Renal Lymphangiogenesis Research Articles

Impact of lymphangiogenesis on renal phosphate handling following kidney injury

Overconsumption of inorganic phosphate (Pi) causes tubular cell damage and kidney dysfunction, and hyperphosphatemia can lead to Ca2+ retention, vascular calcification, and cardiovascular disease. Conversely, kidney injury and chronic kidney disease (CKD) cause hyperphosphatemia and Pi retention. We hypothesized that the damage caused by chronic Pi consumption likely induces kidney lymphangiogenesis and that changes to renal lymphatic density may alter kidney mineral handling. We identified significant lymphangiogenesis and increased expression of the lymphatic growth factors VEGF-C/D in kidneys of mice fed a 2% Pi diet for 2 months and as early as three weeks of diet. We then utilized “KidVD” mice, a mouse model of kidney-specific inducible expression of VEGF-D, to expand renal lymphatic density and test Pi handling. Mineral handling was largely normal in male and female KidVD mice compared to littermates on Pi diet. In a Pi-driven CKD model mice were injured with 10 mg/kg cisplatin dose, allowed to recover for 2 weeks, then challenged with chow or 2% Pi diet for 3 weeks with VEGF-D induction. This resulted in Pi retention in all mice. Pi handling in both male and female KidVD mice were normal to controls. Female KidVD, however, demonstrated significantly increased Ca2+ excretion compared to controls. Circulating FGF23 levels were significantly elevated in male and female with VEGF-D overexpression. Gene expression of Fgfr1 was also significantly increased in male and female KidVD mice. Renal lymphangiogenesis thus appears to specifically impact Ca2+ retention through the FGF23-Fgfr1 axis. National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (R01 DK119497). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Novel avenues to control blood pressure: targeting the renal lymphatic system.

Hypertension is associated with the activation of the immune and lymphatic systems as well as lymphangiogenesis. The changes in the lymphatic system are considered an adaptive response to mitigate the deleterious effects of immune and inflammatory cells on the cardiovascular system. In the article recently published in Clinical Science by Goodlett and collaborators, evidence is shown that inducing renal lymphangiogenesis after the establishment of hypertension in mice is an effective maneuver to reduce systemic arterial blood pressure. In this commentary, we will briefly review what is known about the relationship between the activation of the immune and lymphatic systems, and the resulting effects on systemic blood pressure, summarize the findings published by Goodlett and collaborators, and discuss the impact of their findings on the field.

Role of the Renal Lymphatic System in Heart Failure.

The lymphatic system plays a major but overlooked role in maintaining fluid homeostasis. Given the unique fluid homeostasis functions of the kidneys, dysregulation of the renal lymphatic system underlies the development of self-propagating congestive pathomechanisms. In this review, we outline the roles of the renal lymphatic system in heart failure (HF). Studies have uncovered several pathomechanisms involving the renal lymphatic system in congestive states, such as impaired interstitial draining by the renal lymphatic system, impaired structure and valves of renal lymphatics, lymphatic-induced increase in renal reabsorption of water and sodium, and development of albuminuria with proteinuria-induced renal lymphangiogenesis. These self-propagating mechanisms result in "renal tamponade" with manifestations of cardiorenal syndrome and inappropriate renal response to diuretics. Dysregulation of the renal lymphatic system is integral to the development and progression of congestion in HF. Targeting renal lymphatics may provide a novel pathway to treat intractable congestion.

Genetically inducing renal lymphangiogenesis attenuates hypertension in mice.

Hypertension (HTN) is associated with renal proinflammatory immune cell infiltration and increased sodium retention. We reported previously that renal lymphatic vessels, which are responsible for trafficking immune cells from the interstitial space to draining lymph nodes, increase in density under hypertensive conditions. We also demonstrated that augmenting renal lymphatic density can prevent HTN in mice. Whether renal lymphangiogenesis can treat HTN in mice is unknown. We hypothesized that genetically inducing renal lymphangiogenesis after the establishment of HTN would attenuate HTN in male and female mice from three different HTN models. Mice with inducible kidney-specific overexpression of VEGF-D (KidVD) experience renal lymphangiogenesis upon doxycycline administration. HTN was induced in KidVD+ and KidVD- mice by subcutaneous release of angiotensin II, administration of the nitric oxide synthase inhibitor L-NAME, or consumption of a 4% salt diet following a L-NAME priming and washout period. After a week of HTN stimuli treatment, doxycycline was introduced. Systolic blood pressure (SBP) readings were taken weekly. Kidney function was determined from urine and serum measures. Kidneys were processed for RT-qPCR, flow cytometry, and imaging. Mice that underwent renal-specific lymphangiogenesis had significantly decreased SBP and renal proinflammatory immune cells. Additionally, renal lymphangiogenesis was associated with a decrease in sodium transporter expression and increased fractional excretion of sodium, indicating improved sodium handling efficiency. These findings demonstrate that augmenting renal lymphangiogenesis can treat HTN in male and female mice by improving renal immune cell trafficking and sodium handling.

Abstract P110: Differential Gene Expression Associated With Ccn1-positive Renal Cells In Murine Hypertensive Models

Hypertension affects roughly half of the U.S. population and is characterized by detrimental pro-inflammatory changes that alter renal function and the cardiovascular system over time. The kidney is a major regulator of blood pressure, however, the specific contributions of renal cell types, including lymphatic endothelial cells (LECs), to hypertension have not been thoroughly explored. Previous work from our lab has shown an increase in renal lymphangiogenesis in response to hypertension and that augmentation of renal lymphangiogenesis can reduce blood pressure. CCN1/Cyr61 is an extracellular matrix protein secreted by multiple cell types, particularly LECs and fibroblasts, which contributes to integrin signaling pathways. CCN1 is also known to be involved in lymphangiogenesis, and has previously been implicated in GWAS studies of hypertension. Given these connections, we performed single cell RNA sequencing using angiotensin II and salt sensitive mouse models of hypertension, which were analyzed separately as well as aggregated into one hypertensive group. Kidneys were turned to a single cell suspension and separated for CD31+/Podoplanin+ cells meant to better represent LECs, which are a small fraction of renal cells. Sequencing was performed and the gene reads were run through the Cell Ranger, Seurat, FGSEA, and Slingshot pipelines, then CCN1+ cells were isolated digitally and analyzed separately. In the AngII model, a total of 60 genes (28 up, 32 down) were differentially expressed (p<0.01) between the renal CCN1+ cells in the hypertensive mice compared to controls, while the salt sensitive model had a total of 331 differentially expressed genes (305 up, 26 down). When the hypertension groups were merged, there were a total of 600 differentially expressed genes (503 up, 97 down) with GSEA showing 48 pathways with enrichment scores >3 or <-3. Additionally, the cellular profile of CCN1+ cells shifted, branching out from a fibroblast-like population to other cell types such as endothelium and tubular epithelium. Overall, the expression of CCN1+ renal cells demonstrates notable changes in response to hypertension in murine models and encourages further study of the mechanisms behind CCN1’s role in the development and maintenance of hypertension.

CD137L-macrophage induce lymphatic endothelial cells autophagy to promote lymphangiogenesis in renal fibrosis

Renal lymphangiogenesis is a new field of international nephrology in recent years and plays an important role in the progression of chronic renal disease. CD137 was originally described as a surface molecule present on activated T and NK cells and detected on hypoxic endothelial cells and inflamed blood vessels, but its function on lymphatic endothelial cells remains unclear. We investigated the relationships among CD137, lymphangiogenesis and macrophages, which are involved in interstitial fibrosis. Similar to other chronic inflammatory diseases, we found lymphangiogenesis and expression of CD137 in the renal tissue of patients with IgA nephropathy. CD137-positive lymphatic vessels were involved in the development process of IgA nephropathy and positively correlated with serum creatinine, serum urea nitrogen, serum uric acid, and urinary 24 h total protein. The expression of these indicators was negatively correlated with eGFR, plasma albumin, and HB. In mouse models of UUO, we verified that CD137 expression was significantly elevated during lymphangiogenesis and that its ligand CD137L was released by macrophages after VEGF-C stimulation in the kidney. In vitro, recombinant CD137L significantly enhanced LEC proliferation, migration and tube formation, and these effects were inhibited by CD137 siRNA. Mechanistically, the CD137L interaction with CD137 induced the transition from LC3-I to LC3-II and the expression of Atg5, Atg7, Atg12 and p62 proteins by activating the PI3K/AKT/mTOR pathway to promote autophagy. Knockdown of Atg5 and Atg7 blocked CD137L-induced autophagy. Thus, we propose that CD137L secretion by macrophages interacts with CD137 on lymphatic endothelial cells to prompt lymphangiogenesis in the kidney, which further drives fibrogenic responses. Our findings suggest that inhibition of the CD137-CD137L pathway is a novel therapeutic approach for obstructive nephropathy.

A Kidney-Targeted Nanoparticle to Augment Renal Lymphatic Density Decreases Blood Pressure in Hypertensive Mice.

Chronic interstitial inflammation and renal infiltration of activated immune cells play an integral role in hypertension. Lymphatics regulate inflammation through clearance of immune cells and excess interstitial fluid. Previously, we demonstrated increasing renal lymphangiogenesis prevents hypertension in mice. We hypothesized that targeted nanoparticle delivery of vascular endothelial growth factor-C (VEGF-C) to the kidney would induce renal lymphangiogenesis, lowering blood pressure in hypertensive mice. A kidney-targeting nanoparticle was loaded with a VEGF receptor-3-specific form of VEGF-C and injected into mice with angiotensin II-induced hypertension or LNAME-induced hypertension every 3 days. Nanoparticle-treated mice exhibited increased renal lymphatic vessel density and width compared to hypertensive mice injected with VEGF-C alone. Nanoparticle-treated mice exhibited decreased systolic blood pressure, decreased pro-inflammatory renal immune cells, and increased urinary fractional excretion of sodium. Our findings demonstrate that pharmacologically expanding renal lymphatics decreases blood pressure and is associated with favorable alterations in renal immune cells and increased sodium excretion.

Increased Plasma GDF15 Is Associated with Altered Levels of Soluble VEGF Receptors 1 and 2 in Symptomatic Multiple Myeloma

Introduction: In multiple myeloma, there is an increase in bone marrow microvascular density and enhanced renal lymphangiogenesis. Increased levels of the proangiogenic protein growth differentiation factor-15 (GDF15) have previously been reported to be associated with poor prognosis in myeloma. A possible association between GDF15 and the soluble forms of vascular endothelial growth factor receptors (sVEGFR) 1 and 2 has not yet been investigated, and a role for these receptors in pathological angiogenesis in myeloma is still to be defined. Methods: Plasma levels of GDF15 and sVEGFR1 and 2 were determined by ELISA in patients with smouldering multiple myeloma (sMM), patients with symptomatic multiple myeloma (abbreviated as MM), and healthy controls. The levels were compared between the three groups, and correlation coefficients were calculated, as were Kaplan-Meier curves for GDF15 and sVEGFR1 and sVEGFR2. Results: Levels of GDF15 were significantly higher in MM than in both patients with sMM and controls. A gradual decrease in mean sVEGFR1 concentration was observed, with MM > sMM > controls. Mean sVEGFR2 was lower in patients with MM than in controls. There was a positive correlation between GDF15 and sVEGFR1, and GDF15 correlated negatively with sVEGFR2. High GDF15 (>3 ng/mL) was associated with poor prognosis. Conclusion: In multiple myeloma, increased expression of GDF15 correlates positively with sVEGFR1 and negatively with sVEGFR2. It is possible that the altered levels of sVEGFR1 and 2 contribute to the increased angio- and lymphangiogenesis observed in myeloma.

Abstract P132: A Kidney-Targeted Nanoparticle To Augment Renal Lymphatic Density Decreases Blood Pressure In Mice With L-NAME- And Angiotensin II-Induced Hypertension

Chronic interstitial inflammation and renal infiltration of activated immune cells play an integral role in hypertension. Lymphatic vessels attenuate inflammation by trafficking activated immune cells and excess fluid from the interstitial space to lymph nodes. Previously, our laboratory demonstrated that genetically inducing renal lymphangiogenesis could treat hypertension in three different mouse models. In an effort to translate these findings into a clinical treatment, we hypothesized that a targeted nanoparticle could deliver the pro-lymphangiogenic factor VEGF-C156S to the kidney, induce lymphangiogenesis, and lower blood pressure in hypertensive mice. A micellar nanoparticle was developed with the capacity to deliver protein to the kidney, as demonstrated through delivery trials. This nanoparticle was loaded with VEGF-C156S and injected into mice with LNAME-induced hypertension (LHTN) or angiotensin II-induced hypertension (AIIHTN) via tail vein every 3 days. Compared to hypertensive mice injected with VEGF-C156S only (no nanoparticle) every 3 days, nanoparticle-treated mice exhibited a significantly lower systolic blood pressure (SBP) after 4 injections (LHTN SBP: 160±5 vs. 120±3 mmHg, p<0.001; AIIHTN SBP: 150±8 vs. 126±6 mmHg, p=0.03). Immunolabeled kidney sections from nanoparticle-treated LHTN mice showed a significant increase in podoplanin+ pixels, corresponding to an increase in lymphatic vessel density (p<0.01). A 5-fold increase in renal gene expression of podoplanin in nanoparticle-treated LHTN mice further supported this finding (p=0.01). Flow cytometric analysis of the nanoparticle-treated LHTN mice showed decreased renal CD45+F4/80+CD11c- cells, while AIIHTN mice revealed decreased levels of renal CD45+CD3e+, CD45+CD4+CD8-, and CD45+F4/80+CD11c+ cells (p<0.01, p=0.03, and p<0.001, respectively) when compared to their respective hypertensive groups. These data support our previous findings that expanding the renal lymphatic vasculature can treat existing hypertension by reducing renal immune cells. The results of this study may provide clinicians with a renal lymphatic-targeted therapeutic for treating hypertensive patients.

Abstract P3020: Therapeutic Induction of Renal Lymphatic Expansion Attenuates Blood Pressure in Mice With L-NAME Hypertension

Renal immune cell infiltration and accompanying inflammation activates renal sodium transporters leading to sodium retention and hypertension. Lymphatic vessels traffic interstitial immune cells to the draining lymph nodes and help resolve inflammation. We have previously demonstrated that genetically inducing renal lymphangiogenesis prevents hypertension associated with a reduction in renal immune cell accumulation; however, it is unknown whether augmenting renal lymphatics can treat hypertension. Our hypothesis was that augmenting renal lymphatics after hypertension is established will lower blood pressure during L-NAME-induced hypertension (LHTN). Transgenic mice that overexpress the lymphangiogenic signal VEGF-D only in the kidney upon doxycycline administration (KidVD+ mice) and KidVD- littermates were administered L-NAME (0.5 mg/mL) in their drinking water for four weeks with doxycycline initiated at the second week of L-NAME. Treatment with L-NAME for one week induced LHTN in both KidVD- and KidVD+ mice (SBP: 134±4 and 142±5 mmHg, respectively). However, doxycycline-induced renal lymphangiogenesis significantly decreased blood pressure in KidVD+ mice compared to KidVD- mice after four weeks of L-NAME (SBP: 127±5 vs. 151±6 mmHg; p<0.05). This was associated with a significant decrease in renal CD11c + F4/80 - monocytes and a significant increase in 24-hour urinary sodium excretion and fractional excretion of sodium in KidVD+ mice. Urinary volume over 24 hours was also increased in KidVD+ mice (1.7±0.8 vs. 3±0.9 mL/24 hours; p<0.05); however, this was not associated with a change in glomerular filtration rate. Thus, augmenting renal lymphatics lowers blood pressure in mice with LHTN and this is associated with a decrease in renal monocyte accumulation and an increase in urinary sodium excretion. Augmenting renal lymphatics may be a promising anti-hypertensive and natriuretic therapeutic strategy.

Augmenting Renal Lymphatic Density Prevents Angiotensin II-Induced Hypertension in Male and Female Mice.

Renal inflammation and immune cell infiltration are characteristic of several forms of hypertension. Our laboratory has previously demonstrated that renal-inflammation-associated lymphangiogenesis occurs in salt-sensitive and nitric-oxide-inhibition-induced hypertension. Moreover, enhancing renal lymphatic density prevented the development of these two forms of hypertension. Here, we investigated the effects of angiotensin II-induced hypertension on renal lymphatic vessel density in male and female mice. Wild-type and genetically engineered male and female mice were infused with angiotensin II for 2 or 3 weeks. Isolated splenocytes and peritoneal macrophages from mice, and commercially available mouse lymphatic endothelial cells were used for in vitro studies. Compared to vehicle controls, angiotensin II-infused male and female mice had significantly increased renal lymphatic vessel density in association with pro-inflammatory immune cells in the kidneys of these mice. Direct treatment of lymphatic endothelial cells with angiotensin II had no effect as they lack angiotensin II receptors; however, angiotensin II treatment of splenocytes and peritoneal macrophages induced secretion of the lymphangiogenic growth factor VEGF-C in vitro. Utilizing our genetic mouse model of inducible renal lymphangiogenesis, we demonstrated that greatly augmenting renal lymphatic density prior to angiotensin II infusion prevented the development of hypertension in male and female mice and this was associated with a reduction in renal CD11c+F4/80- monocytes. Renal lymphatics play a significant role in renal immune cell trafficking and blood pressure regulation, and represent a novel avenue of therapy for hypertension.

Contribution of the Renal Lymphatic Circulation to the Development of Salt‐Sensitive Hypertension

More than one billion people worldwide suffer from hypertension, which is a leading cause of stroke and cardiovascular disease. According to the National Heart, Lung, and Blood Institute (NHLBI) Working Group, an estimated 40% to 85% of hypertensive patients exhibit salt‐sensitivity, which increases the risk for cardiovascular events and mortality. Salt‐sensitive hypertension is known to be caused by an imbalance between sodium retention and excretion in the kidney. However, the mechanisms that enable the kidney to deliver excessive re‐absorbed salt and volume back to the systemic circulation remain obscure. Interestingly, recent studies indicate that the peripheral lymphatics act as a “salt sink” to store excessive salt and volume, thereby maintaining sodium homeostasis to regulate blood pressure. This finding led us to consider whether renal lymphatics contribute to excessive sodium retention in the kidney during the development of salt‐sensitive hypertension. To test this hypothesis, we initially evaluated lymph‐angiogenesis in the kidneys of rats with or without salt‐sensitive hypertension. Real‐time PCR, immunohistochemistry and Western blots revealed increased expression of the lymphatic‐specific marker VEGFR3 in the kidneys of Dahl salt‐sensitive (DSS) rats fed a high‐salt diet compared to either DSS rats fed regular chow or Dahl salt‐resistant (DSR) rats with or without high‐salt intake. To explore the impact of renal lymph‐angiogenesis on blood pressure, we surgically ligated the renal collecting lymph vessel (LV) in DSS rats. Ligation of the renal LV in DSS rats attenuated the increase in systolic blood pressure caused by a high‐salt diet. Notably, a growing body of evidence has implicated macrophages in lymph‐angiogenesis. Indeed, we also observed that a high‐salt diet promoted renal infiltration of macrophages, which stimulated renal lymph‐angiogenesis via upregulating VEGFR3 and its ligand VEGFC. Collectively, our data suggest that in response to a high‐salt diet, kidney‐infiltrated macrophages stimulate renal lymph‐angiogenesis, which appears to support the development of salt‐sensitive hypertension.Support or Funding InformationSupported by NIH grants T32 GM106999 (BRG) and R21 CA187325‐01A1 (NJR), and a Medical Research Endowment Award (SM) from the Univ. of Arkansas for Medical Sciences.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of lymphatic proliferation by the selective VEGFR-3 inhibitor SAR131675 ameliorates diabetic nephropathy in db/db mice

Recent studies have demonstrated that chronic inflammation-induced lymphangiogenesis plays a crucial role in the progression of various renal diseases, including diabetic nephropathy. SAR131675 is a selective vascular endothelial cell growth factor receptor-3 (VEGFR-3)-tyrosine kinase inhibitor that acts as a ligand for VEGF-C and VEGF-D to inhibit lymphangiogenesis. In this study, we evaluated the effect of SAR131675 on renal lymphangiogenesis in a mouse model of type 2 diabetes. Male C57BLKS/J db/m and db/db mice were fed either a regular chow diet or a diet containing SAR131675 for 12 weeks from 8 weeks of age. In addition, we studied palmitate-induced lymphangiogenesis in human kidney-2 (HK2) cells and RAW264.7 monocytes/macrophages, which play a major role in lymphangiogenesis in the kidneys. SAR131475 ameliorated dyslipidemia, albuminuria, and lipid accumulation in the kidneys of db/db mice, with no significant changes in glucose and creatinine levels and body weight. Diabetes-induced systemic inflammation as evidenced by increased systemic monocyte chemoattractant protein-1 and tumor necrosis factor-α level was decreased by SAR131475. SAR131475 ameliorated the accumulation of triglycerides and free fatty acids and reduced inflammation in relation to decreased chemokine expression and pro-inflammatory M1 macrophage infiltration in the kidneys. Downregulation of VEGF-C and VEGFR-3 by SAR131475 inhibited lymphatic growth as demonstrated by decreased expression of LYVE-1 and podoplanin that was further accompanied by reduced tubulointerstitial fibrosis, and inflammation in relation to improvement in oxidative stress and apoptosis. Treatment with SAR131475 improved palmitate-induced increase in the expression of VEGF-C, VEGFR-3, and LYVE-1, along with improvement in cytosolic and mitochondrial oxidative stress in RAW264.7 and HK2 cells. Moreover, the enhanced expression of M1 phenotypes in RAW264.7 cells under palmitate stress was reduced by SAR131475 treatment. The results suggest that modulation of lymphatic proliferation in the kidneys is a new treatment approach for type 2 diabetic nephropathy and that SAR131675 is a promising therapy to ameliorate renal damage by reducing lipotoxicity-induced lymphangiogenesis.

Angiotensin inhibition in the developing kidney; tubulointerstitial effect.

Renin-angiotensin system (RAS) blockade during nephrogenesis causes a broad range of renal mal-development. Here, we hypothesized that disruption of renal lymphangiogenesis may contribute to tubulointerstitial alterations after RAS blockade during kidney maturation. Newborn rat pups were treated with enalapril (30 mg/kg/day) or vehicle for 7 days after birth. Lymphangiogenesis was assessed via immunostaining and/or immunoblots for vascular endothelial growth factor (VEGF)-C, VEGF receptor (VEGFR)-3, Podoplanin, and Ki-67. The intrarenal expression of fibroblast growth factor (FGF)-1, FGF-2, FGF receptor (R)-1, α-smooth muscle actin (α-SMA), and fibroblast-specific protein (FSP)-1 was also determined. Sirius Red staining was performed to evaluate interstitial collagen deposition. On postnatal day 8, renal lymphangiogenesis was disrupted by neonatal enalapril treatment. The expression of podoplanin and Ki-67 decreased in enalapril-treated kidneys. While the expression of VEGF-C was decreased, the levels of VEGFR-3 receptor increased following enalapril treatment. Enalapril treatment also reduced the renal expression of FGF-1, FGF-2, and FGFR-1. Enalapril-treated kidneys exhibited profibrogenic properties with increased expression of α-SMA and FSP-1 and enhanced deposition of interstitial collagen. Enalapril treatment during postnatal renal maturation can disrupt renal lymphangiogenesis along with tubulointerstitial changes, which may result in a pro-fibrotic environment in the developing rat kidney.

Abstract 024: Genetically Inducing Renal Lymphangiogenesis Prevents Angiotensin II-Induced Hypertension in Mice

Angiotensin II (AII)-dependent hypertension (AIIHTN) is associated with renal immune cell infiltration and inflammation. Lymphatic vessels drain interstitial fluid and traffic immune cells to draining lymph nodes; however, the role of renal lymphatics in AIIHTN is unknown. Our hypotheses were that: 1) renal lymphatic vessel density is increased in mice with AIIHTN, and 2) that further augmenting renal lymphatic vessels will prevent AIIHTN. Male and female mice were infused with AII (490 ng/kg/min) or saline for 2 or 3 weeks by subdermal osmotic mini pumps. Male and female mice with AIIHTN had markedly increased renal lymphatic vessel density compared to controls. AIIHTN males had significantly increased renal gene expression of the lymphatic vessel markers Lyve1, Pdpn, and Vegfr3, while Pdpn and the lymphangiogenic signal Vegfc were increased significantly in AIIHTN females. Kidneys of AIIHTN males had significantly increased F4/80+ macrophages at 2 weeks and F4/80+ macrophages and CD3e+ T cells at 3 weeks as determined by flow cytometry. Unlike in males, renal CD11c+ dendritic cells were increased significantly in females. Renal mRNA levels of the pro-inflammatory cyto/chemokines Tnfa , Il1b , Mcp1 , and Cxcl13 were elevated significantly in males at 2 and 3 weeks and in females at 3 weeks. To determine whether augmenting renal lymphatic vessels prior to AII infusion could prevent AIIHTN, we used transgenic mice that overexpress the lymphangiogenic signal VEGF-D only in the kidney under the control of doxycycline (KidVD+ mice) and thus exhibit renal-specific lymphangiogenesis. Doxycycline initiated 1 week prior to 3-week AII infusion prevented AIIHTN in KidVD+ mice while KidVD- mice still developed AIIHTN (Males SBP: 122±2 vs. 161±3 mmHg; p<0.05; Females SBP: 114±1 vs. 131±4 mmHg; p<0.05). KidVD+ AIIHTN mice had significantly decreased renal levels of CD11c+ dendritic cells and CD8+ T cells. Renal gene expression of Tnfa and Il1b were normalized in all KidVD+ mice. These data demonstrate that renal lymphatic vessel density is increased in AIIHTN and that genetically inducing renal lymphangiogenesis prior to AII infusion can prevent AIIHTN by reducing renal immune cells and inflammation.

Roles of the TGF-β⁻VEGF-C Pathway in Fibrosis-Related Lymphangiogenesis.

Lymphatic vessels drain excess tissue fluids to maintain the interstitial environment. Lymphatic capillaries develop during the progression of tissue fibrosis in various clinical and pathological situations, such as chronic kidney disease, peritoneal injury during peritoneal dialysis, tissue inflammation, and tumor progression. The role of fibrosis-related lymphangiogenesis appears to vary based on organ specificity and etiology. Signaling via vascular endothelial growth factor (VEGF)-C, VEGF-D, and VEGF receptor (VEGFR)-3 is a central molecular mechanism for lymphangiogenesis. Transforming growth factor-β (TGF-β) is a key player in tissue fibrosis. TGF-β induces peritoneal fibrosis in association with peritoneal dialysis, and also induces peritoneal neoangiogenesis through interaction with VEGF-A. On the other hand, TGF-β has a direct inhibitory effect on lymphatic endothelial cell growth. We proposed a possible mechanism of the TGF-β–VEGF-C pathway in which TGF-β promotes VEGF-C production in tubular epithelial cells, macrophages, and mesothelial cells, leading to lymphangiogenesis in renal and peritoneal fibrosis. Connective tissue growth factor (CTGF) is also involved in fibrosis-associated renal lymphangiogenesis through interaction with VEGF-C, in part by mediating TGF-β signaling. Further clarification of the mechanism might lead to the development of new therapeutic strategies to treat fibrotic diseases.

Immune cell trafficking, lymphatics and hypertension.

Activated immune cell infiltration into organs contributes to the development and maintenance of hypertension. Studies targeting specific immune cell populations or reducing their inflammatory signalling have demonstrated a reduction in BP. Lymphatic vessels play a key role in immune cell trafficking and in resolving inflammation, but little is known about their role in hypertension. Studies from our laboratory and others suggest that inflammation-associated or induction of lymphangiogenesis is organ protective and anti-hypertensive. This review provides the basis for hypertension as a disease of chronic inflammation in various tissues and highlights how renal lymphangiogenesis is a novel regulator of kidney health and BP. LINKED ARTICLES: This article is part of a themed section on Immune Targets in Hypertension. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.12/issuetoc.

Abstract 353: Aldehyde Scavenging Reduces Renal Injury Role of IsoLG-modified apoAI

Reactive aldehydes, including isolevuglandins (IsoLG) are lipoxidation products that can modify lipoproteins and promote cellular dysfunction. Previously, we showed that proteinuric injury increases urinary apoAI and causes renal lymphangiogenesis. We now investigate the role of IsoLG and effects of IsoLG scavenging on renal injury and lymphatics. Nphs1-hCD25 mice (NEP25) expressing podocyte-specific human CD25 become proteinuric after injection of immunotoxin (LMB2). NEP25 mice were treated with the aldehyde scavenger, PPM (1g/L) or vehicle from the onset of proteinuria until sacrifice (2 weeks). We assessed proteinuria [measured as albumin:creatinine ratio (ACR)], urinary apoAI, IsoLG, KIM-1 (marker of tubular injury), and the renal expression of lymphatic markers (LYVE-1 and podoplanin). In vitro , we assessed the effects of apoAI or modified apoAI (IsoLG-apoAI) +/- PPM on proximal tubular cells (PTC) and lymphatic endothelial cells (LEC). As expected, LMB2 significantly increased ACR and urinary KIM-1. Proteinuric injury also significantly increased urinary IsoLG (262%). Similar to our previous data, proteinuric kidneys increased expression of apoAI, VEGF-C, podoplanin and LYVE-1 compared to wild type mice. In vitro , PTC uptake of IsoLG-apoAI was significantly increased (39%), an effect that was abrogated by exposure to PPM (22%). In LECs, compared to apoAI, IsoLG-apoAI significantly increased viability (27%) and migration (13%), effects that were abrogated by exposure to PPM. In vivo , proteinuric NEP25 mice treated with PPM showed significantly reduced ACR (22%), urinary KIM-1 (30%) and IsoLG excretion (64%). We conclude that aldehyde scavenger lessens proteinuric renal damage through mechanisms that include reducing proximal tubule uptake of IsoLG-modified apoAI and preserving lymphatic endothelial cell functionality.

Abstract P339: Genetically Induced Renal Lymphangiogenesis Prevents the Development of L-NAME Hypertension in Mice

In humans and experimental animals, persistent immune system activation, accumulation of immune cells in the kidney, and subsequent inflammation plays an essential role in the development of hypertension (HTN). To reduce inflammation, lymphatic vessels drain extracellular fluid from the interstitium and traffic immune cells to draining lymph nodes. However, little is known about the connection between hypertension and renal lymphatic vessels. We hypothesized that renal lymphatic vessel density would increase in mice with L-NAME HTN and that genetically induced renal lymphangiogenesis would prevent this increase in blood pressure. L-NAME (0.5 mg/mL) was administered in the drinking water for two weeks and caused HTN (SBP: 153±3 vs. 103±3 mmHg; p<0.05) and renal lymphatic vessel dilation compared to control mice. Kidneys from mice with L-NAME HTN had significantly increased gene expression of the lymphangiogenic marker Vegfc , macrophage marker Adgre1 (F4/80), dendritic cell marker Cd11c , Th1 cell marker Tbx21 , and the pro-inflammatory cytokine Il6 . Blood pressure decreased after a two-week washout period following L-NAME (SBP: 113±2 mmHg) which was associated with a decrease in renal gene expression of Adgre1 (F4/80) and Cd11c , however renal lymphatic vessels remained dilated. To determine if augmenting renal lymphatic vessel density prior to L-NAME treatment would prevent HTN, we used transgenic mice that in response to doxycycline undergo kidney-specific VEGF-D overexpression (KidVD+ mice) and renal lymphangiogenesis. Doxycycline (200 mg/L) was administered in the drinking water of KidVD+ and KidVD- mice for four weeks with L-NAME being added during the final three weeks. Starting doxycycline one week prior to L-NAME prevented HTN in KidVD+ mice while slightly decreasing SBP in KidVD- mice (SBP: 112±4 vs. 134±2 mmHg; p<0.05). Renal gene expression of the Th17 cell marker Rorc was decreased and the lymphatic chemokine markers Ccl21 and Ccl19 were increased significantly in KidVD+ mice. These data together demonstrate that L-NAME HTN can alter the size of renal lymphatic vessels and genetically augmenting renal lymphatic vessel density prior to L-NAME can prevent the development of HTN.

Abstract 098: Augmenting Renal Lymphatic Vessel Density Prevents Salt-sensitive Hypertension in Mice

Salt-sensitive hypertension (SSHTN) is associated with renal immune cell infiltration and interstitial inflammation. Lymphatic vessels drain the interstitial compartment and traffic immune cells to draining lymph nodes; however little is known about the role of lymphatics and immune cell trafficking in the kidney during SSHTN. Our hypotheses were that renal lymphatic vessel density is increased in mice with SSHTN and that further augmenting renal lymphatic vessels will prevent SSHTN. SSHTN mice were made by administering L-NAME for two weeks, followed by a two week washout, and then were fed a 4% high salt diet for three weeks. Compared to control mice, mice with SSHTN (SBP: 103±3 vs. 136±2 mmHg; p<0.05) had markedly increased renal lymphatic vessel density. Kidneys of SSHTN mice had significantly increased gene expression of the lymphatic vessel marker Lyve1 , the macrophage marker Adgre1 (F4/80), the Th1 cell marker Tbx21 , and the pro-inflammatory cytokine Il6 while expression of the immune cell-lymphatic chemokine receptor Ccr7 was decreased significantly. Mice solely fed a 4% salt diet for three weeks did not exhibit hypertension or increased renal lymphatic vessel density. To determine whether augmenting renal lymphatic vessels prior to the high salt diet could prevent SSHTN, we used transgenic mice that overexpress the lymphangiogenic signal VEGF-D only in the kidney under the control of doxycycline (KidVD+ mice) and thus exhibit renal lymphangiogenesis. Doxycycline initiated one week prior to the high salt diet prevented SSHTN in KidVD+ mice while having no effect on blood pressure in KidVD- mice (SBP: 117±4 vs. 139±5 mmHg; p<0.05). Renal gene expression of Tbx21 was decreased in KidVD+ mice while Ccr7 gene expression was increased significantly. These data demonstrate that renal lymphatic vessel density is increased in SSHTN and that augmenting renal lymphatic vessel density prior to a high salt diet can prevent SSHTN by improving renal immune cell exfiltration.