*2.3. Kidney Injury Diminished the Lymphatic Vascular Response to a High-Sodium Environment and NKCC Inhibition by Furosemide*

To gain further insight into the effects of kidney injury on renal lymphatic physiology, we compared the pumping dynamics of control vessels with vessels from a PAN-injured rat in a normal sodium environment (Figure 7). PAN-injured vessels had a significant increase in EDD (Figure 7B), which contributed to a marked decrease in contraction amplitude (Figure 7D) and ejection fraction (Figure 7E) compared with control vessels. Next, to investigate how a high-sodium environment affects vessels in the setting of kidney injury, we compared the lymphatic dynamics in the vessels of PAN-injured rats before and after exposure to a high-sodium environment (Figure 8). PAN-injured lymphatic vessels had a distinct response to a high-sodium environment compared with the response of control vessels (Figure 3), exhibiting a decreased EDD, and a decreased ejection fraction. This

suggests that a high-sodium environment and PAN injury both result in reduced ejection fraction, albeit by different mechanisms.

**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.

Since injured lymphatic vessels appear to have weaker intrinsic compensatory responses to the high-sodium environment likely prevailing in a disease setting, we examined the effects of the NKCC1 inhibitor furosemide in PAN-injured and control vessels. Control vessels treated with furosemide had a pronounced concentration-dependent decrease in ejection fraction. In contrast, furosemide had more subtle effects on the ejection fraction of PAN-injured vessels, with PAN vessels being significantly less affected by furosemide at physiologically relevant doses (Figure 9). Interestingly, there was no statistical difference in the effects of furosemide on PAN vessels in a normal or high-sodium environment. These results indicate that analogous to a high-sodium environment and PAN-induced kidney injury, furosemide exerts directionally similar moderating effects on lymphatic dynamics that may affect renal interstitial clearance.

**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 highsodium 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.
