Single-Cell RNA Sequencing Identifies Response of Renal Lymphatic Endothelial Cells to Acute Kidney Injury.

SIGNIFICANCE STATEMENT: The renal lymphatic vasculature and the lymphatic endothelial cells that make up this network play important immunomodulatory roles during inflammation. How lymphatics respond to AKI may affect AKI outcomes. The authors used single-cell RNA sequencing to characterize mouse renal lymphatic endothelial cells in quiescent and cisplatin-injured kidneys. lymphatic endothelial cell gene expression changes were confirmed in ischemia-reperfusion injury and in cultured lymphatic endothelial cells, validating renal lymphatic endothelial cells single-cell RNA sequencing data. This study is the first to describe renal lymphatic endothelial cell heterogeneity and uncovers molecular pathways demonstrating lymphatic endothelial cells regulate the local immune response to AKI. These findings provide insights into previously unidentified molecular pathways for lymphatic endothelial cells and roles that may serve as potential therapeutic targets in limiting the progression of AKI. BACKGROUND: The inflammatory response to AKI likely dictates future kidney health. Lymphatic vessels are responsible for maintaining tissue homeostasis through transport and immunomodulatory roles. Owing to the relative sparsity of lymphatic endothelial cells in the kidney, past sequencing efforts have not characterized these cells and their response to AKI. METHODS: Here, we characterized murine renal lymphatic endothelial cell subpopulations by single-cell RNA sequencing and investigated their changes in cisplatin AKI 72 hours postinjury. Data were processed using the Seurat package. We validated our findings by quantitative PCR in lymphatic endothelial cells isolated from both cisplatin-injured and ischemia-reperfusion injury, by immunofluorescence, and confirmation in in vitro human lymphatic endothelial cells. RESULTS: We have identified renal lymphatic endothelial cells and their lymphatic vascular roles that have yet to be characterized in previous studies. We report unique gene changes mapped across control and cisplatin-injured conditions. After AKI, renal lymphatic endothelial cells alter genes involved in endothelial cell apoptosis and vasculogenic processes as well as immunoregulatory signaling and metabolism. Differences between injury models were also identified with renal lymphatic endothelial cells further demonstrating changed gene expression between cisplatin and ischemia-reperfusion injury models, indicating the renal lymphatic endothelial cell response is both specific to where they lie in the lymphatic vasculature and the kidney injury type. CONCLUSIONS: In this study, we uncover lymphatic vessel structural features of captured populations and injury-induced genetic changes. We further determine that lymphatic endothelial cell gene expression is altered between injury models. How lymphatic endothelial cells respond to AKI may therefore be key in regulating future kidney disease progression.

VEGFR-3 signaling in macrophages: friend or foe in disease?

Lymphatic vessels have been increasingly appreciated in the context of immunology not only as passive conduits for immune and cancer cell transport but also as key in local tissue immunomodulation. Targeting lymphatic vessel growth and potential immune regulation often takes advantage of vascular endothelial growth factor receptor-3 (VEGFR-3) signaling to manipulate lymphatic biology. A receptor tyrosine kinase, VEGFR-3, is highly expressed on lymphatic endothelial cells, and its signaling is key in lymphatic growth, development, and survival and, as a result, often considered to be "lymphatic-specific" in adults. A subset of immune cells, notably of the monocyte-derived lineage, have been identified to express VEGFR-3 in tissues from the lung to the gut and in conditions as varied as cancer and chronic kidney disease. These VEGFR-3+ macrophages are highly chemotactic toward the VEGFR-3 ligands VEGF-C and VEGF-D. VEGFR-3 signaling has also been implicated in dictating the plasticity of these cells from pro-inflammatory to anti-inflammatory phenotypes. Conversely, expression may potentially be transient during monocyte differentiation with unknown effects. Macrophages play critically important and varied roles in the onset and resolution of inflammation, tissue remodeling, and vasculogenesis: targeting lymphatic vessel growth and immunomodulation by manipulating VEGFR-3 signaling may thus impact macrophage biology and their impact on disease pathogenesis. This mini review highlights the studies and pathologies in which VEGFR-3+ macrophages have been specifically identified, as well as the activity and polarization changes that macrophage VEGFR-3 signaling may elicit, and affords some conclusions as to the importance of macrophage VEGFR-3 signaling in disease.

Synthesis and Characterization of Epigallocatechin Gallate-mediated Hydroxyapatite.

INTRODUCTION: Hydroxyapatite is a significant material that finds its application in the field of dental and bone tissue engineering. METHOD: The formulation of nanohydroxyapatite with the aid of bioactive compounds has gained importance in recent years due to the beneficial activity contributed by them. The present work focuses on the formulation of nanohydroxyapatite synthesis using epigallocatechin gallate, an active biochemical component of green tea. RESULT: The prepared epigallocatechin gallate-mediated nanohydroxyapatite (epi-HAp) was nanoglobular in shape and composed of calcium, phosphorous, carbon and oxygen, which was confirmed by Scanning electron microscope- energy dispersive X-ray analysis (SEM-EDX). The Attenuated Total Reflection-Infra red spectroscopy (ATR-IR) and X-ray photoelectron spectroscopy (XPS) assured that the reduction and stabilisation of nanohydroxyapatite were mediated by epigallocatechin gallate. CONCLUSION: The epi-HAp exhibited anti-inflammatory behaviour along with nil effect on cytotoxicity. To be precise, the epi-HAp can be an effective biomaterial in bone and dental applications.

Single-cell RNA sequencing identifies response of renal lymphatic endothelial cells to acute kidney injury.

The inflammatory response to acute kidney injury (AKI) likely dictates future renal health. Lymphatic vessels are responsible for maintaining tissue homeostasis through transport and immunomodulatory roles. Due to the relative sparsity of lymphatic endothelial cells (LECs) in the kidney, past sequencing efforts have not characterized these cells and their response to AKI. Here we characterized murine renal LEC subpopulations by single-cell RNA sequencing and investigated their changes in cisplatin AKI. We validated our findings by qPCR in LECs isolated from both cisplatin-injured and ischemia reperfusion injury, by immunofluorescence, and confirmation in in vitro human LECs. We have identified renal LECs and their lymphatic vascular roles that have yet to be characterized in previous studies. We report unique gene changes mapped across control and cisplatin injured conditions. Following AKI, renal LECs alter genes involved endothelial cell apoptosis and vasculogenic processes as well as immunoregulatory signaling and metabolism. Differences between injury models are also identified with renal LECs further demonstrating changed gene expression between cisplatin and ischemia reperfusion injury models, indicating the renal LEC response is both specific to where they lie in the lymphatic vasculature and the renal injury type. How LECs respond to AKI may therefore be key in regulating future kidney disease progression.

Anticancer Activity of Samarium-Coated Magnesium Implants for Immunocompromised Patients.

The removal of tumors in the osseous tissue leads to functional disorders. To overcome this issue, biodegradable implants are used to replace the damaged part of the system. In the study, samarium oxide was coated on the anodic layer of AZ31 magnesium alloy. The potentiodynamic polarization, electrochemical impedance, and localized electrochemical impedance spectroscopy studies were carried out for the samarium-coated magnesium alloy. The corrosion resistance of the coating improved several folds than the bare alloy. The apatite formed on the 3rd day of immersion in the simulated body fluid showed cuboid and triangular structures, whereas on the 7th day, it exhibited a sea sponge-like appearance. The coating exhibited inherent anticancer and antibacterial properties. Our work suggests that the samarium coating is expected to be a promising orthopedic implant for preventing tumor relapse and metastasis.

Sustained obesity reduces litter size by decreasing proteins regulating folliculogenesis and ovulation in rats - A cafeteria diet model.

Global rise in obesity at early age due to overconsumption of energy-dense food is the major health problem which increases the exposure to obesity over longer duration. Recently we reported that the severity of ovarian dysfunction depends on the duration of obesity. In the present study, we examined the consequences of sustained obesity on reproductive outcome and the underlying mechanism. Sprague Dawley female rats (21 days old) were fed ad libitum with a standard diet (control group) and a cafeteria diet (Obese group) for 32 weeks. We observed hypoprolactinemia, sub-fecundity, sub-fertility, delayed conception and macrosomic pups of reduced litter size in sustained obese rats. The observed decrease in the number of ovarian follicles (primordial, primary, secondary and antral follicles) and corpus luteum indicates impairment in folliculogenesis and ovulation. This impairment might be due to decreased level of ovarian proteins (PRLR, AR, GDF-9, OCT-4, COX-2, PPARγ, ER and PR subtypes) in obese rats. We conclude that sustained obesity impaired folliculogenesis and ovulation thereby increased the severity of reproductive deficits.

Association between duration of obesity and severity of ovarian dysfunction in rat-cafeteria diet approach.

Consumption of unhealthy, energy-dense palatable food during early age leads to obesity in children and the onset of obesity during childhood has a profound effect on the reproductive health of women. In this study, the mechanism underlying diet-induced obesity on ovarian dysfunction was studied by exposing rats to cafeteria diet (CAFD) for two different durations. For that purpose, 21-day-old female Sprague Dawley rats were fed ad libitum with a standard diet (control group) and a cafeteria diet (CAFD group) for a period of 20 weeks (20 W) and 32 weeks (32 W). We observed obesity, hyperglycemia, hyperlipidemia, hyperleptinemia and hypoadiponectinemia in CAFD fed groups. Hyperinsulinemia, hypergonadotrophism, hypertestosteronemia and hyperprogesteronemia were observed in the 20 W-CAFD group. Conversely, in the 32 W-CAFD group hypersecretion declined to hyposecretion. The levels of estradiol remained low during both time periods. The duration of estrous cycle was extended in the CAFD fed rats. The ovary weight was higher in the 20 W-CAFD fed rats but it was drastically reduced over a longer duration cafeteria diet feeding. In the 20 W-CAFD fed rats, the protein levels of LHR, StAR, CYP11A1, 3ß-HSD and 17ß-HSD were increased but FSHR and CYP19A1 levels were decreased in the ovary. On the other hand, gonadotropin receptor and the protein levels of steroidogenic enzymes were decreased in the ovary of 32 W-CAFD fed rats. We conclude that the duration of energy-dense diet consumption has differential regulatory mechanism in altering the ovarian steroid production. In 20 W-CAFD fed rats, hypergonadotropic condition was observed whereas, 32 W-CAFD consumption induced hypogonadotropic hypogonadism.