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.

IL-1 receptor signaling in podocytes limits susceptibility to glomerular damage.

Interleukin (IL)-1 receptor type 1 (IL-1R1) activation triggers a proinflammatory signaling cascade that can exacerbate kidney injury. However, the functions of podocyte IL-1R1 in glomerular disease remain unclear. To study the role of IL-1R1 signaling in podocytes, we selectively ablated podocyte IL-1R1 in mice (PKO mice). We then subjected PKO mice and wild-type controls to two glomerular injury models: nephrotoxic serum (NTS)- and adriamycin-induced nephropathy. Surprisingly, we found that IL-1R1 activation in podocytes limited albuminuria and podocyte injury during NTS- and adriamycin-induced nephropathy. Moreover, deletion of IL-1R1 in podocytes drove podocyte apoptosis and glomerular injury through diminishing Akt activation. Activation of Akt signaling abrogated the differences in albuminuria and podocyte injury between wild-type and PKO mice during NTS. Thus, IL-1R1 signaling in podocytes limits susceptibility to glomerular injury via an Akt-dependent signaling pathway. These data identify an unexpected protective role for IL-1R1 signaling in podocytes in the pathogenesis of glomerular disease.NEW & NOTEWORTHY The present study establishes that activation of the receptor for interleukin-1 limits susceptibility to damage to the kidney glomerulus in preclinical mouse models by stimulating Akt signaling cascades inside the podocyte.

A20 in Myeloid Cells Protects Against Hypertension by Inhibiting Dendritic Cell-Mediated T-Cell Activation.

RATIONALE: The ubiquitin-editing protein A20 in dendritic cells (DCs) suppresses NF-κB (nuclear factor-κB) signaling and constrains DC-mediated T-cell stimulation, but the role of A20 in modulating the hypertensive response requires elucidation. OBJECTIVE: Here, we tested the hypothesis that A20 in CD11c-expressing myeloid cells mitigates Ang II (angiotensin II)-induced hypertension by limiting renal T-cell activation. METHODS AND RESULTS: Mice with heterozygous deletion of A20 in CD11c-expressing myeloid cells (DC ACT[Cd11c-Cre+A20flox/wt]) have spontaneous DC activation but have normal baseline blood pressures. In response to low-dose chronic Ang II infusion, DC ACT mice compared with WT (wild type) controls had an exaggerated hypertensive response and augmented proportions of CD62LloCD44hi effector memory T lymphocytes in the kidney lymph node. After 10 days of Ang II, DC ACT kidneys had increased numbers of memory effector CD8+, but not CD4+ T cells, compared with WTs. Moreover, the expressions of TNF-α (tumor necrosis factor-α) and IFN-γ (interferon-γ) were upregulated in the DC ACT renal CD8+ T cells but not CD4+ T cells. Saline challenge testing revealed enhanced renal fluid retention in the DC ACT mice. DC ACT kidneys showed augmented protein expression of γ-epithelial sodium channel and NHE3 (sodium-hydrogen antiporter 3). DC ACT mice also had greater reductions in renal blood flow following acute injections with Ang II and enhanced oxidant stress in the vasculature as evidenced by higher circulating levels of malondialdehyde compared with WT controls. To directly test whether enhanced T-cell activation in the DC ACT cohort was responsible for their exaggerated hypertensive response, we chronically infused Ang II into lymphocyte-deficient DC ACT Rag1 (recombination activating protein 1)-deficient (Rag1-/-) mice and WT (Cd11c-Cre-A20flox/wt) Rag1-/- controls. The difference in blood pressure elevation accruing from DC activation was abrogated on the Rag1-/- strain. CONCLUSIONS: Following stimulation of the renin-angiotensin system, A20 suppresses DC activation and thereby mitigates T-cell-dependent blood pressure elevation.

TNF-α in T lymphocytes attenuates renal injury and fibrosis during nephrotoxic nephritis.

Nephrotoxic serum nephritis (NTN) models immune-mediated human glomerulonephritis and culminates in kidney inflammation and fibrosis, a process regulated by T lymphocytes. TNF-α is a key proinflammatory cytokine that contributes to diverse forms of renal injury. Therefore, we posited that TNF-α from T lymphocytes may contribute to NTN pathogenesis. Here, mice with T cell-specific deletion of TNF-α (TNF TKO) and wild-type (WT) control mice were subjected to the NTN model. At 14 days after NTN, kidney injury and fibrosis were increased in kidneys from TNF TKO mice compared with WT mice. PD1+CD4+ T cell numbers and mRNA levels of IL-17A were elevated in NTN kidneys of TNF TKO mice, suggesting that augmented local T helper 17 lymphocyte responses in the TNF TKO kidney may exaggerate renal injury and fibrosis. In turn, we found increased accumulation of neutrophils in TNF TKO kidneys during NTN. We conclude that TNF-α production in T lymphocytes mitigates NTN-induced kidney injury and fibrosis by inhibiting renal T helper 17 lymphocyte responses and infiltration of neutrophils.

The transcription factor Twist1 in the distal nephron but not in macrophages propagates aristolochic acid nephropathy.

Tubulointerstitial disease in the kidney culminates in renal fibrosis that portents organ failure. Twist1, a basic helix-loop-helix protein 38 transcription factor, regulates several essential biological functions, but inappropriate Twist1 activity in the kidney epithelium can trigger kidney fibrogenesis and chronic kidney disease. By contrast, Twist1 in circulating myeloid cells may constrain inflammatory injury by attenuating cytokine generation. To dissect the effects of Twist1 in kidney tubular versus immune cells on renal inflammation following toxin-induced renal injury, we subjected mice with selective deletion of Twist1 in renal epithelial cells or macrophages to aristolochic acid-induced chronic kidney disease. Ablation of Twist1 in the distal nephron attenuated kidney damage, interstitial fibrosis, and renal inflammation after aristolochic acid exposure. However, macrophage-specific deletion of Twist1 did not impact the development of aristolochic acid-induced nephropathy. In vitro studies confirmed that Twist1 in renal tubular cells underpins their susceptibility to apoptosis and propensity to generate pro-fibrotic mediators in response to aristolochic acid. Moreover, co-culture studies revealed that Twist1 in renal epithelia augmented the recruitment and activation of pro-inflammatory CD64+ macrophages. Thus, Twist1 in the distal nephron rather than in infiltrating macrophages propagates chronic inflammation and fibrogenesis during aristolochic acid-induced nephropathy.

Stimulating Type 1 Angiotensin Receptors on T Lymphocytes Attenuates Renal Fibrosis.

Most forms of chronic kidney disease culminate in renal fibrosis that heralds organ failure. In contrast to the protective effects of globally blocking type 1 angiotensin (AT1) receptors throughout the body, activating AT1 receptors directly on immune cells may serve protective functions. However, the effects of stimulating the T-cell AT1 receptor on the progression of renal fibrosis remain unknown. In this study, mice with T-cell-specific deletion of the dominant murine AT1 receptor isoform Lck-Cre Agtraflox/flox [total knockout (TKO)] and wild-type (WT) controls were subjected to the unilateral ureteral obstruction model of kidney fibrosis. Compared with WT controls, obstructed kidneys from TKO mice at day 14 had increased collagen 1 deposition. CD4+ T cells, CD11b+Ly6Chi myeloid cells, and mRNA levels of Th1 inflammatory cytokines are elevated in obstructed TKO kidneys, suggesting that augmented Th1 responses in the TKO mice may exaggerate renal fibrosis by driving proinflammatory macrophage differentiation. In turn, T-bet deficient (T-bet knockout) mice lacking Th1 responses have attenuated collagen deposition after unilateral ureteral obstruction. We conclude that activating the AT1 receptor on T cells mitigates renal fibrogenesis by inhibiting Th1 differentiation and renal accumulation of profibrotic macrophages.

KLF4 in Macrophages Attenuates TNFα-Mediated Kidney Injury and Fibrosis.

BACKGROUND: Polarized macrophage populations can orchestrate both inflammation of the kidney and tissue repair during CKD. Proinflammatory M1 macrophages initiate kidney injury, but mechanisms through which persistent M1-dependent kidney damage culminates in fibrosis require elucidation. Krüppel-like factor 4 (KLF4), a zinc-finger transcription factor that suppresses inflammatory signals, is an essential regulator of macrophage polarization in adipose tissues, but the effect of myeloid KLF4 on CKD progression is unknown. METHODS: We used conditional mutant mice lacking KLF4 or TNFα (KLF4's downstream effector) selectively in myeloid cells to investigate macrophage KLF4's role in modulating CKD progression in two models of CKD that feature robust macrophage accumulation, nephrotoxic serum nephritis, and unilateral ureteral obstruction. RESULTS: In these murine CKD models, KLF4 deficiency in macrophages infiltrating the kidney augmented their M1 polarization and exacerbated glomerular matrix deposition and tubular epithelial damage. During the induced injury in these models, macrophage-specific KLF4 deletion also exacerbated kidney fibrosis, with increased levels of collagen 1 and α-smooth muscle actin in the injured kidney. CD11b+Ly6Chi myeloid cells isolated from injured kidneys expressed higher levels of TNFα mRNA versus wild-type controls. In turn, mice bearing macrophage-specific deletion of TNFα exhibited decreased glomerular and tubular damage and attenuated kidney fibrosis in the models. Moreover, treatment with the TNF receptor-1 inhibitor R-7050 during nephrotoxic serum nephritis reduced damage, fibrosis, and necroptosis in wild-type mice and mice with KLF4-deficient macrophages, and abrogated the differences between the two groups in these parameters. CONCLUSIONS: These data indicate that macrophage KLF4 ameliorates CKD by mitigating TNF-dependent injury and fibrosis.

Twist1 in Infiltrating Macrophages Attenuates Kidney Fibrosis via Matrix Metallopeptidase 13-Mediated Matrix Degradation.

BACKGROUND: Following an acute insult, macrophages regulate renal fibrogenesis through the release of various factors that either encourage the synthesis of extracellular matrix synthesis or the degradation of matrix via endocytosis, proteolysis, or both. However, the roles of infiltrating versus resident myeloid cells in these opposing processes require elucidation. The transcription factor Twist1 controls diverse essential cellular functions through induction of several downstream targets, including matrix metalloproteinases (MMPs). In macrophages, Twist1 can influence patterns of cytokine generation, but the role of macrophage Twist1 in renal fibrogenesis remains undefined. METHODS: To study Twist1 functions in different macrophage subsets during kidney scar formation, we used two conditional mutant mouse models in which Twist1 was selectively ablated either in infiltrating, inflammatory macrophages or in resident tissue macrophages. We assessed fibrosis-related parameters, matrix metallopeptidase 13 (MMP13, or collagen 3, which catalyzes collagen degradation), inflammatory cytokines, and other factors in these Twist1-deficient mice compared with wild-type controls after subjecting the animals to unilateral ureteral obstruction. We also treated wild-type and Twist1-deficient mice with an MMP13 inhibitor after unilateral ureteral obstruction. RESULTS: Twist1 in infiltrating inflammatory macrophages but not in resident macrophages limited kidney fibrosis after ureteral obstruction by driving extracellular matrix degradation. Moreover, deletion of Twist1 in infiltrating macrophages attenuated the expression of MMP13 in CD11b+Ly6Clo myeloid cells. Inhibition of MMP13 abrogated the protection from renal fibrosis afforded by macrophage Twist1. CONCLUSIONS: Twist1 in infiltrating myeloid cells mitigates interstitial matrix accumulation in the injured kidney by promoting MMP13 production, which drives extracellular matrix degradation. These data highlight the complex cell-specific actions of Twist1 in the pathogenesis of kidney fibrosis.

Protein kinase Cα drives fibroblast activation and kidney fibrosis by stimulating autophagic flux.

Kidney fibrosis is a histological hallmark of chronic kidney disease and arises in large part through extracellular matrix deposition by activated fibroblasts. The signaling protein complex mTOR complex 2 (mTORC2) plays a critical role in fibroblast activation and kidney fibrosis. Protein kinase Cα (PKCα) is one of the major sub-pathways of mTORC2, but its role in fibroblast activation and kidney fibrosis remains to be determined. Here, we found that transforming growth factor ß1 (TGFß1) activates PKCα signaling in cultured NRK-49F cells in a time-dependent manner. Blocking PKCα signaling with the chemical inhibitor Go6976 or by transfection with PKCα siRNA largely reduced expression of the autophagy-associated protein lysosomal-associated membrane protein 2 (LAMP2) and also inhibited autophagosome-lysosome fusion and autophagic flux in the cells. Similarly to chloroquine, Go6976 treatment and PKCα siRNA transfection also markedly inhibited TGFß1-induced fibroblast activation. In murine fibrotic kidneys with unilateral ureteral obstruction (UUO) nephropathy, PKCα signaling is activated in the interstitial myofibroblasts. Go6976 administration largely blocked autophagic flux in fibroblasts in the fibrotic kidneys and attenuated the UUO nephropathy. Together, our findings suggest that blocking PKCα activity may retard autophagic flux and thereby prevent fibroblast activation and kidney fibrosis.

Dynamic contrast-enhanced MRI promotes early detection of toxin-induced acute kidney injury.

Acute kidney injury (AKI) is a common cause of morbidity and mortality in hospitalized patients. Nevertheless, there is limited ability to diagnose AKI in its earliest stages through the collection of structural and functional information. Magnetic resonance imaging (MRI) is increasingly being used to provide structural and functional data that characterize the injured kidney. Dynamic contrast-enhanced (DCE) MRI is an imaging modality with robust spatial and temporal resolution; however, its ability to detect changes in kidney function following AKI has not been determined. We hypothesized that DCE MRI would detect a prolongation in contrast transit time following toxin-induced AKI earlier than commonly used serum and tissue biomarkers. To test our hypothesis, we injected mice with either vehicle or cisplatin (30 mg/kg) and performed DCE MRI at multiple time points. We found that commonly used kidney injury biomarkers, including creatinine, blood urea nitrogen, and neutrophil gelatinase-associated lipocalin, did not rise until day 2 following cisplatin. Tissue levels of the proinflammatory cytokines and chemokines, tumor necrosis factor-α, interleukin (IL)-1ß, IL-1α, IL-6, C-C motif chemokine ligand 2, and C-X-C motif chemokine ligand 2 similarly did not upregulate until day 2 following cisplatin. However, the time to peak intensity of contrast in the renal collecting system was already prolonged at day 1 following cisplatin compared with vehicle-treated mice. This intensity change mirrored changes in kidney injury as measured by histological analysis and in transporter expression in the proximal tubule. Taken together, DCE MRI is a promising preclinical imaging modality that is useful for assessing functional capacity of the kidney in the earliest stages following AKI.

Interleukin-1 receptor activation aggravates autosomal dominant polycystic kidney disease by modulating regulated necrosis.

Autosomal dominant polycystic kidney disease (ADPKD) is associated with increased chemokines, cytokines, and growth factors in the diseased kidney. We found that both isoforms of IL-1, IL-1α and IL-1ß, were upregulated in ADPKD tissues. Here, we used a unique murine ADPKD model with selective deletion of polycystin-1 (pkd1) in the kidney (KPKD1) to study the role of IL-1 signaling in ADPKD progression. In KPKD mice, genetic deletion of the IL-1 receptor [IL-1 receptor (IL-1R) knockout (KO)] prolongs survival and attenuates cyst volume. Compared with IL-1R wild-type KPKD1 kidneys, IL-1R KO KPKD1 kidneys have upregulated TNF-α gene expression, with consequent elevations in markers for TNF-dependent regulated necrosis. We further observed that regulated necrosis was increased in ADPKD tissues from both humans and mice. To confirm that enhanced necroptosis is protective in ADPKD, we treated KPKD1 mice with an inhibitor of regulated necrosis (Nec-1). Regulated necrosis suppression augments kidney weights, suggesting that regulated necrosis is required to limit kidney growth in ADPKD. Thus, IL-1R activation drives ADPKD progression by paradoxically limiting regulated necrosis.

Opposing actions of renal tubular- and myeloid-derived porcupine in obstruction-induced kidney fibrosis.

Wnt/ß-catenin signaling is essential in the pathogenesis of renal fibrosis. We previously reported inhibition of the Wnt O-acyl transferase porcupine, required for Wnt secretion, dramatically attenuates kidney fibrosis in the murine unilateral ureteral obstruction model. Here, we investigated the tissue-specific contributions of porcupine to renal fibrosis and inflammation in ureteral obstruction using mice with porcupine deletion restricted to the kidney tubular epithelium or infiltrating myeloid cells. Obstruction of the ureter induced the renal mRNA expression of porcupine and downstream targets, ß-catenin, T-cell factor, and lymphoid enhancer factor in wild type mice. Renal tubular specific deficiency of porcupine reduced the expression of collagen I and other fibrosis markers in the obstructed kidney. Moreover, kidneys from obstructed mice with tubule-specific porcupine deficiency had reduced macrophage accumulation with attenuated expression of myeloid cytokine and chemokine mRNA. In co-culture with activated macrophages, renal tubular cells from tubular-specific porcupine knockout mice had blunted induction of fibrosis mediators compared with wild type renal tubular cells. In contrast, macrophages from macrophage-specific porcupine deficient mice in co-culture with wild type renal tubular cells had markedly enhanced expression of pro-fibrotic cytokines compared to wild type macrophages. Consequently, porcupine deletion specifically within macrophages augmented renal scar formation following ureteral obstruction. Thus, our experiments suggest a benefit of interrupting Wnt secretion specifically within the kidney epithelium while preserving Wnt O-acylation in infiltrating myeloid cells during renal fibrogenesis.

Role of T-cell activation in salt-sensitive hypertension.

The contributions of T lymphocytes to the pathogenesis of salt-sensitive hypertension has been well established. Under hypertensive stimuli, naive T cells develop into different subsets, including Th1, Th2, Th17, Treg, and cytotoxic CD8+ T cells, depending on the surrounding microenviroment in organs. Distinct subsets of T cells may play totally different roles in tissue damage and hypertension. The underlying mechanisms by which hypertensive stimuli activate naive T cells involve many events and different organs, such as neoantigen presentation by dendritic cells, high salt concentration, and the milieu of oxidative stress in the kidney and vasculature. Infiltrating and activated T subsets in injured organs, in turn, exert considerable impacts on tissue dysfunction, including sodium retention in the kidney, vascular stiffness, and remodeling in the vasculature. Therefore, a thorough knowledge of T-cell actions in hypertension may provide novel insights into the development of new therapeutic strategies for patients with hypertension.

Wnt/ß-Catenin-Promoted Macrophage Alternative Activation Contributes to Kidney Fibrosis.

The Wnt/ß-catenin pathway is crucial in normal development and throughout life, but aberrant activation of this pathway has been linked to kidney fibrosis, although the mechanisms involved remain incompletely determined. Here, we investigated the role of Wnt/ß-catenin in regulating macrophage activation and the contribution thereof to kidney fibrosis. Treatment of macrophages with Wnt3a exacerbated IL-4- or TGFß1-induced macrophage alternative (M2) polarization and the phosphorylation and nuclear translocation of STAT3 in vitro Conversely, inhibition of Wnt/ß-catenin signaling prevented these IL-4- or TGFß1-induced processes. In a mouse model, induced deletion of ß-catenin in macrophages attenuated the fibrosis, macrophage accumulation, and M2 polarization observed in the kidneys of wild-type littermates after unilateral ureter obstruction. This study shows that activation of Wnt/ß-catenin signaling promotes kidney fibrosis by stimulating macrophage M2 polarization.

Rictor/mammalian target of rapamycin complex 2 promotes macrophage activation and kidney fibrosis.

Mammalian target of rapamycin (mTOR) signalling controls many essential cellular functions. However, the role of Rictor/mTOR complex 2 (mTORC2) in regulating macrophage activation and kidney fibrosis remains largely unknown. We report here that Rictor/mTORC2 was activated in macrophages from the fibrotic kidneys of mice. Ablation of Rictor in macrophages reduced kidney fibrosis, inflammatory cell accumulation, macrophage proliferation and polarization after unilateral ureter obstruction or ischaemia/reperfusion injury. In bone marrow-derived macrophages (BMMs), deletion of Rictor or blockade of protein kinase Cα inhibited cell migration. Additionally, deletion of Rictor or blockade of Akt abolished interleukin-4-stimulated or transforming growth factor (TGF)-ß1-stimulated macrophage M2 polarization. Furthermore, deletion of Rictor downregulated TGF-ß1-stimulated upregulation of multiple profibrotic cytokines, including platelet-derived growth factor, vascular endothelial growth factor and connective tissue growth factor, in BMMs. Conditioned medium from TGF-ß1-pretreated Rictor-/- macrophages stimulated fibroblast activation less efficiently than that from TGF-ß1-pretreated Rictor+/+ macrophages. These results demonstrate that Rictor/mTORC2 signalling can promote macrophage activation and kidney fibrosis. Targeting this signalling pathway in macrophages may shine light on ways to protect against kidney fibrosis in patients with chronic kidney diseases. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Rictor/mTORC2 signaling mediates TGFß1-induced fibroblast activation and kidney fibrosis.

The mammalian target of rapamycin (mTOR) was recently identified in two structurally distinct multiprotein complexes: mTORC1 and mTORC2. Previously, we found that Rictor/mTORC2 protects against cisplatin-induced acute kidney injury, but the role and mechanisms for Rictor/mTORC2 in TGFß1-induced fibroblast activation and kidney fibrosis remains unknown. To study this, we initially treated NRK-49F cells with TGFß1 and found that TGFß1 could activate Rictor/mTORC2 signaling in cultured cells. Blocking Rictor/mTORC2 signaling with Rictor or Akt1 small interfering RNAs markedly inhibited TGFß1-induced fibronection and α-smooth muscle actin expression. Ensuing western blotting or immunostaining results showed that Rictor/mTORC2 signaling was activated in kidney interstitial myofibroblasts from mice with unilateral ureteral obstruction. Next, a mouse model with fibroblast-specific deletion of Rictor was generated. These knockout mice were normal at birth and had no obvious kidney dysfunction or kidney morphological abnormality within 2 months of birth. Compared with control littermates, the kidneys of Rictor knockout mice developed less interstitial extracellular matrix deposition and inflammatory cell infiltration at 1 or 2 weeks after ureteral obstruction. Thus our study suggests that Rictor/mTORC2 signaling activation mediates TGFß1-induced fibroblast activation and contributes to the development of kidney fibrosis. This may provide a therapeutic target for chronic kidney diseases.

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.

Twist1 in T Lymphocytes Augments Kidney Fibrosis after Ureteral Obstruction.

Background: Twist1 is a basic helix-loop-helix domain-containing transcription factor that participates in diverse cellular functions, including epithelial-mesenchymal transition and the cellular immune response. Although Twist1 plays critical roles in the initiation and progression of kidney diseases, the effects of Twist1 in the T lymphocyte on the progression of renal fibrosis require elucidation. Methods: 129/SvEv mice with a floxed allele for the gene encoding Twist1 or TNFα were bred with CD4-Cre mice to yield CD4-Cre+ Twist1flox/flox (Twist1-TKO) or CD4-Cre+ TNFflox/flox (TNF-TKO) mice with robust, but selective, deletion of Twist1 or TNFα mRNA in T cells, respectively. Twist1 TKO, TNF TKO, and WT controls underwent UUO with assessment of kidney fibrosis and T-cell phenotype at 14 days. Results: Compared with WT controls, obstructed kidneys from Twist1 TKO mice had attenuated extracellular matrix deposition. Despite this diminished fibrosis, Twist1 TKO obstructed kidneys contained more CD8+ T cells than in WTs. These intrarenal CD8+ T cells exhibited greater activation and higher levels of TNFα expression than those from WT obstructed kidneys. Further, we found that selective deletion of TNFα from T cells exaggerated renal scar formation and injury after UUO, highlighting the capacity of T-cell TNF to constrain fibrosis in the kidney. Conclusions: Twist1 in T cells promotes kidney fibrogenesis, in part, by curtailing the renal accumulation of TNF-elaborating T cells.

Annexin A1 Tripeptide Mimetic Increases Sirtuin-3 and Augments Mitochondrial Function to Limit Ischemic Kidney Injury.

Background: Acute kidney injury (AKI) is one of the most common organ failures following surgery. We have developed a tripeptide mimetic (ANXA1sp) of the parent annexin A1 molecule that shows promise as an organ protectant limiting cellular stress; however, its potential as a kidney protective agent remains unexplored, and its mechanism of action is poorly understood. Our hypothesis was that ANXA1sp would limit kidney injury following surgical ischemic kidney injury. Methods: In a blinded fashion, wildtype mice were assigned to receive vehicle control or ANXA1sp one hour prior to and one hour after kidney vascular clamping. Our primary outcomes were markers of kidney injury and function as measured by serum creatinine and histologic injury scoring of kidney tissue sections. Immunofluorescence microscopy, real-time PCR, and Western blot were used to assess cell death, oxidative stress, and mitochondrial biomarkers. An in vitro model of oxygen-glucose deprivation in immortalized kidney tubule cells was used. Results: ANXA1sp given prior to and after ischemic kidney injury abrogated ischemic kidney injury. ANXA1sp limited cell death both in vivo and in vitro and abrogated oxidative stress following ischemia. ANXA1sp significantly increased the expression of markers associated with protective mitophagy and limited the expression of markers associated with detrimental mitochondrial fission. ANXA1sp upregulated the expression of the mitochondrial protectant sirtuin-3 (SIRT3) in the mitochondria of kidney tubular cells. Silencing of SIRT3 reversed ANXA1sp-mediated protection against hypoxic cell death. Conclusions: ANXA1sp limits kidney injury, upregulates SIRT3, and preserves mitochondrial integrity following ischemic kidney injury. ANXA1sp holds considerable promise as a perioperative kidney protectant prior to ischemia inducing surgery and kidney transplantation.