Epithelial N-methyl-D-aspartate (NMDA) receptors mediate renal vasodilation by affecting kidney autoregulation

Abstract

Background: N-methyl-D-aspartate receptor (NMDAR) are amino acid receptors that are well studied in brain physiology; however, their role in kidney is poorly understood. Nonetheless, NMDAR inhibitors can increase serum K+ and reduce GFR, which suggests they have an important physiological role in the kidney. We hypothesized that NMDARs in the distal nephron induce afferent-arteriole vasodilation through the vasodilator mechanism connecting-tubule-glomerular feedback (CNTGF) that involves ENaC activation.

Methods and results: Using a tubule-specific transcriptome database combined with molecular biology and microscopy techniques, we showed kidney expression of NMDAR subunits along the nephron and specifically in ENaC-positive cells. This receptor is expressed in both male and female mice, with higher abundance in females (p=0.02). Microperfusing NMDAR agonists into the connecting tubule induced afferent-arteriole vasodilation (EC₅₀ 10.7 vs. 24.5 mM; p<0.001) that was blunted or eliminated with the use of NMDAR blocker MK-801 or with the ENaC inhibitor Benzamil, indicating a dependence on CNTGF of the NMDAR-induced vasodilation. In vivo, we confirmed this CNTGF-associated vasodilation using kidney micropuncture (Stop-flow pressure 37.9±2.6 vs. 28.6±1.9 mmHg, NMDAR agonist vs vehicle; p<0.01). We explored NMDAR and ENaC channel interaction by using mpkCCD cells and split-open connecting tubules. We observed increased amiloride-sensitive current following NMDAR activation that was prevented by MK-801 (1.14 vs. 0.4 μAmp; p=0.03). In split-open tubules, NMDAR activation increased ENaC activity (Npo Vehicle vs. NMDA; p=0.04).

Conclusion: NMDARs are expressed along the nephron, including ENaC-positive cells, with higher expression in females. Epithelial NMDAR mediates renal vasodilation through the connecting-tubule-glomerular feedback, by increasing ENaC activity.

Keywords: Acute Kidney Injury; Connecting tubule-glomerular feedback; ENaC; Kidney Autoregulation; NMDA receptors.

Figure 1.

NMDAR RNA expression along the nephron. The mandatory Grin 1 subunit is expressed in all the renal segments, especially in the medulla regions of the nephron. In the medulla region all NMDAR subunit are expressed, while in the cortex in addition to Grin1, Grin2D is the majority expressed in the distal nephron in addition to grin2C and Grin3A in DCT and in less magnitude in CNT and CCD.TMP: Transcript per millon. [PTS1] initial segment of proximal tubule; [PTS2] straight segment of proximal tubule; [PTS3] last segment of the proximal straight tubule in the outer stripe of outer medulla; [DTL1] short descending limb of the loop of Henle; [DTL2] long descending limb of the loop of Henle in the outer medulla; [DTL3] long descending limb of the loop of Henle in the inner medulla; [ATL]) thin ascending limb of the loop of Henle; [MTAL] medullary thick ascending limb of the loop of Henle; [CTAL] cortical thick ascending limb of the loop of Henle; [DCT] distal convoluted tubule; [CNT] connecting tubule, cortical collecting duct [CCD], outer medullary collecting duct [OMCD], and inner medullary collecting duct [IMCD])

Figure 2.

Kidney expression of NMDAR type 1 (GlutN1). GlutN1 is expressed in both cortical (Figure 2A) and medullary region of the kidney (Figure 2B) where strong signal was detected. In the cortex, GlutN1 is expressed several nephron segments and glomerulus (Figure 2C). At higher magnification, GlutN1 disposicition is even in the cytoplasm with perinuclear reinforcement in some cells (star) and while in other segments shows an apical expression (arrows). Figure 2C and 2D shows GlutN1 expression (red) and ENaC alpha subunit (Green) in cortex. Figure 2E shows the co-expression of both protein (yellow) in dotal nephron. Scale bar= 20 μm.

Figure 3.

Sex difference in NMDAR type 1 and its interaction with ENaC. Figure 3A compare the expression of GlutN1 in cortex of male and female mice. Male mice exibit a weak staining in comparison to female. Figure 3B shows total kidney expression quantification between male and female. Figure 3 C Figure 3C shows benzamil-sensitive transepithelial current using mpkCCD cells under the presence of NMDAR agonist, MK801 (non-selective blocker) and a selective GLutN2C/D inhibitor (997-74) The presence of NMDA increase the transepithelial current, which is blunted when the non-selective blocker is used and totally abolished whith the use of the selective inhibitor. Figure 2D shows an increase ENaC activity (NPo) at exprense of the open probability of the channel with not changes in the amount functional channels. AU: Arbitrary units (GlutN1/Actine ratio). Δ trasnepithelial current difference. *p<0.05

Figure 4.

NMDAR induce afferent arteriole vasodilation. Panel A-D represent the afferent arteriole (Af-Art) diameter change after the intratubular infusion of increasing sodium chloride concentration combined or not with different agonist and pharmacological inhibitors in the connecting tubule (CNT) (open circles). Co-infusion of sodium chloride with glutamate and glycine increase the Af-art vasodilation, by shifting the curve to the left (closed circle). Figure 4B demonstrate that the addition of the non-selective NMDAR blocker MK801 restore the curve to the right (closed circles). Figure 4C shows that the co-infusion of the ENaC blocker benzamil totally avoid the Af-Art vasodilation in the presence of amino acids, indicating the total dependence on ENaC and CNTGF for the amino acid induced vasodilation (closed circles). Figure 4D shows no effect of MK801 on Af-Art vasodilation in absence of amino acids. Panel E-H represent the Stop-flow pressure (SFP) results in-vivo during micropuncture. Figure 4E shows a decrease in SFP, a surrogate of Af-Art constriction after increasing distal flow of sodium chloride. When a mix of amino acids is added to the perfusate, SFP is almost constant, indicating a reduced vasoconstriction response. Figure 4F, demonstrate that the diminished vasoconstrictive response is due to an exaggerated CNTGF activation, because when benzamil (CNTGF blocker) is infused, the vasoconstrictive response due to TGF is unmasked (closed circles). Figure 4G shows the CNTGF activity in the absence of amino acids. By comparing the CNTGF magnitude with amino acid infusion versus vehicle, we observed that CNTGF is significantly enhanced (Figure 4H).