*2.2. High Sodium Environment Changed Contractility of Renal Lymphatic Vessels Involving the NKCC1 Transporter*

To determine the effects of a high-sodium environment on renal lymphatic function, we measured the vasodynamics of renal afferent collecting lymphatic vessels. Similar to studies in afferent skin lymphatics [19], the contractility was assessed in renal afferent vessels isolated from normal rats exposed to normal buffer containing 143 mmol Na+ Krebs solution and compared with dynamics following exposure to Krebs solution containing a sodium concentration of 185 mmol. Compared with the physiologic buffer, a high-sodium environment had little effect on lymphatic contraction frequency. However, although high sodium caused only subtle changes in end diastolic diameter (EDD), a pronounced increase in end systolic diameter (ESD) contributed to reduced contraction amplitude and ejection fraction compared with the physiologic buffer (Figure 3).

**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<sup>+</sup> 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.

NKCC1 regulates blood vessel dynamics, and our previous study confirmed expression of NKCC1 in lymphatic endothelial cells. However, it is unknown whether NKCC1 has a role in modulating microenvironmental influences on lymphatic vessel dynamics. This is interesting, as lymphatic vessels were recently reported to regulate sodium homeostasis. Renal lymphangiogenesis in mice with kidney-specific overexpression of VEGF-D increased urinary sodium excretion and reduced systemic blood pressure in salt-loaded hypertensive mice but not normotensive basal conditions [20]. The mechanism was linked to downregulation of sodium transporters, namely, total NCC and ENaCα in tubular epithelial cells. NKCC1 expression and renal lymphatic function were not evaluated. Our immunohistochemical staining verified prominent expression of NKCC1 in afferent renal lymphatic vessels (Figure 4A). Moreover, NKCC1 gene expression was increased in vessels from PAN-injured rats compared with controls (Figure 4B). Also, LECs exposed to a high-sodium environment had elevated NKCC1 mRNA compared with cells maintained in media with physiological levels of sodium (Figure 5A). Similar upregulation in NCKK1 mRNA occurred in response to urea that is equimolar to high sodium exposure. Since NKCC activity is determined by phosphorylation, we assessed phosphorated-NKCC1 protein. Our results show that a high-sodium environment significantly reduced expression of phosphorated-NKCC1 protein while the hyperosmolar urea control did not (Figure 5A). Furthermore, as NKCC activity is linked to phosphorylation of WNK-SPAK/OSR1 signaling cascade, we also examined expression of this upstream kinase [21,22]. Our data show that a high-sodium environment also reduced phosphorylated SPAK compared with the baseline sodium control group (Figure 5C) [21]. Among the vasoactive factors, eNOS is a major endothelial-derived mechanism regulating lymphatic dynamics, which is regulated by activity of NKCC1 [23]. Exposing LECs to a high-sodium environment caused a significant reduction in eNOS activity as measured by the amount of phosphorylated eNOS protein (Figure 6A). In contrast, increased osmolarity with urea did not significantly alter the endothelial eNOS activity although the eNOS activity was significantly higher than in cells exposed to a high-sodium environment, echoing reduced p-eNOS levels shown in cardiac tissues of rats fed a high-salt diet [24]. To determine the consequences of reduced NO signaling on renal lymphatic vessel pumping dynamics, we exposed isolated vessels to L-NAME in order to inhibit eNOS activity. This caused a significant increase in contraction frequency, but reduced EDD, magnitude of contraction, and ejection fraction (Figure 6B).

**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 extrarenal 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**) Highsodium 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.
