Direct Tumor Killing and Immunotherapy through Anti-SerpinB9 Therapy.

Cancer therapies kill tumors either directly or indirectly by evoking immune responses and have been combined with varying levels of success. Here, we describe a paradigm to control cancer growth that is based on both direct tumor killing and the triggering of protective immunity. Genetic ablation of serine protease inhibitor SerpinB9 (Sb9) results in the death of tumor cells in a granzyme B (GrB)-dependent manner. Sb9-deficient mice exhibited protective T cell-based host immunity to tumors in association with a decline in GrB-expressing immunosuppressive cells within the tumor microenvironment (TME). Maximal protection against tumor development was observed when the tumor and host were deficient in Sb9. The therapeutic utility of Sb9 inhibition was demonstrated by the control of tumor growth, resulting in increased survival times in mice. Our studies describe a molecular target that permits a combination of tumor ablation, interference within the TME, and immunotherapy in one potential modality.

Orphan nuclear receptor COUP-TFII enhances myofibroblast glycolysis leading to kidney fibrosis.

Recent studies demonstrate that metabolic disturbance, such as augmented glycolysis, contributes to fibrosis. The molecular regulation of this metabolic perturbation in fibrosis, however, has been elusive. COUP-TFII (also known as NR2F2) is an important regulator of glucose and lipid metabolism. Its contribution to organ fibrosis is undefined. Here, we found increased COUP-TFII expression in myofibroblasts in human fibrotic kidneys, lungs, kidney organoids, and mouse kidneys after injury. Genetic ablation of COUP-TFII in mice resulted in attenuation of injury-induced kidney fibrosis. A non-biased proteomic study revealed the suppression of fatty acid oxidation and the enhancement of glycolysis pathways in COUP-TFII overexpressing fibroblasts. Overexpression of COUP-TFII in fibroblasts also induced production of alpha-smooth muscle actin (αSMA) and collagen 1. Knockout of COUP-TFII decreased glycolysis and collagen 1 levels in fibroblasts. Chip-qPCR revealed the binding of COUP-TFII on the promoter of PGC1α. Overexpression of COUP-TFII reduced the cellular level of PGC1α. Targeting COUP-TFII serves as a novel treatment approach for mitigating fibrosis in chronic kidney disease and potentially fibrosis in other organs.

Association of Coding Variants in Hydroxysteroid 17-beta Dehydrogenase 14 (HSD17B14) with Reduced Progression to End Stage Kidney Disease in Type 1 Diabetes.

BACKGROUND: Rare variants in gene coding regions likely have a greater impact on disease-related phenotypes than common variants through disruption of their encoded protein. We searched for rare variants associated with onset of ESKD in individuals with type 1 diabetes at advanced kidney disease stage. METHODS: Gene-based exome array analyses of 15,449 genes in five large incidence cohorts of individuals with type 1 diabetes and proteinuria were analyzed for survival time to ESKD, testing the top gene in a sixth cohort (n=2372/1115 events all cohorts) and replicating in two retrospective case-control studies (n=1072 cases, 752 controls). Deep resequencing of the top associated gene in five cohorts confirmed the findings. We performed immunohistochemistry and gene expression experiments in human control and diseased cells, and in mouse ischemia reperfusion and aristolochic acid nephropathy models. RESULTS: Protein coding variants in the hydroxysteroid 17-ß dehydrogenase 14 gene (HSD17B14), predicted to affect protein structure, had a net protective effect against development of ESKD at exome-wide significance (n=4196; P value=3.3 × 10-7). The HSD17B14 gene and encoded enzyme were robustly expressed in healthy human kidney, maximally in proximal tubular cells. Paradoxically, gene and protein expression were attenuated in human diabetic proximal tubules and in mouse kidney injury models. Expressed HSD17B14 gene and protein levels remained low without recovery after 21 days in a murine ischemic reperfusion injury model. Decreased gene expression was found in other CKD-associated renal pathologies. CONCLUSIONS: HSD17B14 gene is mechanistically involved in diabetic kidney disease. The encoded sex steroid enzyme is a druggable target, potentially opening a new avenue for therapeutic development.

Nephrotoxicity Assessment with Human Kidney Tubuloids using Spherical Nucleic Acid-Based mRNA Nanoflares.

Drug-induced nephrotoxicity represents an important cause of acute kidney injury with associated patient morbidity and mortality and is often responsible for termination of drug development, after extensive resource allocation. We have developed a human kidney tubuloid system that phenocopies, in 3D culture, kidney proximal tubules, a primary injury site of most nephrotoxicants. Traditional end point assays are often performed on 2D cultures of cells that have lost their differentiated phenotype. Herein, we pair a tubuloid system with Nanoflare (NF) mRNA nanosensors to achieve a facile, real-time assessment of drug nephrotoxicity. Using kidney injury molecule-1 (KIM-1) mRNA as a model injury biomarker, we verify NF specificity in engineered and adenovirus-transfected cells and confirm their efficacy to report tubular cell injury by aristolochic acid and cisplatin. The system also facilitates nephrotoxicity screening as demonstrated with 10 representative anticancer moieties. 5-Fluorouracil and paclitaxel induce acute tubular injury, as reflected by an NF signal increase.

Urine podoplanin heralds the onset of ischemia-reperfusion injury of the kidney.

Ischemia-reperfusion injury represents one of the most common causes of acute kidney injury, a serious and often deadly condition that affects up to 20% of all hospitalized patients in the United States. However, the current standard assay used universally for the diagnosis of acute kidney injury, serum creatinine, does not detect renal damage early in its course. Serendipitously, we found that the immunofluorescent signal of the constitutive podocyte marker podoplanin fades in the glomerulus and intensifies in the tubulointerstitial compartment of the kidney shortly after ischemia-reperfusion injury in 8- to 10-wk-old male C57Bl/6j mice. Therefore, we sought to define the appearance and course of the podoplanin-positive signal in the kidney after ischemia-reperfusion injury. The tubulointerstitial podoplanin-positive signal increased as early as 2 h but persisted for 7 days after ischemia-reperfusion injury. In addition, the strength of this tubulointerstitial signal was directly proportional to the severity of ischemia, and its location shifted from the tubules to interstitial cells over time. Finally, we detected podoplanin in the urine of mice after ischemia, and we observed that an increase in the urine podoplanin-to-creatinine ratio correlated strongly with the onset of renal ischemia-reperfusion injury. Our findings indicate that the measurement of urine podoplanin harbors promising potential for use as a novel biomarker for the early detection of ischemia-reperfusion injury of the kidney.

KIM-1-/TIM-1-mediated phagocytosis links ATG5-/ULK1-dependent clearance of apoptotic cells to antigen presentation.

Phagocytosis of apoptotic cells by both professional and semi-professional phagocytes is required for resolution of organ damage and maintenance of immune tolerance. KIM-1/TIM-1 is a phosphatidylserine receptor that is expressed on epithelial cells and can transform the cells into phagocytes. Here, we demonstrate that KIM-1 phosphorylation and association with p85 results in encapsulation of phagosomes by lipidated LC3 in multi-membrane organelles. KIM-1-mediated phagocytosis is not associated with increased ROS production, and NOX inhibition does not block LC3 lipidation. Autophagy gene expression is required for efficient clearance of apoptotic cells and phagosome maturation. KIM-1-mediated phagocytosis leads to pro-tolerogenic antigen presentation, which suppresses CD4 T-cell proliferation and increases the percentage of regulatory T cells in an autophagy gene-dependent manner. Taken together, these data reveal a novel mechanism of epithelial biology linking phagocytosis, autophagy and antigen presentation to regulation of the inflammatory response.

CD74 Deficiency Mitigates Systemic Lupus Erythematosus-like Autoimmunity and Pathological Findings in Mice.

CD74 mediates MHC class-II antigenic peptide loading and presentation and plays an important role in the pathogenesis of autoimmune diseases, including systemic lupus erythematosus. C57BL/6 Faslpr mice that develop spontaneous lupus-like autoimmunity and pathology showed elevated CD74 expression in the inflammatory cell infiltrates and the adjacent tubular epithelial cells (TECs) in kidneys affected by lupus nephritis but negligible levels in kidneys from age-matched wild-type mice. The inflammatory cytokine IFN-γ or IL-6 induced CD74 expression in kidney TECs in vitro. The presence of kidney TECs from Faslpr mice, rather than from wild-type mice, produced significantly stronger histones, dsDNA, and ribonucleoprotein-Smith Ag complex-induced CD4+ T cell activation. Splenocytes from CD74-deficient FaslprCd74-/- mice had muted responses in a MLR and to the autoantigen histones. Compared with FaslprCd74+/+ mice, FaslprCd74-/- mice had reduced kidney and spleen sizes, splenic activated T cells and B cells, serum IgG and autoantibodies, urine albumin/creatinine ratio, kidney Periodic acid-Schiff score, IgG and C3 deposition, and serum IL-6 and IL-17A levels, but serum IL-2 and TGF-ß levels were increased. Study of chronic graft-versus-host C57BL/6 mice that received donor splenocytes from B6.C-H2bm12 /KhEg mice and those that received syngeneic donor splenocytes yielded similar observations. CD74 deficiency reduced lupus-like autoimmunity and kidney pathology in chronic graft-versus-host mice. This investigation establishes the direct participation of CD74 in autoimmunity and highlights a potential role for CD74 in kidney TECs, together with professional APCs in systemic lupus erythematosus.

Mitochonic Acid 5 Binds Mitochondria and Ameliorates Renal Tubular and Cardiac Myocyte Damage.

Mitochondrial dysfunction causes increased oxidative stress and depletion of ATP, which are involved in the etiology of a variety of renal diseases, such as CKD, AKI, and steroid-resistant nephrotic syndrome. Antioxidant therapies are being investigated, but clinical outcomes have yet to be determined. Recently, we reported that a newly synthesized indole derivative, mitochonic acid 5 (MA-5), increases cellular ATP level and survival of fibroblasts from patients with mitochondrial disease. MA-5 modulates mitochondrial ATP synthesis independently of oxidative phosphorylation and the electron transport chain. Here, we further investigated the mechanism of action for MA-5. Administration of MA-5 to an ischemia-reperfusion injury model and a cisplatin-induced nephropathy model improved renal function. In in vitro bioenergetic studies, MA-5 facilitated ATP production and reduced the level of mitochondrial reactive oxygen species (ROS) without affecting activity of mitochondrial complexes I-IV. Additional assays revealed that MA-5 targets the mitochondrial protein mitofilin at the crista junction of the inner membrane. In Hep3B cells, overexpression of mitofilin increased the basal ATP level, and treatment with MA-5 amplified this effect. In a unique mitochondrial disease model (Mitomice with mitochondrial DNA deletion that mimics typical human mitochondrial disease phenotype), MA-5 improved the reduced cardiac and renal mitochondrial respiration and seemed to prolong survival, although statistical analysis of survival times could not be conducted. These results suggest that MA-5 functions in a manner differing from that of antioxidant therapy and could be a novel therapeutic drug for the treatment of cardiac and renal diseases associated with mitochondrial dysfunction.

High-resolution renal perfusion mapping using contrast-enhanced ultrasonography in ischemia-reperfusion injury monitors changes in renal microperfusion.

Alterations in renal microperfusion play an important role in the development of acute kidney injury with long-term consequences. Here we used contrast-enhanced ultrasonography as a novel method for depicting intrarenal distribution of blood flow. After infusion of microbubble contrast agent, bubbles were collapsed in the kidney and postbubble destruction refilling was measured in various regions of the kidney. Local perfusion was monitored in vivo at 15, 30, 45, 60 minutes and 24 hours after 28 minutes of bilateral ischemia in 12 mice. High-resolution, pixel-by-pixel analysis was performed on each imaging clip using customized software, yielding parametric perfusion maps of the kidney, representing relative blood volume in each pixel. These perfusion maps revealed that outer medullary perfusion decreased disproportionately to the reduction in the cortical and inner medullary perfusion after ischemia. Outer medullary perfusion was significantly decreased by 69% at 60 minutes postischemia and remained significantly less (40%) than preischemic levels at 24 hours postischemia. Thus, contrast-enhanced ultrasonography with high-resolution parametric perfusion maps can monitor changes in renal microvascular perfusion in space and time in mice. This novel technique can be translated to clinical use in man.

Defect in regulatory B-cell function and development of systemic autoimmunity in T-cell Ig mucin 1 (Tim-1) mucin domain-mutant mice.

Tim-1, a type I transmembrane glycoprotein, consists of an IgV domain and a mucin domain. The IgV domain is essential for binding Tim-1 to its ligands, but little is known about the role of the mucin domain, even though genetic association of TIM-1 with atopy/asthma has been linked to the length of mucin domain. We generated a Tim-1-mutant mouse (Tim-1(Δmucin)) in which the mucin domain was deleted genetically. The mutant mice showed a profound defect in IL-10 production from regulatory B cells (Bregs). Associated with the loss of IL-10 production in B cells, older Tim-1(Δmucin) mice developed spontaneous autoimmunity associated with hyperactive T cells, with increased production of IFN-γ and elevated serum levels of Ig and autoantibodies. However, Tim-1(Δmucin) mice did not develop frank systemic autoimmune disease unless they were crossed onto the Fas-mutant lpr mice on a C57BL/6 background. Tim-1(Δmucin)lpr mice developed accelerated and fulminant systemic autoimmunity with accumulation of abnormal double-negative T cells and autoantibodies to a number of lupus-associated autoantigens. Thus, Tim-1 plays a critical role in maintaining suppressive Breg function, and our data also demonstrate an unexpected role of the Tim-1 mucin domain in regulating Breg function and maintaining self-tolerance.

Conformational change in transfer RNA is an early indicator of acute cellular damage.

Tissue damage by oxidative stress is a key pathogenic mechanism in various diseases, including AKI and CKD. Thus, early detection of oxidative tissue damage is important. Using a tRNA-specific modified nucleoside 1-methyladenosine (m1A) antibody, we show that oxidative stress induces a direct conformational change in tRNA structure that promotes subsequent tRNA fragmentation and occurs much earlier than DNA damage. In various models of tissue damage (ischemic reperfusion, toxic injury, and irradiation), the levels of circulating tRNA derivatives increased rapidly. In humans, the levels of circulating tRNA derivatives also increased under conditions of acute renal ischemia, even before levels of other known tissue damage markers increased. Notably, the level of circulating free m1A correlated with mortality in the general population (n=1033) over a mean follow-up of 6.7 years. Compared with healthy controls, patients with CKD had higher levels of circulating free m1A, which were reduced by treatment with pitavastatin (2 mg/d; n=29). Therefore, tRNA damage reflects early oxidative stress damage, and detection of tRNA damage may be a useful tool for identifying organ damage and forming a clinical prognosis.

Nephroprotective effects of TVP1022, a non-MAO inhibitor S-isomer of rasagiline, in an experimental model of diabetic renal ischemic injury.

Ischemic acute kidney injury (iAKI) in diabetes mellitus is associated with a rapid deterioration of kidney function, more than in nondiabetic subjects. TVP1022, a non-MAO inhibitor S-isomer of rasagiline, possesses antioxidative and antiapoptotic activities. The current study examines the effects of TVP1022 and tempol on iAKI in diabetic rats. Diabetes was induced by streptozotocin. iAKI was induced by clamping the left renal artery for 30 min in both diabetic and nondiabetic rats. The right intact kidney served as a control. Forty-eight hours following ischemia, urinary flow (V), sodium excretion (UNaV), and glomerular filtration rate (GFR) in both ischemic and nonischemic kidneys were determined. The nephroprotective effects of tempol and TVP1022 were examined in these rats. Hematoxylin and eosin staining, 4-hydroxynonenal (4-HNE) immunofluorescence, and nitrotyrosine immunohistochemistry were performed on renal tissues of the various experimental groups. Compared with normoglycemic rats, iAKI in diabetic animals caused more profound reductions in V, UNaV, and GFR. Tempol and TVP1022 treatment increased GFR two- and four-fold in diabetic ischemic kidney, respectively. Besides hemodynamic perturbations, iAKI markedly increased renal immunoreactive 4-HNE and nitrotyrosine staining in both diabetic and nondiabetic rats. Moreover, iAKI increased medullary necrosis, congestion, and casts. Noteworthy, these increases were to a larger extent in ischemic diabetic kidneys. TVP1022, and to a lesser extent tempol, decreased nitrotyrosine and 4-HNE immunoreactivities and necrosis and cast formation in the renal medulla. TVP1022 treatment improves renal dysfunction and histological changes in an iAKI diabetic model and suggests a role for TVP1022 therapy in kidney injury.

Histone acetyl transferase (HAT) HBO1 and JADE1 in epithelial cell regeneration.

HBO1 acetylates lysine residues of histones and is involved in DNA replication and gene transcription. Two isoforms of JADE1, JADE1S and JADE1L, bind HBO1 and promote acetylation of histones in chromatin context. We characterized the role of JADE1-HBO1 complexes in vitro and in vivo during epithelial cell replication. Down-regulation of JADE1 by siRNA diminished the rate of DNA synthesis in cultured cells, decreased endogenous HBO1 protein expression, and prevented chromatin recruitment of replication factor Mcm7, demonstrating that JADE1 is required for cell proliferation. We used a murine model of acute kidney injury to examine expression of HBO1-JADE1S/L in injured and regenerating epithelial tissue. In control kidneys, JADE1S, JADE1L, and HBO1 were expressed in nuclei of proximal and distal tubular epithelial cells. Ischemia and reperfusion injury resulted in an initial decrease in JADE1S, JADE1L, and HBO1 protein levels, which returned to baseline during renal recovery. HBO1 and JADE1S recovered as cell proliferation reached its maximum, whereas JADE1L recovered after bulk proliferation had ceased. The temporal expression of JADE1S correlated with the acetylation of histone H4 on lysines 5 and 12, but not with acetylation of histone H3 on lysine 14, demonstrating that the JADE1S-HBO1 complex specifically marks H4 during epithelial cell proliferation. These data implicate JADE1-HBO1 complex in acute kidney injury and suggest distinct roles for JADE1 isoforms during epithelial cell recovery.

Kim-1/Tim-1 and immune cells: shifting sands.

Kim-1/Tim-1 is an apoptotic-cell phagocytosis and scavenger receptor that is most highly upregulated in proximal tubular epithelium in acute and chronic kidney injury. While Kim-1/Tim-1 has been proposed to be a costimulatory molecule for immune cells, its potential immunological role has been controversial. In the presence of very high epithelial cell expression, understanding the influence of immune cell Kim-1/Tim-1 expression in kidney injury relies on a better definition of its functional significance in immune cells and better characterization of antibodies used to probe function.

Targeted proximal tubule injury triggers interstitial fibrosis and glomerulosclerosis.

Chronic kidney disease (CKD) remains one of the leading causes of death in the developed world, and acute kidney injury (AKI) is now recognized as a major risk factor in its development. Understanding the factors leading to CKD after acute injury are limited by current animal models of AKI, which concurrently target various kidney cell types including epithelial, endothelial, and inflammatory cells. Here, we developed a mouse model of kidney injury using the Six2-Cre-LoxP technology to selectively activate expression of the simian diphtheria toxin (DT) receptor in renal epithelia derived from the metanephric mesenchyme. By adjusting the timing and dose of DT, a highly selective model of tubular injury was created to define the acute and chronic consequences of isolated epithelial injury. The DT-induced sublethal tubular epithelial injury was confined to the S1 and S2 segments of the proximal tubule rather than being widespread in the metanephric mesenchyme-derived epithelial lineage. Acute injury was promptly followed by inflammatory cell infiltration and robust tubular cell proliferation, leading to complete recovery after a single toxin insult. In striking contrast, three insults to renal epithelial cells at 1-week intervals resulted in maladaptive repair with interstitial capillary loss, fibrosis, and glomerulosclerosis, which was highly correlated with the degree of interstitial fibrosis. Thus, selective epithelial injury can drive the formation of interstitial fibrosis, capillary rarefaction, and potentially glomerulosclerosis, substantiating a direct role for damaged tubule epithelium in the pathogenesis of CKD.

Focal adhesion kinase signaling mediates acute renal injury induced by ischemia/reperfusion.

Renal ischemia/reperfusion (I/R) injury is associated with cell matrix and focal adhesion remodeling. Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase that localizes at focal adhesions and regulates their turnover. Here, we investigated the role of FAK in renal I/R injury, using a novel conditional proximal tubule-specific fak-deletion mouse model. Tamoxifen treatment of FAK(loxP/loxP)//γGT-Cre-ER(T2) mice caused renal-specific fak recombination (FAK(ΔloxP/ΔloxP)) and reduction of FAK expression in proximal tubules. In FAK(ΔloxP/ΔloxP) mice compared with FAK(loxP/loxP) controls, unilateral renal ischemia followed by reperfusion resulted in less tubular damage with reduced tubular cell proliferation and lower expression of kidney injury molecule-1, which was independent from the postischemic inflammatory response. Oxidative stress is involved in the pathophysiology of I/R injury. Primary cultured mouse renal cells were used to study the role of FAK deficiency for oxidative stress in vitro. The conditional fak deletion did not affect cell survival after hydrogen peroxide-induced cellular stress, whereas it impaired the recovery of focal adhesions that were disrupted by hydrogen peroxide. This was associated with reduced c-Jun N-terminal kinase-dependent phosphorylation of paxillin at serine 178 in FAK-deficient cells, which is required for focal adhesion turnover. Our findings support a role for FAK as a novel factor in the initiation of c-Jun N-terminal kinase-mediated cellular stress response during renal I/R injury and suggest FAK as a target in renal injury protection.

Ischemic injury enhances dendritic cell immunogenicity via TLR4 and NF-kappa B activation.

Ischemic (isc) injury during the course of transplantation enhances the immunogenicity of allografts and thus results in poorer graft outcome. Given the central role of dendritic cells (DCs) in mounting alloimmune responses, activation of donor DCs by ischemia may have a primary function in the increased immunogenicity of isc allografts. In this study, we sought to investigate the effect of ischemia on DC activity in vitro. Following induction of ischemia, bone marrow-derived DCs were shown to augment allogeneic T cell proliferation as well as the IFN-gamma response. Isc DCs produced greater levels of IL-6, and isc insult was concurrent with NF-kappaB activation. TLR4 ligation was also shown to occur in isc DCs, most likely in response to the endogenous ligand heat shock protein 70, which was found to be elevated in DCs following isc injury, and lack of TLR4 abrogated the observed effects of isc DCs. As compared with control DCs, isc DCs injected into the footpads of mice demonstrated enhanced migration, which was concomitant with increased recipient T cell activity. Moreover, isc DCs underwent a greater degree of apoptosis in the lymph nodes of injected mice, which may further demonstrate enhanced immunogenicity of isc DCs. We thus show that isc injury of DCs enhances DC function, augments the allogeneic T cell response, and occurs via ligation of TLR4, followed by activation of NF-kappaB. These data may serve to identify novel therapeutic targets to attenuate graft immunogenicity following ischemia.

KIM-1/TIM-1 is a Receptor for SARS-CoV-2 in Lung and Kidney.

SARS-CoV-2 precipitates respiratory distress by infection of airway epithelial cells and is often accompanied by acute kidney injury. We report that Kidney Injury Molecule-1/T cell immunoglobulin mucin domain 1 (KIM-1/TIM-1) is expressed in lung and kidney epithelial cells in COVID-19 patients and is a receptor for SARS-CoV-2. Human and mouse lung and kidney epithelial cells express KIM-1 and endocytose nanoparticles displaying the SARS-CoV-2 spike protein (virosomes). Uptake was inhibited by anti-KIM-1 antibodies and TW-37, a newly discovered inhibitor of KIM-1-mediated endocytosis. Enhanced KIM-1 expression by human kidney tubuloids increased uptake of virosomes. KIM-1 binds to the SARS-CoV-2 Spike protein in vitro . KIM-1 expressing cells, not expressing angiotensin-converting enzyme 2 (ACE2), are permissive to SARS-CoV-2 infection. Thus, KIM-1 is an alternative receptor to ACE2 for SARS-CoV-2. KIM-1 targeted therapeutics may prevent and/or treat COVID-19.

Characterization of CD41+ cells in the lymph node.

Lymph nodes (LNs) are the critical sites of immunity, and the stromal cells of LNs are crucial to their function. Our understanding of the stromal compartment of the LN has deepened recently with the characterization of nontraditional stromal cells. CD41 (integrin αIIb) is known to be expressed by platelets and hematolymphoid cells. We identified two distinct populations of CD41+Lyve1+ and CD41+Lyve1- cells in the LNs. CD41+Lyve1- cells appear in the LN mostly at the later stages of the lives of mice. We identified CD41+ cells in human LNs as well. We demonstrated that murine CD41+ cells express mesodermal markers, such as Sca-1, CD105 and CD29, but lack platelet markers. We did not observe the presence of platelets around the HEVs or within proximity to fibroblastic reticular cells of the LN. Examination of thoracic duct lymph fluid showed the presence of CD41+Lyve1- cells, suggesting that these cells recirculate throughout the body. FTY720 reduced their trafficking to lymph fluid, suggesting that their egress is controlled by the S1P1 pathway. CD41+Lyve1- cells of the LNs were sensitive to radiation, suggestive of their replicative nature. Single cell RNA sequencing data showed that the CD41+ cell population in naïve mouse LNs expressed largely stromal cell markers. Further studies are required to examine more deeply the role of CD41+ cells in the function of LNs.

Kidney injury molecule-1 is a phosphatidylserine receptor that confers a phagocytic phenotype on epithelial cells.

Following injury, the clearance of apoptotic and necrotic cells is necessary for mitigation and resolution of inflammation and tissue repair. In addition to macrophages, which are traditionally assigned to this task, neighboring epithelial cells in the affected tissue are postulated to contribute to this process. Kidney injury molecule-1 (KIM-1 or TIM-1) is an immunoglobulin superfamily cell-surface protein not expressed by cells of the myeloid lineage but highly upregulated on the surface of injured kidney epithelial cells. Here we demonstrate that injured kidney epithelial cells assumed attributes of endogenous phagocytes. Confocal images confirm internalization of apoptotic bodies within KIM-1-expressing epithelial cells after injury in rat kidney tubules in vivo. KIM-1 was directly responsible for phagocytosis in cultured primary rat tubule epithelial cells and also porcine and canine epithelial cell lines. KIM-1 was able to specifically recognize apoptotic cell surface-specific epitopes phosphatidylserine, and oxidized lipoproteins, expressed by apoptotic tubular epithelial cells. Thus, KIM-1 is the first nonmyeloid phosphatidylserine receptor identified to our knowledge that transforms epithelial cells into semiprofessional phagocytes.