The lymphatic circulation has been rarely considered as a contributor to cardiovascular pathologies or the target of medication side effects. For example, pharmacological openers of adenosine triphosphate-sensitive K+ (KATP) channels are effective antihypertensive agents, but off-target effects, including severe peripheral edema limit their clinical usefulness. It is presumed that the arterial dilation induced by KATP channel openers (KCOs) increases capillary pressure to promote filtration edema. However, KATP channels also are expressed by lymphatic muscle cells (LMCs), raising the possibility that KCOs also attenuate lymph flow to increase interstitial fluid. Aim 1 of the present dissertation explored the effect of KCOs on lymphatic contractile function and lymph flow. In isolated rat mesenteric lymph vessels (LVs), the prototypic KATP channel opener cromakalim (0.01–3 µmol/L) progressively inhibited rhythmic contractions and intraluminal flow. Minoxidil sulfate and diazoxide (0.01–100 µmol/L) had similar effects at clinically relevant plasma concentrations. High speed in-vivo imaging of the rat mesenteric lymphatic circulation revealed that superfusion of LVs with cromakalim and minoxidil sulfate (0.01-10 µmol/L) maximally decreased lymph flow in vivo by 38.4% and 27.4%, respectively. Real-time PCR and flow cytometry identified the abundant KATP channel subunits in LMCs as the pore-forming Kir6.1/6.2 and regulatory SUR2 subunits. Patch-clamp studies detected cromakalim-elicited unitary K+ currents in cell-attached patches of LMCs with a single-channel conductance of 52.1 pS, a property consistent with Kir6.1/6.2 tetrameric channels. Collectively, our findings indicate that KCOs attenuate lymph flow at clinically relevant plasma concentrations as a potential contributing mechanism to peripheral edema. In addition to medication side effects, the role of the lymphatic circulation may also have been over-looked in disease states. In Aim 2, we sought to determine the contribution of the lymphatic circulation to salt-sensitive hypertension. Recent reports indicate that in response to high salt, the lymphatic circulation within the dermis expands by lymphangiogenesis. Therefore, we hypothesized that in a salt-sensitive Dahl rat model, we would observe an increase in lymphangiogenesis markers within the kidney following 4 weeks of a high salt diet. Indeed, we found that Dahl salt-sensitive (DSS) rats fed a high salt (HS) diet had 2.0 ± 0.3-fold and 6.1 ± 0.7-fold higher gene expression of vascular endothelial growth factor receptor-C (VEGF-C) and its receptor vascular endothelial growth factor receptor-3 (VEGFR-3), respectively, within the kidney compared to Dahl salt-resistant (DSR) rats fed a normal salt (NS) diet. Similarly, Western blot analysis of kidneys of DSS rats fed a HS diet exhibited a higher protein expression of the lymphangiogenesis markers, VEGFR-3 and LYVE-1, compared to DSS rats fed a NS diet and DSR rats fed either a NS or HS diet. Next, we hypothesized that the expansion of the renal lymphatic vasculature in DSS rats fed a HS diet contributed to the development of hypertension. In fact, previous reports indicate that the renal lymphatic circulation may be responsible for returning more salt to the systemic circulation than the renal vein. We decided to test this hypothesis by ligating the collecting renal LVs draining the kidney and monitoring the effect on systemic blood pressure. First, we performed proof-of-concept studies in Sprague-Dawley (SD) rats. Renal LV ligation (RLL) of the left kidney decreased systolic blood pressure by 7.8 ± 0.8 mm Hg (n = 2) compared to rats exposed to sham surgery. After successfully performing the surgery in SD rats, we performed the same experiments in DSS rats fed a HS diet to induce hypertension. In DSS rats, RLL also significantly decreased systolic blood pressure, but did not halt the progression of hypertension. Two different time points were used to test this method. First, the DSS rats were fed a HS diet for 9 days before the RLL surgeries were performed. This resulted in a significant decrease in systolic blood pressure (24.4 ± 0.83 mm Hg) compared to sham animals. However, the decrease in blood pressure did not normalize blood pressure levels. In these animals, the RLL surgery was performed after the DSS rats were already execrably hypertensive. Therefore, we decided to perform the same experiment at an earlier time point after the induction of salt-sensitive hypertension by a HS diet. Accordingly, DSS rats were fed a HS diet for 5 days instead of 9 days before RLL to induce a less pronounced increase in blood pressure prior to the RLL surgery. Again, RLL caused a significant decrease in systolic blood pressure (14.1 ± 1.2 mm Hg) compared to similar rats exposed to sham surgery, but this decrease only partially normalized blood pressure, and again, was not as robust as anticipated. However, our data do provide evidence that the renal lymphatic vasculature contributes to salt-sensitive hypertension in DSS rats. Finally, earlier reports indicated that RLL induced a higher sodium urinary output and higher urine excretion. We anticipated that we would see similar results in our DSS model. Initially we performed a pilot study in which we ligated renal collecting LVs of left kidneys of normotensive SD rats fed a HS diet and measured urinary output using metabolic cages. This proof-of-concept study (n=2) in SD rats revealed that RLL resulted in a 30.2% increase in urine output compared to a 2% increase in urine output in sham-operated animals (n=1). When we performed the same experiments in DSS rats, however, we saw no significant difference in urine output between DSS rats subjected to RLL and sham-operated DSS rats. Similarly, we found no significant differences in urinary K+ or Cl- content between these animal groups. Unfortunately, the RLL group had a higher baseline urinary Na+ compared to sham animals, which confounded interpretation of these data. Following RLL, the Na+ urinary output in the RLL actually showed a significant reduction. Despite these negative results, we did see that RLL decreased systemic blood pressure in both SD rats, and in DSS rats who were fed a HS diet. Therefore, based on these results, the renal lymphatic circulation does appear to have some intrinsic effect on blood pressure, but the exact mechanism was not defined by our studies.
Based on our data from Aim 2 as well as data previously published indicating that a HS diet induces lymphangiogenesis in the kidney, we proposed Aim 3 as a mechanistic aim to determine the molecular mechanism of renal lymphangiogenesis during HS intake. Previous studies report that dermal lymphatics act as a “salt sink” to prevent excess salt from reentering the systemic circulation during a HS diet. We wanted to see if a similar “lymphangiogenesis event” occurred in the kidney. Contrary to the outcome of lymphatic expansion in the dermis, we hypothesized that a “lymphangiogenesis event” in the kidney results in the return of more salt and volume to systemic circulation. To determine the molecular mechanism of lymphangiogenesis, which was postulated to rely, in part, on macrophage transdifferentiation to lymphatic endothelial cells, we decided to utilize a macrophage cell line: RAW264.7. As determined using real time PCR, addition of 40 mmol/L NaCl to the cell culture media for 18 hours significantly increased gene expression of the lymphatic endothelial cell-specific markers, VEGFR-3 1.5–fold (n=3) and LYVE-1 5.5–fold (n=3), while significantly decreasing the gene expression of macrophage markers F4/80 and CD11b (n =3) 0.46-fold and 0.32-fold, respectively. Next, we utilized flow cytometry to determine if addition of 40 mmol/L NaCl to the culture media would increase VEGFR-3 protein expression in the RAW264.7 cells. Indeed, we found that addition of 40 mmol/L NaCl to the cell culture significantly increased the expression of VEGFR-3 protein while concomitantly decreasing the protein abundance of the macrophage-specific marker F4/80 (n=3). Interestingly, the addition of 40 mmol/L NaCl to the cell culture media significantly increased the gene expression of CD5L 4.5-fold. This gene is up-regulated under inflammatory conditions and is known to contribute to the pathogenesis of various disease states, including metabolic syndrome and atherosclerosis. When CD5L expression was down-regulated with siRNA, we found that the expected increase in gene expression of VEGFR-3 was attenuated, implying that CD5L may play a role as a proximal factor in the lymphangiogenesis pathway. Finally, we wanted to investigate the impact of a HS diet on the rhythmic contractions of isolated and cannulated LVs from DSS rats. DSS rats on a HS diet had shown increased expression of lymphatic endothelial cell markers, and now we sought to determine if a HS diet also led to functional changes in the collecting LVs draining the kidneys. In these studies, renal collecting LVs were dissected from 4 groups of rats: DSR rats on a NS diet or HS diet, and DSS rats on a NS diet or HS diet for 4 weeks. Diameter recordings of rhythmic contractions were performed at increasing intraluminal pressures (in 2 mm Hg increments) from 0 mm Hg to 10 mm Hg, but we failed to detect any differences in lymphatic contractile parameters within a rat strain due to diet. However, we found that the rhythmic contractions of LVs from DSS rats fed a HS diet showed a reduced frequency of contraction compared to DSR rats fed a HS diet at pressures 4, 6, and 10 mm Hg and DSS rats fed a NS diet at 8 and 10 mm Hg. Interestingly, we observed a preservation of lymph flow at the higher intraluminal pressures of 8 and 10 mm Hg in LVs from DSS rats compared to DSR rats. Thus, there appear to be differences between the rhythmic contractions in LVs from DSS and DSR rats, and even within the same rat strain NS and HS diets, but the findings are complex and no conclusions can be drawn about their translational importance without further ex vivo and in vivo studies.
In conclusion, we believe that the lymphatic system is a dynamic vasculature that is sensitive to medications at clinically relevant plasma concentrations and is highly responsive to pathological conditions. Our finding that KCOs inhibit lymphatic rhythmic contractions and attenuate lymph flow is likely only one example of the impact of prescription drugs on the function of the lymphatic circulation. Similarly, our studies in the Dahl salt-sensitive rat model of hypertension indicate that surgical ligation of the renal lymphatic collecting vessels can mitigate hypertension induced by a high salt diet. Additionally, the renal lymphatic system undergoes lymphangiogenesis during salt-induced hypertension in DSS rats, which may involve transdifferentiation of macrophages into lymphatic endothelial cells, and the collecting LVs from these animals show abnormalities of contractile function. The latter studies have raised many questions about the role of the lymphatic circulation in regulating blood pressure during salt-sensitive hypertension and other forms of hypertension that our laboratory and others will continue to seek to answer in future studies.
|Advisor:||Rusch, Nancy J., Mu, Shengyu|
|Commitee:||Davis, Michael J., Rhee, Sung, Weinkopff, Tiffany|
|School:||University of Arkansas for Medical Sciences|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 82/6(E), Dissertation Abstracts International|
|Subjects:||Toxicology, Pharmacology, Surgery, Cellular biology, Medicine, Public health|
|Keywords:||KATP channels, Lymph vessels, Lymphatic system, Salt-sensitivity, Vasculature, Medication sensitivity, Plasma concentrations, Prescription drugs, Hypertension, Surgical ligation, Endothelial cells, Regulating blood pressure, Ex vivo, In vivo|
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