Gasdermin D drives focal crystalline thrombotic microangiopathy by accelerating immunothrombosis and necroinflammation.

ABSTRACT: Thrombotic microangiopathy (TMA) is characterized by immunothrombosis and life-threatening organ failure but the precise underlying mechanism driving its pathogenesis remains elusive. In this study, we hypothesized that gasdermin D (GSDMD), a pore-forming protein that serves as the final downstream effector of the pyroptosis/interleukin-1ß (IL-1ß) pathway, contributes to TMA and its consequences by amplifying neutrophil maturation and subsequent necrosis. Using a murine model of focal crystalline TMA, we found that Gsdmd deficiency ameliorated immunothrombosis, acute tissue injury, and failure. Gsdmd-/- mice exhibited a decrease in mature IL-1ß, as well as in neutrophil maturation, ß2-integrin activation, and recruitment to TMA lesions, in which they formed reduced neutrophil extracellular traps in both arteries and interstitial tissue. The GSDMD inhibitor disulfiram dose-dependently suppressed human neutrophil pyroptosis in response to cholesterol crystals. Experiments with GSDMD-deficient, human-induced, pluripotent stem cell-derived neutrophils confirmed the involvement of GSDMD in neutrophil ß2-integrin activation, maturation, and pyroptosis. Both prophylactic and therapeutic administration of disulfiram protected the mice from focal TMA, acute tissue injury, and failure. Our data identified GSDMD as a key mediator of focal crystalline TMA and its consequences, including ischemic tissue infarction and organ failure. GSDMD could potentially serve as a therapeutic target for the systemic forms of TMA.

Sex dimorphism in kidney health and disease: mechanistic insights and clinical implication.

Sex is a key variable in the regulation of human physiology and pathology. Many diseases disproportionately affect one sex: autoimmune diseases, such as systemic lupus erythematosus, are more common in women but more severe in men, while the incidence of other disorders such as gouty arthritis and malignant cancers is higher in men. Besides the pathophysiology, sex may also influence the efficacy of therapeutics: participants in clinical trials are still predominately men, and side effects of drugs are more common in women than in men. Sex dimorphism is a prominent feature of kidney physiology and function, and consequently affects the predisposition to many adult kidney diseases. These differences subsequently influence the response to immune stimuli, hormones and therapies. It is highly likely that these responses differ between the sexes. Therefore, it becomes imperative to consider sex differences in translational science from basic science to preclinical research to clinical research and trials. Underrepresentation of one sex in preclinical animal studies or clinical trials remains an issue and key reported outcomes of such studies ought to be presented separately. Without this, it remains difficult to tailor the management of kidney disease appropriately and effectively. In this review, we provide mechanistic insights into sex differences in rodents and in humans, both in kidney health and disease, highlight the importance of considering sex differences in the design of any preclinical animal or clinical study, and propose guidance how to optimal design and conduct preclinical animal studies in future research.

Soluble uric acid inhibits ß2 integrin-mediated neutrophil recruitment in innate immunity.

Neutrophils are key players during host defense and sterile inflammation. Neutrophil dysfunction is a characteristic feature of the acquired immunodeficiency during kidney disease. We speculated that the impaired renal clearance of the intrinsic purine metabolite soluble uric acid (sUA) may account for neutrophil dysfunction. Indeed, hyperuricemia (HU, serum UA of 9-12 mg/dL) related or unrelated to kidney dysfunction significantly diminished neutrophil adhesion and extravasation in mice with crystal- and coronavirus-related sterile inflammation using intravital microscopy and an air pouch model. This impaired neutrophil recruitment was partially reversible by depleting UA with rasburicase. We validated these findings in vitro using either neutrophils or serum from patients with kidney dysfunction-related HU with or without UA depletion, which partially normalized the defective migration of neutrophils. Mechanistically, sUA impaired ß2 integrin activity and internalization/recycling by regulating intracellular pH and cytoskeletal dynamics, physiological processes that are known to alter the migratory and phagocytic capability of neutrophils. This effect was fully reversible by blocking intracellular uptake of sUA via urate transporters. In contrast, sUA had no effect on neutrophil extracellular trap formation in neutrophils from healthy subjects or patients with kidney dysfunction. Our results identify an unexpected immunoregulatory role of the intrinsic purine metabolite sUA, which contrasts the well-known immunostimulatory effects of crystalline UA. Specifically targeting UA may help to overcome certain forms of immunodeficiency, for example in kidney dysfunction, but may enhance sterile forms of inflammation.

Neutrophils-biology and diversity.

Neutrophils, the most abundant white blood cells in the human circulation, play crucial roles in various diseases, including kidney disease. Traditionally viewed as short-lived pro-inflammatory phagocytes that release reactive oxygen species, cytokines and neutrophil extracellular traps, recent studies have revealed their complexity and heterogeneity, thereby challenging this perception. Neutrophils are now recognized as transcriptionally active cells capable of proliferation and reverse migration, displaying phenotypic and functional heterogeneity. They respond to a wide range of signals and deploy various cargo to influence the activity of other cells in the circulation and in tissues. They can regulate the behavior of multiple immune cell types, exhibit innate immune memory, and contribute to both acute and chronic inflammatory responses while also promoting inflammation resolution in a context-dependent manner. Here, we explore the origin and heterogeneity of neutrophils, their functional diversity, and the cues that regulate their effector functions. We also examine their emerging role in infectious and non-infectious diseases with a particular emphasis on kidney disease. Understanding the complex behavior of neutrophils during tissue injury and inflammation may provide novel insights, thereby paving the way for potential therapeutic strategies to manage acute and chronic conditions. By deciphering their multifaceted role, targeted interventions can be developed to address the intricacies of neutrophil-mediated immune responses and improve disease outcomes.

Management of adult patients with podocytopathies: an update from the ERA Immunonephrology Working Group.

The histopathological lesions, minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) are entities without immune complex deposits which can cause podocyte injury, thus are frequently grouped under the umbrella of podocytopathies. Whether MCD and FSGS may represent a spectrum of the same disease remains a matter of conjecture. Both frequently require repeated high-dose glucocorticoid therapy with alternative immunosuppressive treatments reserved for relapsing or resistant cases and response rates are variable. There is an unmet need to identify patients who should receive immunosuppressive therapies as opposed to those who would benefit from supportive strategies. Therapeutic trials focusing on MCD are scarce, and the evidence used for the 2021 Kidney Disease: Improving Global Outcomes (KDIGO) guideline for the management of glomerular diseases largely stems from observational and pediatric trials. In FSGS, the differentiation between primary forms and those with underlying genetic variants or secondary forms further complicates trial design. This article provides a perspective of the Immunonephrology Working Group (IWG) of the European Renal Association (ERA) and discusses the KDIGO 2021 Clinical Practice Guideline for the Management of Glomerular Diseases focusing on the management of MCD and primary forms of FSGS in the context of recently published evidence, with a special emphasis on the role of rituximab, cyclophosphamide, supportive treatment options and ongoing clinical trials in the field.

Both hyperglycemia and hyperuricemia aggravate acute kidney injury during cholesterol embolism syndrome despite opposite effects on kidney infarct size.

Kidney cholesterol crystal embolism (CCE) occurs in advanced atherosclerosis and induces a thrombotic (micro)angiopathy, a drop in the glomerular filtration rate (GFR), and an ischemic kidney infarction with necroinflammation. We speculated that common metabolic comorbidities such as diabetes or hyperuricemia would independently modulate each of these distinct pathophysiological processes. To test this, experimental CCE was induced by injecting cholesterol crystals into the left kidney artery of mice and thrombotic angiopathy, GFR drop, and infarct size were analyzed after 24 hours in the presence of hyperglycemia (about 500 mg/dL) or hyperuricemia (about 8 mg/dL) or their absence. In healthy mice, unilateral CCE caused diffuse thrombotic angiopathy in interlobar, arcuate and interlobular arteries, followed by a 50% or less drop in GFR compared to baseline and a variable degree of ischemic kidney necrosis. Hyperglycemia but not hyperuricemia aggravated thrombotic angiopathy although both caused a GFR decline, albeit via different mechanisms. Hyperglycemia aggravated GFR loss by increasing necroinflammation and infarct size, while the antioxidative effects of hyperuricemia reasonably attenuated necroinflammation and infarct size but induced a diffuse vasoconstriction in affected and unaffected kidney tissue. Thus, both hyperglycemia or hyperuricemia aggravate CCE-induced acute kidney failure despite having opposite effects on ischemic necroinflammation and infarction.

Secondary Immunodeficiency Related to Kidney Disease (SIDKD)-Definition, Unmet Need, and Mechanisms.

Kidney disease is a known risk factor for poor outcomes of COVID-19 and many other serious infections. Conversely, infection is the second most common cause of death in patients with kidney disease. However, little is known about the underlying secondary immunodeficiency related to kidney disease (SIDKD). In contrast to cardiovascular disease related to kidney disease, which has triggered countless epidemiologic, clinical, and experimental research activities or interventional trials, investments in tracing, understanding, and therapeutically targeting SIDKD have been sparse. As a call for more awareness of SIDKD as an imminent unmet medical need that requires rigorous research activities at all levels, we review the epidemiology of SIDKD and the numerous aspects of the abnormal immunophenotype of patients with kidney disease. We propose a definition of SIDKD and discuss the pathogenic mechanisms of SIDKD known thus far, including more recent insights into the unexpected immunoregulatory roles of elevated levels of FGF23 and hyperuricemia and shifts in the secretome of the intestinal microbiota in kidney disease. As an ultimate goal, we should aim to develop therapeutics that can reduce mortality due to infections in patients with kidney disease by normalizing host defense to pathogens and immune responses to vaccines.

Uremic Toxin Indoxyl Sulfate Promotes Macrophage-Associated Low-Grade Inflammation and Epithelial Cell Senescence.

In this study, we investigated the impact of the uremic toxin indoxyl sulfate on macrophages and tubular epithelial cells and its role in modulating the response to lipopolysaccharide (LPS). Indoxyl sulfate accumulates in the blood of patients with chronic kidney disease (CKD) and is a predictor of overall and cardiovascular morbidity/mortality. To simulate the uremic condition, primary macrophages and tubular epithelial cells were incubated with indoxyl sulfate at low concentrations as well as concentrations found in uremic patients, both alone and upon LPS challenge. The results showed that indoxyl sulfate alone induced the release of reactive oxygen species and low-grade inflammation in macrophages. Moreover, combined with LPS (proinflammatory conditions), indoxyl sulfate significantly increased TNF-α, CCL2, and IL-10 release but did not significantly affect the polarization of macrophages. Pre-treatment with indoxyl sulfate following LPS challenge induced the expression of aryl hydrocarbon receptor (Ahr) and NADPH oxidase 4 (Nox4) which generate reactive oxygen species (ROS). Further, experiments with tubular epithelial cells revealed that indoxyl sulfate might induce senescence in parenchymal cells and therefore participate in the progression of inflammaging. In conclusion, this study provides evidence that indoxyl sulfate provokes low-grade inflammation, modulates macrophage function, and enhances the inflammatory response associated with LPS. Finally, indoxyl sulfate signaling contributes to the senescence of tubular epithelial cells during injury.

Crystal Clots as Therapeutic Target in Cholesterol Crystal Embolism.

RATIONALE: Cholesterol crystal embolism can be a life-threatening complication of advanced atherosclerosis. Pathophysiology and molecular targets for treatment are largely unknown. OBJECTIVE: We aimed to develop a new animal model of cholesterol crystal embolism to dissect the molecular mechanisms of cholesterol crystal (CC)-driven arterial occlusion, tissue infarction, and organ failure. METHODS AND RESULTS: C57BL/6J mice were injected with CC into the left kidney artery. Primary end point was glomerular filtration rate (GFR). CC caused crystal clots occluding intrarenal arteries and a dose-dependent drop in GFR, followed by GFR recovery within 4 weeks, that is, acute kidney disease. In contrast, the extent of kidney infarction was more variable. Blocking necroptosis using mixed lineage kinase domain-like deficient mice or necrostatin-1s treatment protected from kidney infarction but not from GFR loss because arterial obstructions persisted, identifying crystal clots as a primary target to prevent organ failure. CC involved platelets, neutrophils, fibrin, and extracellular DNA. Neutrophil depletion or inhibition of the release of neutrophil extracellular traps had little effects, but platelet P2Y12 receptor antagonism with clopidogrel, fibrinolysis with urokinase, or DNA digestion with recombinant DNase I all prevented arterial occlusions, GFR loss, and kidney infarction. The window-of-opportunity was <3 hours after CC injection. However, combining Nec-1s (necrostatin-1s) prophylaxis given 1 hour before and DNase I 3 hours after CC injection completely prevented kidney failure and infarcts. In vitro, CC did not directly induce plasmatic coagulation but induced neutrophil extracellular trap formation and DNA release mainly from kidney endothelial cells, neutrophils, and few from platelets. CC induced ATP release from aggregating platelets, which increased fibrin formation in a DNase-dependent manner. CONCLUSIONS: CC embolism causes arterial obstructions and organ failure via the formation of crystal clots with fibrin, platelets, and extracellular DNA as critical components. Therefore, our model enables to unravel the pathogenesis of the CC embolism syndrome as a basis for both prophylaxis and targeted therapy.

Soluble Uric Acid Is an Intrinsic Negative Regulator of Monocyte Activation in Monosodium Urate Crystal-Induced Tissue Inflammation.

Although monosodium urate (MSU) crystals are known to trigger inflammation, published data on soluble uric acid (sUA) in this context are discrepant. We hypothesized that diverse sUA preparation methods account for this discrepancy and that an animal model with clinically relevant levels of asymptomatic hyperuricemia and gouty arthritis can ultimately clarify this issue. To test this, we cultured human monocytes with different sUA preparation solutions and found that solubilizing uric acid (UA) by prewarming created erroneous results because of UA microcrystal contaminants triggering IL-1ß release. Solubilizing UA with NaOH avoided this artifact, and this microcrystal-free preparation suppressed LPS- or MSU crystal-induced monocyte activation, a process depending on the intracellular uptake of sUA via the urate transporter SLC2A9/GLUT9. CD14+ monocytes isolated from hyperuricemic patients were less responsive to inflammatory stimuli compared with monocytes from healthy individuals. Treatment with plasma from hyperuricemic patients impaired the inflammatory function of CD14+ monocytes, an effect fully reversible by removing sUA from hyperuricemic plasma. Moreover, Alb-creERT2;Glut9 lox/lox mice with hyperuricemia (serum UA of 9-11 mg/dl) showed a suppressed inflammatory response to MSU crystals compared with Glut9 lox/lox controls without hyperuricemia. Taken together, we unravel a technical explanation for discrepancies in the published literature on immune effects of sUA and identify hyperuricemia as an intrinsic suppressor of innate immunity, in which sUA modulates the capacity of monocytes to respond to danger signals. Thus, sUA is not only a substrate for the formation of MSU crystals but also an intrinsic inhibitor of MSU crystal-induced tissue inflammation.

Only Hyperuricemia with Crystalluria, but not Asymptomatic Hyperuricemia, Drives Progression of Chronic Kidney Disease.

BACKGROUND: The roles of asymptomatic hyperuricemia or uric acid (UA) crystals in CKD progression are unknown. Hypotheses to explain links between UA deposition and progression of CKD include that (1) asymptomatic hyperuricemia does not promote CKD progression unless UA crystallizes in the kidney; (2) UA crystal granulomas may form due to pre-existing CKD; and (3) proinflammatory granuloma-related M1-like macrophages may drive UA crystal-induced CKD progression. METHODS: MALDI-FTICR mass spectrometry, immunohistochemistry, 3D confocal microscopy, and flow cytometry were used to characterize a novel mouse model of hyperuricemia and chronic UA crystal nephropathy with granulomatous nephritis. Interventional studies probed the role of crystal-induced inflammation and macrophages in the pathology of progressive CKD. RESULTS: Asymptomatic hyperuricemia alone did not cause CKD or drive the progression of aristolochic acid I-induced CKD. Only hyperuricemia with UA crystalluria due to urinary acidification caused tubular obstruction, inflammation, and interstitial fibrosis. UA crystal granulomas surrounded by proinflammatory M1-like macrophages developed late in this process of chronic UA crystal nephropathy and contributed to the progression of pre-existing CKD. Suppressing M1-like macrophages with adenosine attenuated granulomatous nephritis and the progressive decline in GFR. In contrast, inhibiting the JAK/STAT inflammatory pathway with tofacitinib was not renoprotective. CONCLUSIONS: Asymptomatic hyperuricemia does not affect CKD progression unless UA crystallizes in the kidney. UA crystal granulomas develop late in chronic UA crystal nephropathy and contribute to CKD progression because UA crystals trigger M1-like macrophage-related interstitial inflammation and fibrosis. Targeting proinflammatory macrophages, but not JAK/STAT signaling, can attenuate granulomatous interstitial nephritis.

Growth Differentiation Factor 15 Ameliorates Anti-Glomerular Basement Membrane Glomerulonephritis in Mice.

Growth differentiation factor 15 (GDF15) is a member of the transforming growth factor-ß (TGF-ß) cytokine family and an inflammation-associated protein. Here, we investigated the role of GDF15 in murine anti-glomerular basement membrane (GBM) glomerulonephritis. Glomerulonephritis induction in mice induced systemic expression of GDF15. Moreover, we demonstrate the protective effects for GDF15, as GDF15-deficient mice exhibited increased proteinuria with an aggravated crescent formation and mesangial expansion in anti-GBM nephritis. Herein, GDF15 was required for the regulation of T-cell chemotactic chemokines in the kidney. In addition, we found the upregulation of the CXCR3 receptor in activated T-cells in GDF15-deficient mice. These data indicate that CXCL10/CXCR3-dependent-signaling promotes the infiltration of T cells into the organ during acute inflammation controlled by GDF15. Together, these results reveal a novel mechanism limiting the migration of lymphocytes to the site of inflammation during glomerulonephritis.

STAT1 regulates macrophage number and phenotype and prevents renal fibrosis after ischemia-reperfusion injury.

Renal ischemia-reperfusion injury (IRI) leads to acute kidney injury or delayed allograft function, which predisposes to fibrosis in the native kidney or kidney transplant. Here we investigated the role of the signal transducer and activator of transcription 1 (STAT1) in inflammatory responses following renal IRI. Our study showed that a subsequent stimulation of Janus-activated kinase 2/STAT1 and Toll-like receptor 4 pathways led to greater STAT1 activation followed by increased cytokine transcription compared with single-pathway stimulation in murine renal tubular cells. Moreover, we observed increased activation of STAT1 under hypoxic conditions. In vivo, STAT1-/- mice displayed less acute tubular necrosis and decreased macrophage infiltration 24 h after renal ischemia. However, investigation of the healing phase (30 days after IRI) revealed significantly more fibrosis in STAT1-/- than in wild-type kidneys. In addition, we demonstrated increased macrophage infiltration in STAT1-/- kidneys. Flow cytometry analysis revealed that STAT1 deficiency drives an alternatively activated macrophage phenotype, which is associated with downregulated cluster of differentiation 80 expression, decreased intracellular reactive oxygen species production, and enhanced ability for phagocytosis. Furthermore, we detected immunohistochemically enhanced STAT1 expression in human renal allograft biopsies with no interstitial fibrosis/tubular atrophy (IF/TA) compared with specimens with severe IF/TA without specific etiology. Thus, STAT1 activation drives macrophages toward an alternatively activated phenotype and enhances fibrogenesis indicating a potential STAT1-driven protective mechanism in tissue repair after ischemic injury.

Immunomodulatory Molecule IRAK-M Balances Macrophage Polarization and Determines Macrophage Responses during Renal Fibrosis.

Activation of various innate immune receptors results in IL-1 receptor-associated kinase (IRAK)-1/IRAK-4-mediated signaling and secretion of proinflammatory cytokines such as IL-12, IL-6, or TNF-α, all of which are implicated in tissue injury and elevated during tissue remodeling processes. IRAK-M, also known as IRAK-3, is an inhibitor of proinflammatory cytokine and chemokine expression in intrarenal macrophages. Innate immune activation contributes to both acute kidney injury and tissue remodeling that is associated with chronic kidney disease (CKD). Our study assessed the contribution of macrophages in CKD and the role of IRAK-M in modulating disease progression. To evaluate the effect of IRAK-M in chronic renal injury in vivo, a mouse model of unilateral ureteral obstruction (UUO) was employed. The expression of IRAK-M increased within 2 d after UUO in obstructed compared with unobstructed kidneys. Mice deficient in IRAK-M were protected from fibrosis and displayed a diminished number of alternatively activated macrophages. Compared to wild-type mice, IRAK-M-deficient mice showed reduced tubular injury, leukocyte infiltration, and inflammation following renal injury as determined by light microscopy, immunohistochemistry, and intrarenal mRNA expression of proinflammatory and profibrotic mediators. Taken together, these results strongly support a role for IRAK-M in renal injury and identify IRAK-M as a possible modulator in driving an alternatively activated profibrotic macrophage phenotype in UUO-induced CKD.

The macrophage phenotype and inflammasome component NLRP3 contributes to nephrocalcinosis-related chronic kidney disease independent from IL-1-mediated tissue injury.

Primary/secondary hyperoxalurias involve nephrocalcinosis-related chronic kidney disease (CKD) leading to end-stage kidney disease. Mechanistically, intrarenal calcium oxalate crystal deposition is thought to elicit inflammation, tubular injury and atrophy, involving the NLRP3 inflammasome. Here, we found that mice deficient in NLRP3 and ASC adaptor protein failed to develop nephrocalcinosis, compromising conclusions on nephrocalcinosis-related CKD. In contrast, hyperoxaluric wild-type mice developed profound nephrocalcinosis. NLRP3 inhibition using the ß-hydroxybutyrate precursor 1,3-butanediol protected such mice from nephrocalcinosis-related CKD. Interestingly, the IL-1 inhibitor anakinra had no such effect, suggesting IL-1-independent functions of NLRP3. NLRP3 inhibition using 1,3-butanediol treatment induced a shift of infiltrating renal macrophages from pro-inflammatory (CD45+F4/80+CD11b+CX3CR1+CD206-) and pro-fibrotic (CD45+F4/80+CD11b+CX3CR1+CD206+TGFß+) to an anti-inflammatory (CD45+F4/80+CD11b+CD206+TGFß-) phenotype, and prevented renal fibrosis. Finally, in vitro studies with primary murine fibroblasts confirmed the non-redundant role of NLRP3 in the TGF-ß signaling pathway for fibroblast activation and proliferation independent of the NLRP3 inflammasome complex formation. Thus, nephrocalcinosis-related CKD involves NLRP3 but not necessarily via intrarenal IL-1 release but rather via other biological functions including TGFR signaling and macrophage polarization. Hence, NLRP3 may be a promising therapeutic target in hyperoxaluria and nephrocalcinosis.

Monosodium Urate Crystals Induce Upregulation of NK1.1-Dependent Killing by Macrophages and Support Tumor-Resident NK1.1+ Monocyte/Macrophage Populations in Antitumor Therapy.

Macrophages display phenotypic and functional heterogeneity dependent on the changing inflammatory microenvironment. Under some conditions, macrophages can acquire effector functions commonly associated with NK cells. In the current study, we investigated how the endogenous danger signal monosodium urate (MSU) crystals can alter macrophage functions. We report that naive, primary peritoneal macrophages rapidly upregulate the expression of the NK cell-surface marker NK1.1 in response to MSU crystals but not in response to LPS or other urate crystals. NK1.1 upregulation by macrophages was associated with mechanisms including phagocytosis of crystals, NLRP3 inflammasome activation, and autocrine proinflammatory cytokine signaling. Further analysis demonstrated that MSU crystal-activated macrophages exhibited NK cell-like cytotoxic activity against target cells in a perforin/granzyme B-dependent manner. Furthermore, analysis of tumor hemopoietic cell populations showed that effective, MSU-mediated antitumor activity required coadministration with Mycobacterium smegmatis to induce IL-1ß production and significant accumulation of monocytes and macrophages (but not granulocytes or dendritic cells) expressing elevated levels of NK1.1. Our findings provide evidence that MSU crystal-activated macrophages have the potential to develop tumoricidal NK cell-like functions that may be exploited to boost antitumor activity in vivo.