KEAP1 Editing Using CRISPR/Cas9 for Therapeutic NRF2 Activation in Primary Human T Lymphocytes.

Oxidant stress modifies T lymphocyte activation and function. Previous work demonstrated that murine T cell-specific kelch like-ECH-associated protein 1 (Keap1) deletion enhances antioxidant capacity and protects from experimental acute kidney injury. In this study, we used CRISPR technology to develop clinically translatable human T cell-specific KEAP1 deletion. Delivery of KEAP1 exon 2 specific Cas9:guide RNA in Jurkat T cells led to significant (∼70%) editing and upregulation of NRF2-regulated antioxidant genes NADPH dehydrogenase quinone 1 (NQO1) (up to 11-fold), heme oxygenase 1 (HO1) (up to 11-fold), and GCLM (up to 2-fold). In primary human T cells, delivery of KEAP1 exon 2 target site 2-specific ATTO 550-labeled Cas9:guide RNA edited KEAP1 in ∼40% cells and significantly (p ≤ 0.04) increased NQO1 (16-fold), HO1 (9-fold), and GCLM (2-fold) expression. To further enrich KEAP1-edited cells, ATTO 550-positive cells were sorted 24 h after electroporation. Assessment of ATTO 550-positive cells showed KEAP1 editing in ∼55% cells. There was no detectable off-target cleavage in the top three predicted genes in the ATTO 550-positive cells. Gene expression analysis found significantly (p ≤ 0.01) higher expression of NQO1 mRNA in ATTO 550-positive cells compared with control cells. Flow cytometric assessment showed increased (p ≤ 0.01) frequency of CD4-, CD25-, and CD69-expressing KEAP1 edited cells whereas frequency of CD8- (p ≤ 0.01) and IL-17- (p ≤ 0.05) expressing cells was reduced compared with control cells. Similar experimental conditions resulted in significant KEAP1 editing, increased antioxidant gene expression, and frequency of CD69 and IL-10 positive cells in highly enriched KEAP1-edited regulatory T cells. KEAP1-edited T cells could potentially be used for treating multiple human diseases.

T Lymphocyte-Specific Activation of Nrf2 Protects from AKI.

T lymphocytes are established mediators of ischemia reperfusion (IR)-induced AKI, but traditional immune principles do not explain their mechanism of early action in the absence of alloantigen. Nrf2 is a transcription factor that is crucial for cytoprotective gene expression and is generally thought to have a key role in dampening IR-induced AKI through protective effects on epithelial cells. We proposed an alternative hypothesis that augmentation of Nrf2 in T cells is essential to mitigate oxidative stress during IR-induced AKI. We therefore generated mice with genetically amplified levels of Nrf2 specifically in T cells and examined the effect on antioxidant gene expression, T cell activation, cytokine production, and IR-induced AKI. T cell-specific augmentation of Nrf2 significantly increased baseline antioxidant gene expression. These mice had a high frequency of intrarenal CD25(+)Foxp3(+) regulatory T cells and decreased frequencies of CD11b(+)CD11c(+) and F4/80(+) cells. Intracellular levels of TNF-α, IFN-γ, and IL-17 were significantly lower in CD4(+) T cells with high Nrf2 expression. Mice with increased T cell expression of Nrf2 were significantly protected from functional and histologic consequences of AKI. Furthermore, adoptive transfer of high-Nrf2 T cells protected wild-type mice from IR injury and significantly improved their survival. These data demonstrate that T cell-specific activation of Nrf2 protects from IR-induced AKI, revealing a novel mechanism of tissue protection during acute injury responses.

Reviving the promise of transcription factor Nrf2-based therapeutics for kidney diseases.

The transcription factor Nrf2 plays an important role in many kidney diseases from acute kidney injury to chronic kidney disease, and there have been preliminary Nrf2-based therapeutic trials in humans. Shelton et al. presents an integrated transcriptomic and proteomic analysis of mouse kidney to reveal Nrf2 targets with potentially important roles in kidney homeostasis and pathophysiology. These results can further our understanding of Nrf2-based mechanisms and help in the development of therapeutics for a wide range of kidney diseases.

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.

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.

T Lymphocytes in Acute Kidney Injury and Repair.

Innate and adaptive immune systems participate in the pathogenesis of acute kidney injury (AKI). Considerable data from different research teams have shown the importance of T lymphocytes in the pathophysiology of AKI and, more recently, prevention and repair. T cells can generate or resolve inflammation by secreting specific cytokines and growth factors as well as interact with other immune and stromal cells to induce kidney injury or promote tissue repair. There also are emerging data on the role of T cells in the progression of AKI to chronic kidney disease and organ cross-talk in AKI. These data set the stage for immunomodulatory therapies for AKI. This review focuses on the major populations of T lymphocytes and their roles as mediators for AKI and repair.

Characterization of kidney CD45intCD11bintF4/80+MHCII+CX3CR1+Ly6C- "intermediate mononuclear phagocytic cells".

Kidney immune cells play important roles in pathogenesis of many diseases, including ischemia-reperfusion injury (IRI) and transplant rejection. While studying murine kidney T cells, we serendipitously identified a kidney mononuclear phagocytic cell (MPC) subset characterized by intermediate surface expression of CD45 and CD11b. These CD45intCD11bint MPCs were further identified as F4/80+MHCII+CX3CR1+Ly6C- cells, comprising ~17% of total CD45+ cells in normal mouse kidney (P < 0.01) and virtually absent from all other organs examined except the heart. Systemic clodronate treatment had more significant depletive effect on the CD45intCD11bint population (77.3%±5.9%, P = 0.03) than on CD45highCD11b+ population (14.8%±16.6%, P = 0.49). In addition, CD45intCD11bint MPCs had higher phagocytic function in the normal kidney (35.6%±3.3% vs. 24.1%±2.2%, P = 0.04), but lower phagocytic capacity in post-ischemic kidney (54.9%±1.0% vs. 67.8%±1.9%, P < 0.01) compared to the CD45highCD11b+ population. Moreover, the CD45intCD11bint population had higher intracellular production of the pro-inflammatory tumor necrosis factor (TNF)-α (58.4%±5.2% vs. 27.3%±0.9%, P < 0.001) after lipopolysaccharide (LPS) stimulation and lower production of the anti-inflammatory interleukin (IL)-10 (7.2%±1.3% vs. 14.9%±2.2%, P = 0.02) following kidney IRI, suggesting a functional role under inflammatory conditions. The CD45intCD11bint cells increased early after IRI, and then abruptly decreased 48h later, whereas CD45highCD11b+ cells steadily increased after IRI before declining at 72h (P = 0.03). We also identified the CD45intCD11bint MPC subtype in human kidney. We conclude that CD45intCD11bint F4/80+MHCII+CX3CR1+Ly6C-population represent a unique subset of MPCs found in both mouse and human kidneys. Future studies will further characterize their role in kidney health and disease.

Role of Immune Cells in Acute Kidney Injury and Repair.

Acute kidney injury (AKI) is a significant problem in both native and transplant kidneys. There have been significant advances in understanding the role of immune cells in the early injury and repair from AKI. In this brief review, we aim to update information on the pathophysiologic impact of various immune cells in AKI, with special emphasis on repair. An improved understanding of the AKI immunopathology will lead to new therapies that prevent AKI, accelerate repair, and prevent the progression of AKI to chronic kidney disease.