Double-negative T cells have a reparative role after experimental severe ischemic acute kidney injury.

T cells mediate organ injury and repair. A proportion of unconventional kidney T cells called double-negative (DN) T cells (TCR+ CD4- CD8-), with anti-inflammatory properties, were previously demonstrated to protect from early injury in moderate experimental AKI. However, their role in repair after AKI has not been studied. We hypothesized that DN T cells mediate repair after severe AKI. C57B6 mice underwent severe (40min) unilateral ischemia-reperfusion injury (IRI). Kidney DN T cells were studied by flow cytometry and compared to gold-standard anti-inflammatory CD4+ Tregs. In vitro effects of DN T cells and Tregs on renal tubular epithelial cell (RTEC) repair after injury were quantified with live-cell analysis. DN T cells, Tregs, CD4 or vehicle were adoptively transferred after severe AKI. Glomerular filtration rate (GFR) was measured using FITC-sinistrin. Fibrosis was assessed with Masson's trichrome staining. Profibrotic genes were measured with qRT-PCR. Percentages and the numbers of DN T cells substantially decreased during repair phase after severe AKI, as well as their activation and proliferation. Both DN T cells and Tregs accelerated RTEC cell repair in vitro. Post-AKI transfer of DN T cells reduced kidney fibrosis and improved GFR, as did Treg transfer. DN T cell transfer lowered TGFß1 and αSMA expression. DN T cells reduced effector-memory CD4+ T cells and IL-17 expression. DN T cells undergo quantitative and phenotypical changes after severe AKI, accelerate RTEC repair in vitro as well as improve GFR and renal fibrosis in vivo. DN T cells have potential as immunotherapy to accelerate repair after AKI.

Targeting immune cell glutamyl-prolyl-transfer RNA synthetase 1 (EPRS1) to prevent fibrosis after tubulointerstitial nephritis.

Glutamyl-prolyl-transfer RNA synthetase 1 is an enzyme that connects glutamic acid and proline to transfer RNA during protein synthesis. In this issue, a study by Kang et al. examined the role of the immune cell glutamyl-prolyl-transfer RNA synthetase 1 in toxin-induced tubulointerstitial nephritis mice. The study demonstrated that blocking glutamyl-prolyl-transfer RNA synthetase 1 may be a therapeutic target to attenuate fibrosis after toxin-induced tubulointerstitial nephritis.

Kidney double positive T cells have distinct characteristics in normal and diseased kidneys.

Multiple types of T cells have been described and assigned pathophysiologic functions in the kidneys. However, the existence and functions of TCR+CD4+CD8+ (double positive; DP) T cells are understudied in normal and diseased murine and human kidneys. We studied kidney DPT cells in mice at baseline and after ischemia reperfusion (IR) and cisplatin injury. Additionally, effects of viral infection and gut microbiota were studied. Human kidneys from patients with renal cell carcinoma were evaluated. Our results demonstrate that DPT cells expressing CD4 and CD8 co-receptors constitute a minor T cell population in mouse kidneys. DPT cells had significant Ki67 and PD1 expression, effector/central memory phenotype, proinflammatory cytokine (IFNγ, TNFα and IL-17) and metabolic marker (GLUT1, HKII, CPT1a and pS6) expression at baseline. IR, cisplatin and viral infection elevated DPT cell proportions, and induced distinct functional and metabolic changes. scRNA-seq analysis showed increased expression of Klf2 and Ccr7 and enrichment of TNFα and oxidative phosphorylation related genes in DPT cells. DPT cells constituted a minor population in both normal and cancer portion of human kidneys. In conclusion, DPT cells constitute a small population of mouse and human kidney T cells with distinct inflammatory and metabolic profile at baseline and following kidney injury.

Kidney Immune Cell Characterization of Humanized Mouse Models.

Experimental studies often fail to translate to clinical practice. Humanized mouse models are an important tool to close this gap. We immunophenotyped the kidneys of NOG (EXL) and NSG mouse strains engrafted with human CD34 + hematopoietic stem cells or PBMCs and compared with immune cell composition of normal human kidney. Human CD34 + hematopoietic stem cell engraftment results in steady renal immune cell populations in mouse kidney with key similarities in composition compared with human kidney. Successful translation of experimental mouse data to human diseases is limited because of biological differences and imperfect disease models. Humanized mouse models are being used to bring murine models closer to humans. However, data for application in renal immune cell-mediated diseases are rare. We therefore studied immune cell composition of three different humanized mouse kidneys and compared them with human kidney. NOG and NOGEXL mice engrafted with human CD34 + hematopoietic stem cells were compared with NSG mice engrafted with human PBMCs. Engraftment was confirmed with flow cytometry, and immune cell composition in kidney, blood, spleen, and bone marrow was analyzed in different models. The results from immunophenotyping of kidneys from different humanized mouse strains were compared with normal portions of human kidneys. We found significant engraftment of human immune cells in blood and kidney of all tested models. huNSG mice showed highest frequencies of hTCR + cells compared with huNOG and huNOGEXL in blood. huNOGEXL was found to have the highest hCD4 + frequency among all tested models. Non-T cells such as hCD20 + and hCD11c + cells were decreased in huNSG mice compared with huNOG and huNOGEXL. Compared with normal human kidney, huNOG and huNOGEXL mice showed representative immune cell composition, rather than huNSG mice. In summary, humanization results in immune cell infiltration in the kidney with variable immune cell composition of tested humanized mouse models and partially reflects normal human kidneys, suggesting potential use for translational studies.

Single cell and spatial transcriptomics analysis of kidney double negative T lymphocytes in normal and ischemic mouse kidneys.

T cells are important in the pathogenesis of acute kidney injury (AKI), and TCR+CD4-CD8- (double negative-DN) are T cells that have regulatory properties. However, there is limited information on DN T cells compared to traditional CD4+ and CD8+ cells. To elucidate the molecular signature and spatial dynamics of DN T cells during AKI, we performed single-cell RNA sequencing (scRNA-seq) on sorted murine DN, CD4+, and CD8+ cells combined with spatial transcriptomic profiling of normal and post AKI mouse kidneys. scRNA-seq revealed distinct transcriptional profiles for DN, CD4+, and CD8+ T cells of mouse kidneys with enrichment of Kcnq5, Klrb1c, Fcer1g, and Klre1 expression in DN T cells compared to CD4+ and CD8+ T cells in normal kidney tissue. We validated the expression of these four genes in mouse kidney DN, CD4+ and CD8+ T cells using RT-PCR and Kcnq5, Klrb1, and Fcer1g genes with the NIH human kidney precision medicine project (KPMP). Spatial transcriptomics in normal and ischemic mouse kidney tissue showed a localized cluster of T cells in the outer medulla expressing DN T cell genes including Fcer1g. These results provide a template for future studies in DN T as well as CD4+ and CD8+ cells in normal and diseased kidneys.

Successful liver transplantation from deceased donors with active COVID-19 infections with undetectable SARS-CoV-2 in donor liver and aorta.

BACKGROUND: The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has had unprecedented effects on society and modern healthcare. In liver transplantation, uncertainty regarding the safety of performing transplants during the early stage of the pandemic resulted in increased waitlist mortality. Additionally, concerns about disease transmission led to avoidance of deceased donors with COVID-19 infections. Several successful case reports describing incidental transplant of organs from donors with COVID-19 infections or intentional transplant of such donors into recipients with current or prior COVID-19 infections prompted the transplant community to re-evaluate that position. While excellent short-term results have been published, little is known about use of donors with active infections and the extent of COVID-19 organ involvement, which may affect long term outcomes. METHODS: We report the successful transplantation of three livers from deceased donors with active COVID-19 infections. Donor liver and aortic tissues were evaluated by sensitive molecular testing for SARS-CoV-2 RNA via in situ hybridization and real-time quantitative reverse transcription PCR. RESULTS: Postoperatively, all patients had excellent allograft function, without clinical or molecular evidence of SARS-CoV-2 transmission in donor tissues. CONCLUSION: This evidence supports the use of liver donors with active COVID-19 infections.

T cell metabolic reprogramming in acute kidney injury and protection by glutamine blockade.

T cells play an important role in acute kidney injury (AKI). Metabolic programming of T cells regulates their function, is a rapidly emerging field, and is unknown in AKI. We induced ischemic AKI in C57BL/6J mice and collected kidneys and spleens at multiple time points. T cells were isolated and analyzed by an immune-metabolic assay. Unbiased machine learning analyses identified a distinct T cell subset with reduced voltage-dependent anion channel 1 and mTOR expression in post-AKI kidneys. Ischemic kidneys showed higher expression of trimethylation of histone H3 lysine 27 and glutaminase. Splenic T cells from post-AKI mice had higher expression of glucose transporter 1, hexokinase II, and carnitine palmitoyltransferase 1a. Human nonischemic and ischemic kidney tissue displayed similar findings to mouse kidneys. Given a convergent role for glutamine in T cell metabolic pathways and the availability of a relatively safe glutamine antagonist, JHU083, effects on AKI were evaluated. JHU083 attenuated renal injury and reduced T cell activation and proliferation in ischemic and nephrotoxic AKI, whereas T cell-deficient mice were not protected by glutamine blockade. In vitro hypoxia demonstrated upregulation of glycolysis-related enzymes. T cells undergo metabolic reprogramming during AKI, and reconstitution of metabolism by targeting T cell glutamine pathway could be a promising novel therapeutic approach.

Microbiome modulation after severe acute kidney injury accelerates functional recovery and decreases kidney fibrosis.

Targeting gut microbiota has shown promise to prevent experimental acute kidney injury (AKI). However, this has not been studied in relation to accelerating recovery and preventing fibrosis. Here, we found that modifying gut microbiota with an antibiotic administered after severe ischemic kidney injury in mice, particularly with amoxicillin, accelerated recovery. These indices of recovery included increased glomerular filtration rate, diminution of kidney fibrosis, and reduction of kidney profibrotic gene expression. Amoxicillin was found to increase stool Alistipes, Odoribacter and Stomatobaculum species while significantly depleting Holdemanella and Anaeroplasma. Specifically, amoxicillin treatment reduced kidney CD4+T cells, interleukin (IL)-17 +CD4+T cells, and tumor necrosis factor-α double negative T cells while it increased CD8+T cells and PD1+CD8+T cells. Amoxicillin also increased gut lamina propria CD4+T cells while decreasing CD8+T and IL-17+CD4+T cells. Amoxicillin did not accelerate repair in germ-free or CD8-deficient mice, demonstrating microbiome and CD8+T lymphocytes dependence for amoxicillin protective effects. However, amoxicillin remained effective in CD4-deficient mice. Fecal microbiota transplantation from amoxicillin-treated to germ-free mice reduced kidney fibrosis and increased Foxp3+CD8+T cells. Amoxicillin pre-treatment protected mice against kidney bilateral ischemia reperfusion injury but not cisplatin-induced AKI. Thus, modification of gut bacteria with amoxicillin after severe ischemic AKI is a promising novel therapeutic approach to accelerate recovery of kidney function and mitigate the progression of AKI to chronic kidney disease.

Acute kidney injury and distant organ dysfunction-network system analysis.

Acute kidney injury (AKI) occurs in about half of critically ill patients and is associated with high in-hospital mortality, increased long-term mortality postdischarge, and subsequent progression to chronic kidney disease. Numerous clinical studies have shown that AKI is often complicated by dysfunction of distant organs, which is a cause of the high mortality incidence associated with AKI. Experimental studies have elucidated many mechanisms of AKI-induced distant organ injury, which include inflammatory cytokines, oxidative stress, and immune responses. This review provides an update on evidence of organ crosstalk and potential therapeutics for AKI-induced organ injuries, and presents the new concept of a systemic organ network that balances homeostasis and involves multi-organ crosstalk beyond that of the kidney with a single distant organ.

Immune Checkpoint Molecule TIGIT Regulates Kidney T Cell Functions and Contributes to AKI.

SIGNIFICANCE STATEMENT: T cells mediate pathogenic and reparative processes during AKI, but the exact mechanisms regulating kidney T cell functions are unclear. This study identified upregulation of the novel immune checkpoint molecule, TIGIT, on mouse and human kidney T cells after AKI. TIGIT-expressing kidney T cells produced proinflammatory cytokines and had effector (EM) and central memory (CM) phenotypes. TIGIT-deficient mice had protection from both ischemic and nephrotoxic AKI. Single-cell RNA sequencing led to the discovery of possible downstream targets of TIGIT. TIGIT mediates AKI pathophysiology, is a promising novel target for AKI therapy, and is being increasingly studied in human cancer therapy trials. BACKGROUND: T cells play pathogenic and reparative roles during AKI. However, mechanisms regulating T cell responses are relatively unknown. We investigated the roles of the novel immune checkpoint molecule T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT) in kidney T cells and AKI outcomes. METHODS: TIGIT expression and functional effects were evaluated in mouse kidney T cells using RNA sequencing (RNA-Seq) and flow cytometry. TIGIT effect on AKI outcomes was studied with TIGIT knockout (TIGIT-KO) mice in ischemia reperfusion (IR) and cisplatin AKI models. Human kidney T cells from nephrectomy samples and single cell RNA sequencing (scRNA-Seq) data from the Kidney Precision Medicine Project were used to assess TIGIT's role in humans. RESULTS: RNA-Seq and flow cytometry analysis of mouse kidney CD4+ T cells revealed increased expression of TIGIT after IR injury. Ischemic injury also increased TIGIT expression in human kidney T cells, and TIGIT expression was restricted to T/natural killer cell subsets in patients with AKI. TIGIT-expressing kidney T cells in wild type (WT) mice had an effector/central memory phenotype and proinflammatory profile at baseline and post-IR. Kidney regulatory T cells were predominantly TIGIT+ and significantly reduced post-IR. TIGIT-KO mice had significantly reduced kidney injury after IR and nephrotoxic injury compared with WT mice. scRNA-Seq analysis showed enrichment of genes related to oxidative phosphorylation and mTORC1 signaling in Th17 cells from TIGIT-KO mice. CONCLUSIONS: TIGIT expression increases in mouse and human kidney T cells during AKI, worsens AKI outcomes, and is a novel therapeutic target for AKI.

T Cell Nrf2/Keap1 Gene Editing Using CRISPR/Cas9 and Experimental Kidney Ischemia-Reperfusion Injury.

Aims: T cells play pathophysiologic roles in kidney ischemia-reperfusion injury (IRI), and the nuclear factor erythroid 2-related factor 2/kelch-like ECH-associated protein 1 (Nrf2/Keap1) pathway regulates T cell responses. We hypothesized that clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated Keap1-knockout (KO) augments Nrf2 antioxidant potential of CD4+ T cells, and that Keap1-KO CD4+ T cell immunotherapy protects from kidney IRI. Results: CD4+ T cell Keap1-KO resulted in significant increase of Nrf2 target genes NAD(P)H quinone dehydrogenase 1, heme oxygenase 1, glutamate-cysteine ligase catalytic subunit, and glutamate-cysteine ligase modifier subunit. Keap1-KO cells displayed no signs of exhaustion, and had significantly lower levels of interleukin 2 (IL2) and IL6 in normoxic conditions, but increased interferon gamma in hypoxic conditions in vitro. In vivo, adoptive transfer of Keap1-KO CD4+ T cells before IRI improved kidney function in T cell-deficient nu/nu mice compared with mice receiving unedited control CD4+ T cells. Keap1-KO CD4+ T cells isolated from recipient kidneys 24 h post IR were less activated compared with unedited CD4+ T cells, isolated from control kidneys. Innovation: Editing Nrf2/Keap1 pathway in murine T cells using CRISPR/Cas9 is an innovative and promising immunotherapy approach for kidney IRI and possibly other solid organ IRI. Conclusion: CRISPR/Cas9-mediated Keap1-KO increased Nrf2-regulated antioxidant gene expression in murine CD4+ T cells, modified responses to in vitro hypoxia and in vivo kidney IRI. Gene editing targeting the Nrf2/Keap1 pathway in T cells is a promising approach for immune-mediated kidney diseases.

Intestinal Microbiota in Experimental Acute Kidney Injury.

Recent studies have demonstrated an important role played by gut microbiota in maintaining intestinal homeostasis and host immune system function. Gut microbiota have been studied in experimental acute kidney injury (AKI) using different mice and rat models exposed to either ischemia or cisplatin-mediated tubular injury. Differences in inflammatory markers and severity of AKI have been observed between germ-free mice, wild-type mice, and mice treated with antibiotics or specific bacteria. Interventions modifying the gut microbiota after experimental AKI have had either beneficial or harmful effects on kidney tubular injury and recovery. These findings provide strong evidence for a modulatory role of gut microbiota during AKI. Ischemic and cis-platin-induced AKI have distinct stool microbial signatures based on 16s sequencing. Future in-depth studies exploring the mechanisms of how the microbiota influence AKI and development of feasible therapeutic options have the potential to improve outcomes in clinical AKI.

TCRαß+ CD4-/CD8- "double negative" T cells in health and disease-implications for the kidney.

Double negative (DN) T cells, one of the least studied T lymphocyte subgroups, express T cell receptor αß but lack CD4 and CD8 coreceptors. DN T cells are found in multiple organs including kidney, lung, heart, gastrointestinal tract, liver, genital tract, and central nervous system. DN T cells suppress inflammatory responses in different disease models including experimental acute kidney injury, and significant evidence supports an important role in the pathogenesis of systemic lupus erythematosus. However, little is known about these cells in other kidney diseases. Therefore, it is important to better understand different functions of DN T cells and their signaling pathways as promising therapeutic targets, particularly with the increasing application of T cell-directed therapy in humans. In this review, we aim to summarize studies performed on DN T cells in normal and diseased organs in the setting of different disease models with a focus on kidney.

Possible kidney-lung cross-talk in COVID-19: in silico modeling of SARS-CoV-2 infection.

BACKGROUND: Publicly available genomics datasets have grown drastically during the past decades. Although most of these datasets were initially generated to answer a pre-defined scientific question, their repurposing can be useful when new challenges such as COVID-19 arise. While the establishment and use of experimental models of COVID-19 are in progress, the potential hypotheses for mechanisms of onset and progression of COVID-19 can be generated by using in silico analysis of known molecular changes during COVID-19 and targets for SARS-CoV-2 invasion. METHODS: Selecting condition: COVID-19 infection leads to pneumonia and mechanical ventilation (PMV) and associated with acute kidney injury (AKI). There is increasing data demonstrating mechanistic links between AKI and lung injury caused by mechanical ventilation. Selecting targets: SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) for cell entry. We hypothesized that expression of ACE2 and TMPRSS2 would be affected in models of AKI and PMV. We therefore evaluated expression of ACE2 and TMPRSS2 as well as other novel molecular players of AKI and AKI-lung cross-talk in the publicly available microarray datasets GSE6730 and GSE60088, which represent gene expression of lungs and kidneys in mouse models of AKI and PMV, respectively. RESULTS: Expression of COVID-19 related genes ACE2 and TMPRSS2 was downregulated in lungs after 6 h of distant AKI effects. The expression of ACE2 decreased further after 36 h, while expression of TMPRSS2 recovered. In kidneys, both genes were downregulated by AKI, but not by distant lung injury. We also identified 53 kidney genes upregulated by PMV; and 254 lung genes upregulated by AKI, 9 genes of which were common to both organs. 3 of 9 genes were previously linked to kidney-lung cross-talk: Lcn2 (Fold Change (FC)Lung (L) = 18.6, FCKidney (K) = 6.32), Socs3 (FCL = 10.5, FCK = 10.4), Inhbb (FCL = 6.20, FCK = 6.17). This finding validates the current approach and reveals 6 new candidates, including Maff (FCL = 7.21, FCK = 5.98). CONCLUSIONS: Using our in silico approach, we identified changes in COVID-19 related genes ACE2 and TMPRSS2 in traditional mouse models of AKI and kidney-lung cross-talk. We also found changes in new candidate genes, which could be involved in the combined kidney-lung injury during COVID-19.

Nrf2 mediates hypoxia-inducible HIF1α activation in kidney tubular epithelial cells.

Nuclear factor erythroid 2-related factor 2 (Nrf2) and hypoxia-inducible factor-1α (HIF1α) transcription factors protect against ischemic acute kidney injury (AKI) by upregulating metabolic and cytoprotective gene expression. In this study, we tested the hypothesis that Nrf2 is required for HIF1α-mediated hypoxic responses using Nrf2-sufficient (wild-type) and Nrf2-deficient (Nrf2-/-) primary murine renal/kidney tubular epithelial cells (RTECs) and human immortalized tubular epithelial cells (HK2 cells) with HIF1 inhibition and activation. The HIF1 pathway inhibitor digoxin blocked hypoxia-stimulated HIF1α activation and heme oxygenase (HMOX1) expression in HK2 cells. Hypoxia-mimicking cobalt (II) chloride-stimulated HMOX1 expression was significantly lower in Nrf2-/- RTECs than in wild-type counterparts. Similarly, hypoxia-stimulated HIF1α-dependent metabolic gene expression was markedly impaired in Nrf2-/- RTECs. Nrf2 deficiency impaired hypoxia-induced HIF1α stabilization independent of increased prolyl 4-hydroxylase gene expression. We found decreased HIF1α mRNA levels in Nrf2-/- RTECs under both normoxia and hypoxia-reoxygenation conditions. In silico analysis and chromatin immunoprecipitation assays demonstrated Nrf2 binding to the HIF1α promoter in normoxia, but its binding decreased in hypoxia-exposed HK2 cells. However, Nrf2 binding at the HIF1α promoter was enriched following reoxygenation, demonstrating that Nrf2 maintains constitutive HIF1α expression. Consistent with this result, we found decreased levels of Nrf2 in hypoxia and that were restored following reoxygenation. Inhibition of mitochondrial complex I prevented hypoxia-induced Nrf2 downregulation and also increased basal Nrf2 levels. These results demonstrate a crucial role for Nrf2 in optimal HIF1α activation in hypoxia and that mitochondrial signaling downregulates Nrf2 levels in hypoxia, whereas reoxygenation restores it. Nrf2 and HIF1α interact to provide optimal metabolic and cytoprotective responses in ischemic AKI.

CD3+CD4-CD8- Double-negative αß T cells attenuate lung ischemia-reperfusion injury.

OBJECTIVE: Lung ischemia-reperfusion injury (IRI) is a common complication after lung transplantation, and immune cells have been implicated in modulating outcomes. We hypothesized that a newly described subset of αß T-cell receptor positive cells; that is, CD4-CD8- (double negative [DN]) T cells, are found in lungs and can protect against lung IRI. METHODS: Ischemia was induced in C57BL/6 mice by left pulmonary artery and vein occlusion for 30 minutes followed by 180 minutes of reperfusion. These mice were paired with sham hilar dissected surgical controls. In mice undergoing IRI, adoptive transfer of DN T cells or conventional T cells was performed 12 hours before occlusion. Flow cytometry was used to quantify T cells and inflammatory cytokines, and apoptotic signaling pathways were evaluated with immunoblotting. Lung injury was assessed with Evans blue dye extravasation. RESULTS: DN T cells were significantly higher (5.29% ± 1% vs 2.21% ± 3%; P < .01) in IRI lungs and secreted higher levels of interleukin-10 (30% ± 5% vs 6% ± 1%; P < .01) compared with surgical sham controls. Immunoblotting, hematoxylin and eosin staining and Evans blue dye demonstrated that adoptive transfer of DN T cells significantly decreased interstitial edema (P < .01) and attenuated apoptosis/cleaved caspase-3 expression in the lungs following lung IRI (P < .01). CONCLUSIONS: DN T cells traffic into lungs during IRI, and have tissue protective functions regulating inflammation and apoptosis. We propose a potential novel immunoregulatory function of DN T cells during lung IRI.

Persistent Interferon Production by Double Negative T Cells and Collapsing Focal Segmental Glomerulosclerosis.

Collapsing glomerulopathy has multiple associations, including viral infections, medications like bisphosphonates and interferon, autoimmune diseases, and genetic predisposition. We report a case of collapsing focal segmental glomerulosclerosis associated with persistently high levels of interferon gamma produced by T-cell receptor αß (+), CD4- CD8- (double negative) T lymphocytes that progressed despite treatment and improvement of other cytokine levels. Double negative T cells are elevated and activated in autoimmune lymphoproliferative syndrome (ALPS). Production of elevated interferon gamma levels from double negative T cells in ALPS despite treatment provides insight to the pathophysiology of collapsing glomerulopathy, guiding future research for collapsing glomerulopathy.