Kidney Injury Causes Accumulation of Renal Sodium That Modulates Renal Lymphatic Dynamics

Abstract

Lymphatic vessels are highly responsive to changes in the interstitial environment. Previously, we showed renal lymphatics express the Na-K-2Cl cotransporter. Since interstitial sodium retention is a hallmark of proteinuric injury, we examined whether renal sodium affects NKCC1 expression and the dynamic pumping function of renal lymphatic vessels. Puromycin aminonucleoside (PAN)-injected rats served as a model of proteinuric kidney injury. Sodium ²³Na/¹H-MRI was used to measure renal sodium and water content in live animals. Renal lymph, which reflects the interstitial composition, was collected, and the sodium analyzed. The contractile dynamics of isolated renal lymphatic vessels were studied in a perfusion chamber. Cultured lymphatic endothelial cells (LECs) were used to assess direct sodium effects on NKCC1. MRI showed elevation in renal sodium and water in PAN. In addition, renal lymph contained higher sodium, although the plasma sodium showed no difference between PAN and controls. High sodium decreased contractility of renal collecting lymphatic vessels. In LECs, high sodium reduced phosphorylated NKCC1 and SPAK, an upstream activating kinase of NKCC1, and eNOS, a downstream effector of lymphatic contractility. The NKCC1 inhibitor furosemide showed a weaker effect on ejection fraction in isolated renal lymphatics of PAN vs controls. High sodium within the renal interstitium following proteinuric injury is associated with impaired renal lymphatic pumping that may, in part, involve the SPAK-NKCC1-eNOS pathway, which may contribute to sodium retention and reduce lymphatic responsiveness to furosemide. We propose that this lymphatic vessel dysfunction is a novel mechanism of impaired interstitial clearance and edema in proteinuric kidney disease.

Keywords: NKCC1 transporter; kidney; lymphatics; sodium.

Figure 1

Proteinuric kidney injury increased renal sodium and water content. (A) Representative sodium ²³Na-MRI in uninjured control (Cont) (open circles) and puromycin (PAN)-injured rats (closed squares). The graph shows mean tissue sodium content localized in the cortex and medulla of the kidneys (arrows). (B) Representative T₂-weighted MRI images and quantitative T₂-relaxation time measurements indicative of renal cortical and medullary water content in Cont and PAN-injured rats. Results are expressed as mean ± SEM. n = 5 to 8 rats per group analyzed by unpaired t test. * p < 0.05.

Figure 2

Proteinuric kidney injury increased sodium concentration in renal lymph. (A) Analysis of renal lymph showed higher sodium concentration in PAN-injured (closed squares) vs control rats (open circles). (B) Analysis of plasma showed no difference in sodium concentration between PAN-injured vs control rats. Results are expressed as mean ± SEM for 6 to 8 rats per group analyzed by unpaired t test. * p < 0.05.

Figure 3

High salt environment altered renal collecting lymphatic vessel pumping function. Extra−renal afferent lymphatic vessels were subjected to a high−sodium buffer (185 mmol Na⁺ Krebs solution). A digital image capture system was used to measure the following vessel pumping parameters: frequency of spontaneous contractions, end diastolic lumen diameter (EDD), end systolic lumen diameter (ESD), contraction amplitude, and ejection fraction. Exposure to a high−sodium environment caused a significant increase in ESD, resulting in a significant decrease in amplitude and ejection fraction. Data points represent the percent change from measurements captured under baseline conditions (143 mmol Na⁺ Krebs solution) and are expressed as mean ± SEM. n = 5 individual vessels isolated from 5 rats. Significance was assessed by analyzing raw measurements using an unpaired t test. * p < 0.05.

Figure 4

NKCC-1 transporter expression in renal lymphatic vessels and vessels with PAN proteinuric injury. (A) Immunostaining of afferent renal lymphatic vessels demonstrated NKCC1 transporter expression, particularly prominent in lymphatic endothelial cells. (B) NKCC1 mRNA levels in extra-renal lymphatic vessels were significantly greater in PAN-injured rats vs uninjured controls. Results are mean ± SEM for 7 rats per group analyzed by unpaired t test * p < 0.05.

Figure 5

High Na⁺ environment regulated NKCC-1 signaling pathway in lymphatic endothelial cells (LECs). (A) Cultured LECs exposed to a high-sodium environment showed greater expression of NKCC1 mRNA, (B) while expression of phosphorated NKCC-1 protein decreased. (C) High-sodium environment decreased protein expression of SPAK, an upstream activating kinase of NKCC1. Experiments were performed independently 3 times using 3 wells per treatment and analyzed by ANOVA followed by Dunnett multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

eNOS modulated lymphatic vessel function. (A) Cultured LECs exposed to high-sodium, but not high-osmolar environment showed reduced eNOS activity. (B) Isolated renal lymphatic vessels challenged with the eNOS inhibitor, L-NAME, exhibited increased contraction frequency and reduced EDD, amplitude, and ejection fraction. EDD, end diastolic diameter; ESD, end systolic diameter. Protein concentration results are expressed as mean ± SEM for 3 samples analyzed by ANOVA followed by Dunnett multiple comparison test. Vessel pumping parameters are expressed as the percent change from measurements captured under baseline conditions (143 mmol Na⁺ Krebs solution) and are expressed as mean ± SEM for 5 individual vessels isolated from 5 rats. Significance was assessed by analyzing raw measurements using an unpaired t test. * p < 0.05.

Figure 7

Kidney injury diminishes lymphatic vessel pumping efficiency. Vasodynamic parameters were measured in renal lymphatic vessels isolated from control and PAN-injured rats. Vessels from PAN-injured rats had significantly increased EDD (B), resulting in reduced contraction amplitude (D) and ejection fraction (E), while contraction frequency (A) and ESD (C) remained unchanged. Datapoints represent raw measurements from individual vessels isolated from 7 to 11 rats per group. Results are expressed as mean ± SEM analyzed by unpaired t test. * p < 0.05 EDD, end diastolic diameter; ESD, end systolic diameter.

Figure 8

Vasodynamic response of PAN-injured lymphatic vessels exposed to a high-sodium environment. PAN-injured vessels exposed to high sodium had a significant decrease in the frequency of spontaneous contractions, EDD, and ejection compared with PAN-injured vessels in a normal sodium environment. Data points represent the percent change from measurements captured under normal sodium conditions and are expressed as mean ± SEM for 6 to 7 individual vessels isolated from 6 to 7 rats. Significance was assessed by analyzing raw measurements using an unpaired t test. * p < 0.05. EDD, end diastolic diameter; ESD, end systolic diameter.

Figure 9

Kidney injury and exposure to elevated sodium blunt lymphatic vessel response to NKCC inhibition by furosemide. Renal lymphatic vessels isolated from PAN-injured rats and normal controls were subjected to increasing concentrations of the NKCC antagonist furosemide. Some PAN vessels were challenged with furosemide in a high-sodium environment. In control vessels, furosemide induces a robust decrease in ejection fraction. In contrast, PAN-injured vessels in normal and high-sodium environments are significantly less sensitive to furosemide. Data points represent the percent change from measurements captured at baseline conditions and are expressed as mean ± SEM for <6 individual vessels isolated from <6 rats. Significance (p < 0.05) was analyzed by ANOVA followed by Dunnett multiple comparisons. * PAN vessels compared with control vessels, ** PAN vessels in high sodium compared with control vessels.