**3. Discussion**

A high-sodium environment is a critical modulator of lymphatic vessels. Although kidneys are central in Na+ homeostasis, little is understood about Na+ effects on renal lymphatics. The current studies provide new insights into regulation of renal lymphatic network by showing (1) proteinuric kidney injury increases renal Na+ by 23Na/1H MRI and direct sampling of renal lymphatic fluid shows elevated Na+ concentration while plasma Na+ is unchanged (2) high Na+ and furosemide inhibition of NKCC1 decrease lymphatic vessel contraction amplitude and ejection fraction in isolated renal lymphatic vessels, (3) a high Na+ environment decreases phosphorated-NKCC1, phosphorylated SPAK, an upstream kinase, and phosphorylated eNOS, a downstream vasoactive factor, and (4) a high Na+ environment together with renal injury contribute to a blunted lymphatic response in PAN-injured kidneys.

Noninvasive imaging by 23Na/1H MRI showed that proteinuric kidney injury leads to accumulation of sodium and water in the in vivo kidneys. This new observation reflects advances in multi-nuclear imaging technology that exploit endogenous 23Na, the second most abundant magnetic nuclei in living systems [25]. Imaging methods are advantageous for longitudinal measurement of tissue sodium before and after intervention, localization of tissue sodium in renal sub-compartments, and comparison of multi-modal data, strategies explored in this study. The findings of this study demonstrate 23Na-MRI quantification of renal sodium as a potential biomarker of renal disease involving lymphatic clearance dysfunction. Imaging results, supported by data, suggest that lymph exiting proteinuric kidneys has significantly higher sodium concertation than the renal lymph of normal, uninjured control rats. Sodium levels in the blood of these proteinuric animals were not different from normal rats. To date, there are only sparse data on the composition of renal lymph, especially in disease settings, although more than 50 years ago, two studies describing partial occlusion of the inferior vena cava model of right heart failure found increased renal lymphatic flow and sodium content [26,27]. More recently, sodium accumulation in the skin of salt-sensitive hypertensive rats was shown to be accompanied by increased sodium concentration in lymph collected from dermal lymphatic vessels, while no change in the circulating level of sodium was observed [4]. These findings reinforce the concept that lymph reflects the composition of the interstitial compartment of the draining organ. Our

data make the original observation that kidney injury leads to renal sodium accumulation, although the study did not localize sodium to any specific interstitial compartment [1]. Sodium accumulation in the interstitium has been linked to the modulation of lymphatic vessels, especially lymphangiogenesis. This has been most extensively studied in the skin of hypertensive animals and humans and involves transcription factor tonicity-responsive enhancer protein (TonEBP)-induced macrophage secretion of vascular endothelial growth factor-C (VEGF-C) [4]. Although kidney injury causes renal lymphangiogenesis and modulates sodium reabsorption and excretion, there have been no studies on the possible effects of accumulating interstitial sodium on renal lymphatic function. We now show that direct exposure of renal lymphatic vessels to a high-sodium environment increases the frequency of contraction in the renal collecting lymphatic vessels and reduces the contraction amplitude, and, to a lesser extent, the ejection fraction (Figure 3). These results complement findings that a high-salt diet, or DOCA treatment that increases sodium in skin and muscle, increases contraction frequency while reducing contraction amplitude [19]. These observations are timely, since strategies to improve interstitial clearance currently target lymphatic network growth, although the efficacy appears to be context-dependent. Thus, activation of the VEGF-C–VEGFR-3 pathway to promote lymphangiogenesis can reduce kidney fibrosis and lessen cystic kidney disease in mice and rats [9]. Also, kidney-specific overexpression of VEGF-D before injury increased lymphatic density and amplified recovery from ischemia-reperfusion damage [28]. In contrast, inhibition of VEGFR-3 reduces kidney lymphangiogenesis, glomerulosclerosis, and tubulointerstitial fibrosis in a mouse model of diabetic kidney disease as well as fibrosis following UUO and ischemia-reperfusion [10]. Our data suggest that high interstitial sodium blunts lymphatic dynamics and may be a critical factor contributing to the efficacy of therapeutic intervention.

Currently, the first-line therapy to reduce sodium overload in a variety of diseases, including kidney disease, is inhibition of NKCC cotransporter with furosemide. Immunohistochemical staining clearly demonstrated NKCC1 in endothelial cells of renal collecting lymphatic vessels, and quantitation of mRNA showed increased gene expression in PAN vessels vs collecting vessels of uninjured kidneys (Figure 5). However, a high-sodium environment significantly reduced phosphorylation of NKCC1 in LECs, (Figure 6). Moreover, a high-sodium environment also reduced phosphorylation of SPAK, the upstream kinase of NKCC1, suggesting sodium dampens lymphatic contractility. Previous studies showed high salt downregulated phosphorylation and ubiquitination of WNK [29], which reduced expression of SPAK and NKCC1. Zeniya et al. showed suppressed phosphorylation of NKCC1 on mouse aortae fed a high-salt diet and stimulated phosphorylation of NKCC1 in mice on a low-salt diet [22]. Similar to our results with direct sodium exposure, a high-salt diet caused a divergent effect on the gene and protein expression of upstream kinases. Together, these data fit well with evidence that, aside from maintaining extracellular fluid volume, sodium acts as a signaling molecule.

NKCC1 activity can contribute to both vasoconstriction and vasodilation. Vasoconstrictors such as norepinephrine, endothelin, and angiotensin II directly activate NKCC1 activity in vascular smooth muscle cells, causing constriction, while NO and sodium nitroprusside inhibit NKCC1, resulting in vasodilation [30,31]. High-sodium environments reduce phosphorylated eNOS, which would predict reduced vasodilation but increased contractility. Indeed, inhibiting NO signaling with L-NAME decreased end diastolic and end systolic vessel diameter, the amplitude of contraction, calculated ejection fraction, and increased contraction frequency in renal lymphatic vessels (Figure 6B). Interestingly, previous studies confirm that a high-salt diet and/or direct exposure of lymphatic vessels to a high-sodium environment increases contraction frequency in skin and muscle lymphatics and inguinal lymphatic vessels of mice and rats [19,32,33].

Our data clearly show that a high-sodium environment directly blunts lymphatic dynamics (Figure 3). Since lymphatic vessels are exquisitely sensitive to environmental stimuli, other molecules within the renal interstitial compartment including vasoactive substances, for example, angiotensin II, may also play a role in lymphatic dynamic functions.

However, comparison with vessels from PAN-injured kidneys exposed to a high-sodium environment revealed that renal injury is an additive contributor to lymphatic dysfunction (Figure 7). Thus, injured vessels exposed to high sodium showed diminution in their ability to respond to a pathological shift in their environment. This constellation of findings predicts impaired drainage of the renal interstitium in settings where a high interstitial sodium environment may prevail, such as in congestive heart failure, cirrhosis, and acute and chronic kidney disease. Moreover, these are the very conditions that show relative resistance to interventions that promote sodium excretion by inhibition of NKCC1. Notably, the ejection fraction in PAN-injured vessels is less affected by increasing concentrations of furosemide (Figure 9). Currently, therapeutic resistance to these agents centers on impaired delivery of the therapeutic to the relevant tubular segment. However, based on our data, we propose that dysfunction of renal lymphatic vessels is related to electrolyte abnormalities in the microenvironment of the kidney.
