusion of NMDAr inhibitor (SBP 96.1±2.5 vs. 126.8±25, p<0.001). Female cohort did not develop salt-sensitive hypertension, indicating sex difference. GluN2C-specific inhibition under normal salt diet displays similar phenomenon as non-selective inhibition (155.2±6 vs 108±1.6 mmHg, p=0.008). RNAscope results confirm GluN2C expression among LS and WT animals with no significant difference among cohorts. Immunofluorescence results demonstrate that WT males display significantly less GluN2C level compared to LS cohort, (437.3±172.3 vs. 1506±360, p<0.01), while the females displayed apparent reciprocal results (WT 1316±371.8 vs LS 590±172.5 p=0.05). Spectral analysis of RBF tracing indicates decrease in connecting tubule glomerular feedback (CNTGF) upon NMDAr inhibitor injection (0.22±0.1 vs 0.08±0.04, p=0.03). Results demonstrate that change in blood pressure in response to NMDAr inhibition on LS mice depends on dietary sodium intake and sex. There is significant difference in translation of GluN2C across genotype and sex, but not in transcription. NMDAr inhibition decreases CNTGF but does not alter total RBF.

Table of Contents

I.     Introduction………………………………………………………………………….1

a.    Structure of the Nephron

b.    Renal Blood Flow

c.    N-methyl-D-aspartate Receptors

d.    Liddle Syndrome

e.    Hypothess & Research Objective

II.   Methodology…………………………………………………………………………7

a.    Incremental Drug Induction and Mice Blood Pressure Monitoring

    i.    Osmotic Minipump Implantation

           ii.    BP-2000 Blood Pressure Analysis System

b.    Localization and Quantification of NMDA Receptor Expression

           i.    RNAscope Imaging

            ii.    Immunofluorescence

c.    Evaluation of Renal Blood Flow and Autoregulatory Mechanism

            i.    Spectral Analysis

d.    Data Analysis

III.          Results……………………………………………………………………………..16

a.    Acute and Subacute Effect of NMDA Receptor Inhibition on Blood Pressure on Normal Salt Diet

b.    MK801 Prevents Salt-Sensitivity in Male Liddle Syndrome Mice

c.    GluN2C Subtype Receptor on Blood Pressure Regulation

          i.    DQP997-74 GluN2C-Specific Inhibitor

         ii.    RNA and Protein Expression

     iii.    Blood Pressure on Grin2C-KO Mice

d.    Implications of NMDA Receptor on Renal Blood Flow and Autoregulation

IV.         Discussion and Conclusion………………………………………………………..25

V.           References……………………………………………………………………………29

VI.         Appendix……………………………………………………………………………..34

a.    RNAscope Technique Validation Supplementary Data

b.    NeuroMab Antibody Supplementary File

c.    Urine Collection and Osmolarity Analysis Supplementary Data

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Keyword	Liddle Syndrome Connecting tubule-glomerular feedback Epithelial Sodium Channel RNAscope Nephron Immunofluorescence Renal blood flow Hypertension NMDA
Committee Chair / Thesis Advisor	Cesar A. Romero, Emory University
Committee Members	Jeanie Park, Emory University Leila Rieder, Emory University

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