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 β-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+TGFβ+) to an anti-inflammatory (CD45+F4/80+CD11b+CD206+TGFβ-) phenotype, and prevented renal fibrosis. Finally, in vitro studies with primary murine fibroblasts confirmed the non-redundant role of NLRP3 in the TGF-β 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.

Significance Statement: The role of asymptomatic hyperuricemia in the progression of CKD has been unclear due to lack of animal models with clinically relevant uric acid (UA) levels. A new mouse model reveals that persistent asymptomatic hyperuricemia (approximately 15 mg/dl) does not cause CKD, nor accelerate progression, unless UA crystallizes in acidic tubular fluid. Crystallization initially causes tubular injury, inflammation, and interstitial fibrosis, and subsequently granulomatous interstitial nephritis with perilesional proinflammatory M1-like macrophage infiltrates. Modulating the M1-like macrophage phenotype, but not JAK/STAT inhibition, attenuates granulomatous nephritis. 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.

The human body is exposed to a wide range of particles of industrial, environmental or internal origin such as asbestos, alum, silica or crystals of urate, calcium phosphate, calcium oxalate, cystine or cholesterol. Phagocytic clearance of such particles involves neutrophils and macrophages. Here we report that neutrophils encountering such particles of diverse sizes and shapes undergo necrotic cell death, a process associated with the formation of neutrophil extracellular trap (NET)-like extracellular DNA. In human neutrophils receptor-interacting protein kinase (RIPK)-1 inhibition with necrostatin-1s or mixed lineage kinase domain-like (MLKL) inhibition with necrosulfonamide abrogated cell death and associated-neutrophil extracellular DNA release induced by all of the aforementioned particles. Similar results were obtained with Mlkl-deficient mice neutrophils for all particles in vitro. Furthermore, Mlkl-deficient mice lacked tophus formation upon injection of MSU crystals into subcutaneous air pouches. These findings imply that nano- or microparticle-induced neutrophil extracellular DNA release is the consequence of neutrophil necroptosis, a regulated form of cell necrosis defined by RIPK1-RIPK3-MLKL signaling. Interestingly, this finding was consistent across different particle sizes and shapes. The RIPK1-RIPK3-MLKL signaling pathway may represent a potential therapeutic target in nano- or microparticle-related diseases (crystallopathies).

Objective: To profile monosodium urate monohydrate (MSU) crystal–recruited monocyte inflammatory function during the course of in vivo differentiation, in a murine model of peritoneal MSU crystal–induced inflammation.Methods: C57BL/6J mice were injected intraperitoneally with MSU crystals, and the peritoneal cells were harvested at different time points. The MSU crystal–recruited monocyte/macrophage population was analyzed for the expression of differentiation and activation markers, cytokine production following MSU crystal restimulation ex vivo and in vivo, expression of NLRP3‐associated proteins (ASC, caspase 1) and pro–interleukin‐1β (proIL‐1β), and phagocytic capacity.Results: Monocytes recruited 8 hours after MSU crystal stimulation (F4/80^lowGr‐1^int7/4+) exhibited poor phagocytic capacity, expressed low levels of proIL‐1β, and failed to produce proinflammatory cytokines in response to MSU crystal restimulation. In the absence of MSU crystal restimulation, differentiating monocytes produced low levels of transforming growth factor β1 ex vivo, and this was abrogated following MSU crystal restimulation. Over time these cells developed a proinflammatory phenotype in vivo, characterized by the production of IL‐1β, tumor necrosis factor α, IL‐6, CCL2 (monocyte chemotactic protein 1), and CXCL1 (cytokine‐induced neutrophil chemoattractant) following ex vivo MSU crystal restimulation, and leading to IL‐1β production and cell infiltration following MSU crystal rechallenge in vivo. Proinflammatory function was associated with differentiation toward a macrophage phenotype (F4/80^highGr‐1–7/4–), an increase in phagocytic capacity, and an increase in the expression of proIL‐1β.Conclusion: MSU crystal–recruited monocytes differentiate into proinflammatory M1‐like macrophages in vivo. This proinflammatory macrophage phenotype is likely to play a key role in perpetuating inflammation in gouty arthritis in the presence of ongoing deposition of fresh MSU crystals.

Endothelial dysfunction is a central pathomechanism in diabetes-associated complications. We hypothesized a pathogenic role in this dysfunction of cathepsin S (Cat-S), a cysteine protease that degrades elastic fibers and activates the protease-activated receptor-2 (PAR2) on endothelial cells. We found that injection of mice with recombinant Cat-S induced albuminuria and glomerular endothelial cell injury in a PAR2-dependent manner. In vivo microscopy confirmed a role for intrinsic Cat-S/PAR2 in ischemia–induced microvascular permeability. In vitro transcriptome analysis and experiments using siRNA or specific Cat-S and PAR2 antagonists revealed that Cat-S specifically impaired the integrity and barrier function of glomerular endothelial cells selectively through PAR2. In human and mouse type 2 diabetic nephropathy, only CD68⁺ intrarenal monocytes expressed Cat-S mRNA, whereas Cat-S protein was present along endothelial cells and inside proximal tubular epithelial cells also. In contrast, the cysteine protease inhibitor cystatin C was expressed only in tubules. Delayed treatment of type 2 diabetic db/db mice with Cat-S or PAR2 inhibitors attenuated albuminuria and glomerulosclerosis (indicators of diabetic nephropathy) and attenuated albumin leakage into the retina and other structural markers of diabetic retinopathy. These data identify Cat-S as a monocyte/macrophage–derived circulating PAR2 agonist and mediator of endothelial dysfunction–related microvascular diabetes complications. Thus, Cat-S or PAR2 inhibition might be a novel strategy to prevent microvascular disease in diabetes and other diseases.