Divergent Actions of Renal Tubular and Endothelial Type 1 IL-1 Receptor Signaling in Toxin-Induced AKI.

SIGNIFICANCE STATEMENT: Activation of the type 1 IL-1 receptor (IL-1R1) triggers a critical innate immune signaling cascade that contributes to the pathogenesis of AKI. However, blockade of IL-1 signaling in AKI has not consistently demonstrated kidney protection. The current murine experiments show that IL-1R1 activation in the proximal tubule exacerbates toxin-induced AKI and cell death through local suppression of apolipoprotein M. By contrast, IL-1R1 activation in endothelial cells ameliorates AKI by restoring VEGFA-dependent endothelial cell viability. Using this information, future delivery strategies can maximize the protective effects of blocking IL-1R1 while mitigating unwanted actions of IL-1R1 manipulation. BACKGROUND: Activation of the type 1 IL-1 receptor (IL-1R1) triggers a critical innate immune signaling cascade that contributes to the pathogenesis of AKI. IL-1R1 is expressed on some myeloid cell populations and on multiple kidney cell lineages, including tubular and endothelial cells. Pharmacological inhibition of the IL-1R1 does not consistently protect the kidney from injury, suggesting there may be complex, cell-specific effects of IL-1R1 stimulation in AKI. METHODS: To examine expression of IL-1 and IL-1R1 in intrinsic renal versus infiltrating immune cell populations during AKI, we analyzed single-cell RNA sequencing (scRNA-seq) data from kidney tissues of humans with AKI and mice with acute aristolochic acid exposure. We then investigated cell-specific contributions of renal IL-1R1 signaling to AKI using scRNA-seq, RNA microarray, and pharmacological interventions in mice with IL-1R1 deletion restricted to the proximal tubule or endothelium. RESULTS: scRNA-seq analyses demonstrated robust IL-1 expression in myeloid cell populations and low-level IL-1R1 expression in kidney parenchymal cells during toxin-induced AKI. Our genetic studies showed that IL-1R1 activation in the proximal tubule exacerbated toxin-induced AKI and cell death through local suppression of apolipoprotein M. By contrast, IL-1R1 activation in endothelial cells ameliorated aristolochic acid-induced AKI by restoring VEGFA-dependent endothelial cell viability and density. CONCLUSIONS: These data highlight opposing cell-specific effects of IL-1 receptor signaling on AKI after toxin exposure. Disrupting pathways activated by IL-1R1 in the tubule, while preserving those triggered by IL-1R1 activation on endothelial cells, may afford renoprotection exceeding that of global IL-1R1 inhibition while mitigating unwanted actions of IL-1R1 blockade.

A macrophage-endothelial immunoregulatory axis ameliorates septic acute kidney injury.

The most common cause of acute kidney injury (AKI) in critically ill patients is sepsis. Kidney macrophages consist of both F4/80hi and CD11bhi cells. The role of macrophage subpopulations in septic AKI pathogenesis remains unclear. As F4/80hi macrophages are reported to contribute to immunomodulation following injury, we hypothesized that selective depletion of F4/80hi macrophages would worsen septic AKI. F4/80hi macrophages were depleted via diphtheria toxin injection in CD11cCre(+)/CX3CR1dtr/wt (F4/80 MKO mice) compared to CD11cCre(-)/CX3CR1dtr/wt (F4/80 MWT) mice. F4/80 MWT and F4/80 MKO mice were subjected to sham or cecal ligation and puncture to induce sepsis. Compared to F4/80 MWT mice, F4/80 MKO mice displayed worsened septic AKI at 24 hours as measured by serum creatinine and histologic injury scoring. Kidneys from F4/80 MKO mice elaborated higher kidney interleukin-6 levels. Mechanistically, single cell RNA sequencing identified a macrophage-endothelial cell immunoregulatory axis that underlies interleukin-6 expression. F4/80hi macrophages expressed interleukin-1 receptor antagonist and limited interleukin-6 expression in endothelial cells. In turn, anti-interleukin-6 therapy ameliorated septic AKI in F4/80 MKO mice. Thus, F4/80hi macrophages express interleukin-1 receptor antagonist and constrain interleukin-6 generation from endothelial cells to limit septic AKI, representing a targetable cellular crosstalk in septic AKI. These findings are particularly relevant owing to the efficacy of anti-interleukin-6 therapies during COVID-19 infection, a disease associated with high rates of AKI and endothelial dysfunction.

Hypoxia signaling in renal pericytes-is it safe to activate?

While excitement has grown for the use of hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors for treating renal anemia, multiple preclinical studies have shown the complex and cell-type-dependent roles of HIFs in kidney disease pathogenesis, including renal fibrosis. Pan et al. now clearly show that activating the HIF signaling in the Gli1-lineage myofibroblasts restores erythropoietin production while not adversely affecting matrix production, mitigating the concerns of exacerbated fibrosis by HIF prolyl hydroxylase inhibitors.

Context-dependent functions of angiopoietin 2 are determined by the endothelial phosphatase VEPTP.

The angiopoietin (ANGPT)-TIE2/TEK signaling pathway is essential for blood and lymphatic vascular homeostasis. ANGPT1 is a potent TIE2 activator, whereas ANGPT2 functions as a context-dependent agonist/antagonist. In disease, ANGPT2-mediated inhibition of TIE2 in blood vessels is linked to vascular leak, inflammation, and metastasis. Using conditional knockout studies in mice, we show TIE2 is predominantly activated by ANGPT1 in the cardiovascular system and by ANGPT2 in the lymphatic vasculature. Mechanisms underlying opposing actions of ANGPT2 in blood vs. lymphatic endothelium are poorly understood. Here we show the endothelial-specific phosphatase VEPTP (vascular endothelial protein tyrosine phosphatase) determines TIE2 response to ANGPT2. VEPTP is absent from lymphatic endothelium in mouse in vivo, permitting ANGPT2/TIE2-mediated lymphangiogenesis. Inhibition of VEPTP converts ANGPT2 into a potent TIE2 activator in blood endothelium. Our data support a model whereby VEPTP functions as a rheostat to modulate ANGPT2 ligand effect on TIE2.

Transcription Factor 21 Is Required for Branching Morphogenesis and Regulates the Gdnf-Axis in Kidney Development.

BACKGROUND: The mammalian kidney develops through reciprocal inductive signals between the metanephric mesenchyme and ureteric bud. Transcription factor 21 (Tcf21) is highly expressed in the metanephric mesenchyme, including Six2-expressing cap mesenchyme and Foxd1-expressing stromal mesenchyme. Tcf21 knockout mice die in the perinatal period from severe renal hypodysplasia. In humans, Tcf21 mRNA levels are reduced in renal tissue from human fetuses with renal dysplasia. The molecular mechanisms underlying these renal defects are not yet known. METHODS: Using a variety of techniques to assess kidney development and gene expression, we compared the phenotypes of wild-type mice, mice with germline deletion of the Tcf21 gene, mice with stromal mesenchyme-specific Tcf21 deletion, and mice with cap mesenchyme-specific Tcf21 deletion. RESULTS: Germline deletion of Tcf21 leads to impaired ureteric bud branching and is accompanied by downregulated expression of Gdnf-Ret-Wnt11, a key pathway required for branching morphogenesis. Selective removal of Tcf21 from the renal stroma is also associated with attenuation of the Gdnf signaling axis and leads to a defect in ureteric bud branching, a paucity of collecting ducts, and a defect in urine concentration capacity. In contrast, deletion of Tcf21 from the cap mesenchyme leads to abnormal glomerulogenesis and massive proteinuria, but no downregulation of Gdnf-Ret-Wnt11 or obvious defect in branching. CONCLUSIONS: Our findings indicate that Tcf21 has distinct roles in the cap mesenchyme and stromal mesenchyme compartments during kidney development and suggest that Tcf21 regulates key molecular pathways required for branching morphogenesis.

Novel ACE2-Fc chimeric fusion provides long-lasting hypertension control and organ protection in mouse models of systemic renin angiotensin system activation.

Angiotensin-converting enzyme 2 (ACE2) is a carboxypeptidase that potently degrades angiotensin II to angiotensin 1-7. Previous studies showed that injection of the enzymatic ectodomain of recombinant ACE2 (rACE2) markedly increases circulatory levels of ACE2 activity, and effectively lowered blood pressure in angiotensin II-induced hypertension. However, due to the short plasma half-life of rACE2, its therapeutic potential for chronic use is limited. To circumvent this, we generated a chimeric fusion of rACE2 and the immunoglobulin fragment Fc segment to increase its plasma stability. This rACE2-Fc fusion protein retained full peptidase activity and exhibited greatly extended plasma half-life in mice, from less than two hours of the original rACE2, to over a week. A single 2.5 mg/kg injection of rACE2-Fc increased the overall angiotensin II-conversion activities in blood by up to 100-fold and enhanced blood pressure recovery from acute angiotensin II induced hypertension seven days after administration. To assess rACE2-Fc given weekly on cardiac protection, we performed studies in mice continuously infused with angiotensin II for 28 days and in a Renin transgenic mouse model of hypertension. The angiotensin II infused mice achieved sustained blood pressure control and reduced cardiac hypertrophy and fibrosis. In chronic hypertensive transgenic mice, weekly injections of rACE2-Fc effectively lowered plasma angiotensin II and blood pressure. Additionally, rACE2-Fc ameliorated albuminuria, and reduced kidney and cardiac fibrosis. Thus, our chimeric fusion strategy for rACE2-Fc is suitable for future development of new renin angiotensin system-based inhibition therapies.

Acute kidney injury to chronic kidney disease transition: insufficient cellular stress response.

PURPOSE OF REVIEW: Recent epidemiological and preclinical mechanistic studies provide strong evidence that acute kidney injury (AKI) and chronic kidney disease (CKD) form an interconnected syndrome. Injured kidneys undergo a coordinated reparative process with an engagement of multiple cell types after injury; however, maladaptation to the injury subjects kidneys to a vicious cycle of fibrogenesis and nephron loss. In this review, we will outline and discuss the pathogenesis of AKI-to-CKD transition with an emphasis on dysregulated 'cellular stress adaptation' as a potential therapeutic target. RECENT FINDINGS: Recent studies identify the crucial role of injured tubular epithelial cells in the transition from AKI to CKD. Damaged tubular cells undergo reactivation of developmental and epithelial-mesenchymal transition signaling, metabolic alteration, and cell-cycle arrest, thereby driving inflammation and fibrogenesis. Recent work highlights that cellular stress-adaptive pathways against hypoxic and oxidative stress provide insufficient protection after severe AKI episode. SUMMARY: Insufficient cellular stress adaptation may underpin the persistent activation of inflammatory and fibrogenic signaling in damaged kidneys. We propose that harnessing cellular stress-adaptive responses will be a promising therapeutic strategy to halt or even reverse the deleterious process of AKI-to-CKD transition.

Transcription factor Nrf2 hyperactivation in early-phase renal ischemia-reperfusion injury prevents tubular damage progression.

Acute kidney injury is a devastating disease with high morbidity in hospitalized patients and contributes to the pathogenesis of chronic kidney disease. An underlying mechanism of acute kidney injury involves ischemia-reperfusion injury which, in turn, induces oxidative stress and provokes organ damage. Nrf2 is a master transcription factor that regulates the cellular response to oxidative stress. Here, we examined the role of Nrf2 in the progression of ischemia-reperfusion injury-induced kidney damage in mice using genetic and pharmacological approaches. Both global and tubular-specific Nrf2 activation enhanced gene expression of antioxidant and NADPH synthesis enzymes, including glucose-6-phosphate dehydrogenase, and ameliorated both the initiation of injury in the outer medulla and the progression of tubular damage in the cortex. Myeloid-specific Nrf2 activation was ineffective. Short-term administration of the Nrf2 inducer CDDO during the initial phase of injury ameliorated the late phase of tubular damage. This inducer effectively protected the human proximal tubular cell line HK-2 from oxidative stress-mediated cell death while glucose-6-phosphate dehydrogenase knockdown increased intracellular reactive oxygen species. These findings demonstrate that tubular hyperactivation of Nrf2 in the initial phase of injury prevents the progression of reactive oxygen species-mediated tubular damage by inducing antioxidant enzymes and NADPH synthesis. Thus, Nrf2 may be a promising therapeutic target for preventing acute kidney injury to chronic kidney disease transition.

Erythropoietin Synthesis in Renal Myofibroblasts Is Restored by Activation of Hypoxia Signaling.

Erythropoietin (Epo) is produced by renal Epo-producing cells (REPs) in a hypoxia-inducible manner. The conversion of REPs into myofibroblasts and coincident loss of Epo-producing ability are the major cause of renal fibrosis and anemia. However, the hypoxic response of these transformed myofibroblasts remains unclear. Here, we used complementary in vivo transgenic and live imaging approaches to better understand the importance of hypoxia signaling in Epo production. Live imaging of REPs in transgenic mice expressing green fluorescent protein from a modified Epo-gene locus revealed that healthy REPs tightly associated with endothelium by wrapping processes around capillaries. However, this association was hampered in states of renal injury-induced inflammation previously shown to correlate with the transition to myofibroblast-transformed renal Epo-producing cells (MF-REPs). Furthermore, activation of hypoxia-inducible factors (HIFs) by genetic inactivation of HIF-prolyl hydroxylases (PHD1, PHD2, and PHD3) selectively in Epo-producing cells reactivated Epo production in MF-REPs. Loss of PHD2 in REPs restored Epo-gene expression in injured kidneys but caused polycythemia. Notably, combined deletions of PHD1 and PHD3 prevented loss of Epo expression without provoking polycythemia. Mice with PHD-deficient REPs also showed resistance to LPS-induced Epo repression in kidneys, suggesting that augmented HIF signaling counterbalances inflammatory stimuli in regulation of Epo production. Thus, augmentation of HIF signaling may be an attractive therapeutic strategy for treating renal anemia by reactivating Epo synthesis in MF-REPs.

Plasticity of renal erythropoietin-producing cells governs fibrosis.

CKD progresses with fibrosis and erythropoietin (Epo)-dependent anemia, leading to increased cardiovascular complications, but the mechanisms linking Epo-dependent anemia and fibrosis remain unclear. Here, we show that the cellular phenotype of renal Epo-producing cells (REPs) alternates between a physiologic Epo-producing state and a pathologic fibrogenic state in response to microenvironmental signals. In a novel mouse model, unilateral ureteral obstruction-induced inflammatory milieu activated NFκB and Smad signaling pathways in REPs, rapidly repressed the Epo-producing potential of REPs, and led to myofibroblast transformation of these cells. Moreover, we developed a unique Cre-based cell-fate tracing method that marked current and/or previous Epo-producing cells and revealed that the majority of myofibroblasts are derived from REPs. Genetic induction of NFκB activity selectively in REPs resulted in myofibroblastic transformation, indicating that NFκB signaling elicits a phenotypic switch. Reversing the unilateral ureteral obstruction-induced inflammatory microenvironment restored the Epo-producing potential and the physiologic phenotype of REPs. This phenotypic reversion was accelerated by anti-inflammatory therapy. These findings demonstrate that REPs possess cellular plasticity, and suggest that the phenotypic transition of REPs to myofibroblasts, modulated by inflammatory molecules, underlies the connection between anemia and renal fibrosis in CKD.

[Renal erythropoietin-producing cells and kidney disease].

Erythropoietin(EPO) is an indispensable erythropoietic hormone, produced mainly from kidneys in adult, and the production declines with progression of chronic kidney disease(CKD). Renal EPO-producing cells(REPs) are peri-tubular interstitial fibroblasts. Dysfunction and myofibroblast transformation of REPs have been reported in rodent models of kidney injuries. Despite the crucial importance of EPO in health and diseases, many aspects of REPs remain to be elucidated because of technical difficulties to investi- gate the cells in vivo. This review will summarize our recent progress in characterization of REPs. We also summarize the role REPs play in kidney fibrosis and their unique character "plasticity". Future therapeutic approach targeting REPs to treat both anemia and fibrosis in CKD will also be discussed.

AXL inhibition suppresses early allograft monocyte-to-macrophage differentiation and prolongs allograft survival.

Innate immune cells are important in the initiation and potentiation of alloimmunity in transplantation. Immediately upon organ anastomosis and reperfusion, recipient monocytes enter the graft from circulation and differentiate to inflammatory macrophages to promote allograft inflammation. However, factors that drive their differentiation to inflammatory macrophages are not understood. Here, we show that the receptor tyrosine kinase AXL was a key driver of early intragraft differentiation of recipient infiltrating monocytes to inflammatory macrophages in the presence of allogeneic stimulation and cell-to-cell contact. In this context, the differentiated inflammatory macrophages were capable of efficient alloantigen presentation and allostimulation of T cells of the indirect pathway. Consequently, early and transient AXL inhibition with the pharmacological inhibitor bemcentinib resulted in a profound reduction of initial allograft inflammation and a significant prolongation of allograft survival in a murine heart transplant model. Our results support further investigation of AXL inhibition as part of an induction regimen for transplantation.

Transcriptional control of glyoxalase 1 by Nrf2 provides a stress-responsive defence against dicarbonyl glycation.

Abnormal cellular accumulation of the dicarbonyl metabolite MG (methylglyoxal) occurs on exposure to high glucose concentrations, inflammation, cell aging and senescence. It is associated with increased MG-adduct content of protein and DNA linked to increased DNA strand breaks and mutagenesis, mitochondrial dysfunction and ROS (reactive oxygen species) formation and cell detachment from the extracellular matrix. MG-mediated damage is countered by glutathione-dependent metabolism by Glo1 (glyoxalase 1). It is not known, however, whether Glo1 has stress-responsive up-regulation to counter periods of high MG concentration or dicarbonyl stress. We identified a functional ARE (antioxidant-response element) in the 5'-untranslated region of exon 1 of the mammalian Glo1 gene. Transcription factor Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2) binds to this ARE, increasing basal and inducible expression of Glo1. Activators of Nrf2 induced increased Glo1 mRNA, protein and activity. Increased expression of Glo1 decreased cellular and extracellular concentrations of MG, MG-derived protein adducts, mutagenesis and cell detachment. Hepatic, brain, heart, kidney and lung Glo1 mRNA and protein were decreased in Nrf2-/- mice, and urinary excretion of MG protein and nucleotide adducts were increased approximately 2-fold. We conclude that dicarbonyl stress is countered by up-regulation of Glo1 in the Nrf2 stress-responsive system, protecting protein and DNA from increased damage and preserving cell function.

Transitional cell states sculpt tissue topology during lung regeneration.

Organ regeneration requires dynamic cell interactions to reestablish cell numbers and tissue architecture. While we know the identity of progenitor cells that replace lost tissue, the transient states they give rise to and their role in repair remain elusive. Here, using multiple injury models, we find that alveolar fibroblasts acquire distinct states marked by Sfrp1 and Runx1 that influence tissue remodeling and reorganization. Unexpectedly, ablation of alveolar epithelial type-1 (AT1) cells alone is sufficient to induce tissue remodeling and transitional states. Integrated scRNA-seq followed by genetic interrogation reveals RUNX1 is a key driver of fibroblast states. Importantly, the ectopic induction or accumulation of epithelial transitional states induce rapid formation of transient alveolar fibroblasts, leading to organ-wide fibrosis. Conversely, the elimination of epithelial or fibroblast transitional states or RUNX1 loss, leads to tissue simplification resembling emphysema. This work uncovered a key role for transitional states in orchestrating tissue topologies during regeneration.

Luminal alkalinization attenuates proteinuria-induced oxidative damage in proximal tubular cells.

A highly acidic environment surrounds proximal tubular cells as a result of their reabsorption of HCO(3)(-). It is unclear whether this luminal acidity affects proteinuria-induced progression of tubular cell damage. Here, we investigated the contribution of luminal acidity to superoxide (O(2)(·-)) production induced by oleic acid-bound albumin (OA-Alb) in proximal tubular cells. Acidic media significantly enhanced OA-Alb-induced O(2)(·-) production in the HK-2 proximal tubular cell line. Simultaneous treatment with both OA-Alb and acidic media led to phosphorylation of the intracellular pH sensor Pyk2. Highly phosphorylated Pyk2 associated with activation of Rac1, an essential subcomponent of NAD(P)H oxidase. Furthermore, knockdown of Pyk2 with siRNA attenuated the O(2)(·-) production induced by cotreatment with OA-Alb and acid. To assess whether luminal alkalinization abrogates proteinuria-induced tubular damage, we studied a mouse model of protein-overload nephropathy. NaHCO(3) feeding selectively alkalinized the urine and dramatically attenuated the accumulation of O(2)(·-)-induced DNA damage and proximal tubular injury. Overall, these observations suggest that luminal acidity aggravates proteinuria-induced tubular damage and that modulation of this acidic environment may hold potential as a therapeutic target for proteinuric kidney disease.

In Vivo Assessment of Ferroptosis and Ferroptotic Stress in Mice.

Ferroptosis is iron-dependent, lipid peroxidation-driven, regulated cell death that is triggered when cellular glutathione peroxidase 4 (GPX4)-mediated cellular defense is insufficient to prevent pathologic accumulation of toxic lipid peroxides. Ferroptosis is implicated in various human pathologies, including neurodegeneration, chemotherapy-resistant cancers, ischemia-reperfusion injury, and acute and chronic kidney diseases. Despite the fact that the ferroptotic process has been rigorously interrogated in multiple preclinical models, the lack of specific and readily available biomarkers to detect ferroptosis in vivo in mouse models makes it challenging to delineate its contribution to key pathologic events in vivo. Critical steps to practically evaluate ferroptosis include, but are not limited to, detecting increased cell death and pathologic accumulation of toxic lipid peroxides and testing augmentation of observed pathologic events by genetic inhibition of the glutathione-GPX4 axis or mitigation of the pathologic process by ferroptosis inhibitors. Here, we describe methods to evaluate these key features of the ferroptotic process in mice in vivo. Specifically, we describe methods to detect toxic lipid peroxides (4-hydroxynonenal) and cell death (based on terminal deoxynucleotidyl transferase dUTP nick end labeling staining) as well as a protocol to pharmacologically inhibit ferroptotic stress using liproxstatin-1. These protocols provide tools for understanding the ferroptotic process in mouse genetic or disease models. © 2022 Wiley Periodicals LLC. Basic Protocol 1: How to use liproxstatin-1 Basic Protocol 2: How to evaluate ferroptosis in mouse kidneys.

Type 1 Angiotensin Receptors on CD11c-Expressing Cells Protect Against Hypertension by Regulating Dendritic Cell-Mediated T Cell Activation.

BACKGROUND: Type 1 angiotensin (AT1) receptors are expressed on immune cells, and we previously found that bone marrow-derived AT1 receptors protect against Ang (angiotensin) II-induced hypertension. CD11c is expressed on myeloid cells derived from the bone marrow, including dendritic cells (DCs) that activate T lymphocytes. Here, we examined the role of AT1 receptors on CD11c+ cells in hypertension pathogenesis. METHODS: Mice lacking the dominant murine AT1 receptor isoform, AT1a, on CD11c+ cells (dendritic cell [DC] AT1aR knockout [KO]) and wild-type (WT) littermates were subjected to Ang II-induced hypertension. Blood pressures were measured by radiotelemetry. RESULTS: DC AT1aR KO mice had exaggerated hypertensive responses to chronic Ang II infusion with enhanced renal accumulation of effector memory T cells and CD40+ DCs. CCL5 (C-C motif chemokine ligand 5) recruits T cells into injured tissues, and CCR7 (C-C motif chemokine receptor 7) facilitates DC and T cell interactions in the kidney lymph node to allow T cell activation. DCs from the hypertensive DC AT1aR KO kidneys expressed higher levels of CCL5 and CCR7. mRNA expressions for CCR7 and tumor necrosis factor-α were increased in CD4+ T cells from the renal lymph nodes of DC AT1aR KO mice. During the second week of Ang II infusion when blood pressures between groups diverged, DC AT1aR KO mice excreted less sodium than WTs. Expressions for epithelial sodium channel subunits were increased in DC AT1aR KO kidneys. CONCLUSIONS: Following activation of the renin angiotensin system, AT1aR stimulation on DCs suppresses renal DC maturation and T cell activation with consequent protection from sodium retention and blood pressure elevation.

Sex differences in resilience to ferroptosis underlie sexual dimorphism in kidney injury and repair.

In both humans and mice, repair of acute kidney injury is worse in males than in females. Here, we provide evidence that this sexual dimorphism results from sex differences in ferroptosis, an iron-dependent, lipid-peroxidation-driven regulated cell death. Using genetic and single-cell transcriptomic approaches in mice, we report that female sex confers striking protection against ferroptosis, which was experimentally induced in proximal tubular (PT) cells by deleting glutathione peroxidase 4 (Gpx4). Single-cell transcriptomic analyses further identify the NFE2-related factor 2 (NRF2) antioxidant protective pathway as a female resilience mechanism against ferroptosis. Genetic inhibition and pharmacological activation studies show that NRF2 controls PT cell fate and plasticity by regulating ferroptosis. Importantly, pharmacological NRF2 activation protects male PT cells from ferroptosis and improves cellular plasticity as in females. Our data highlight NRF2 as a potential therapeutic target to prevent failed renal repair after acute kidney injury in both sexes by modulating cellular plasticity.

Stromal Transcription Factor 21 Regulates Development of the Renal Stroma via Interaction with Wnt/ß-Catenin Signaling.

Background: Kidney formation requires coordinated interactions between multiple cell types. Input from the interstitial progenitor cells is implicated in multiple aspects of kidney development. We previously reported that transcription factor 21 (Tcf21) is required for ureteric bud branching. Here, we show that Tcf21 in Foxd1+ interstitial progenitors regulates stromal formation and differentiation via interaction with ß-catenin. Methods: We utilized the Foxd1Cre;Tcf21f/f murine kidney for morphologic analysis. We used the murine clonal mesenchymal cell lines MK3/M15 to study Tcf21 interaction with Wnt/ß-catenin. Results: Absence of Tcf21 from Foxd1+ stromal progenitors caused a decrease in stromal cell proliferation, leading to marked reduction of the medullary stromal space. Lack of Tcf21 in the Foxd1+ stromal cells also led to defective differentiation of interstitial cells to smooth-muscle cells, perivascular pericytes, and mesangial cells. Foxd1Cre;Tcf21f/f kidney showed an abnormal pattern of the renal vascular tree. The stroma of Foxd1Cre;Tcf21f/f kidney demonstrated marked reduction in ß-catenin protein expression compared with wild type. Tcf21 was bound to ß-catenin both upon ß-catenin stabilization and at basal state as demonstrated by immunoprecipitation in vitro. In MK3/M15 metanephric mesenchymal cells, Tcf21 enhanced TCF/LEF promoter activity upon ß-catenin stabilization, whereas DNA-binding deficient mutated Tcf21 did not enhance TCF/LEF promoter activity. Kidney explants of Foxd1Cre;Tcf21f/f showed low mRNA expression of stromal Wnt target genes. Treatment of the explants with CHIR, a Wnt ligand mimetic, restored Wnt target gene expression. Here, we also corroborated previous evidence that normal development of the kidney stroma is required for normal development of the Six2+ nephron progenitor cells, loop of Henle, and the collecting ducts. Conclusions: These findings suggest that stromal Tcf21 facilitates medullary stroma development by enhancing Wnt/ß-catenin signaling and promotes stromal cell proliferation and differentiation. Stromal Tcf21 is also required for the development of the adjacent nephron epithelia.

Ferroptotic stress promotes the accumulation of pro-inflammatory proximal tubular cells in maladaptive renal repair.

Overwhelming lipid peroxidation induces ferroptotic stress and ferroptosis, a non-apoptotic form of regulated cell death that has been implicated in maladaptive renal repair in mice and humans. Using single-cell transcriptomic and mouse genetic approaches, we show that proximal tubular (PT) cells develop a molecularly distinct, pro-inflammatory state following injury. While these inflammatory PT cells transiently appear after mild injury and return to their original state without inducing fibrosis, after severe injury they accumulate and contribute to persistent inflammation. This transient inflammatory PT state significantly downregulates glutathione metabolism genes, making the cells vulnerable to ferroptotic stress. Genetic induction of high ferroptotic stress in these cells after mild injury leads to the accumulation of the inflammatory PT cells, enhancing inflammation and fibrosis. Our study broadens the roles of ferroptotic stress from being a trigger of regulated cell death to include the promotion and accumulation of proinflammatory cells that underlie maladaptive repair.