Stefanie Steiger

Cathepsin S Cleavage of Protease-Activated Receptor-2 on Endothelial Cells Promotes Microvascular Diabetes Complications

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.

Particles of different sizes and shapes induce neutrophil necroptosis followed by the release of neutrophil extracellular trap-like chromatin

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).

Sodium glucose transporter‐2 inhibition has no renoprotective effects on non‐diabetic chronic kidney disease

Sodium glucose transporter (SGLT)‐2 inhibition has renoprotective effects in diabetic kidney disease. Whether similar effects can be achieved also in non‐diabetic kidney disease is speculative. Chronic kidney disease was induced in C57BL/6N mice by feeding an oxalate‐rich diet for 14 days, known to induce nephrocalcinosis‐related tubular atrophy and interstitial fibrosis without directly affecting the glomerular compartment. Empagliflozin treatment started from day 0 of oxalate feeding had no effect on the decline of glomerular filtration rate, crystal deposition, blood urea nitrogen or serum creatinine levels on day 7 and 14. Tissue morphometry of tubular injury and kidney mRNA levels of kidney injury molecule‐1 or tissue inhibitor of metalloproteinase‐2 were comparable between empagliflozin‐ and vehicle‐treated mice with oxalate nephropathy on day 7 and 14. Similarly, empagliflozin did not affect markers of interstitial fibrosis, including silver, alpha smooth muscle actin (αSMA) and collagen 1 staining, and mRNA levels of fibronectin‐1, collagen 1α1, fibroblast‐specific protein‐1, and transforming growth factor (TGF)‐β2 on day 7 and 14. Thus, the specific renoprotective mechanisms‐of‐action of SGLT2 inhibition in diabetic kidney disease do not apply to chronic oxalosis, a non‐diabetic form of chronic kidney disease.

Molecular Pathophysiology of Gout

Three contradictory clinical presentations of gout have puzzled clinicians and basic scientists for some time: first, the crescendo of sterile inflammation in acute gouty arthritis; second, its spontaneous resolution, despite monosodium urate (MSU) crystal persistence in the synovium; and third, immune anergy to MSU crystal masses observed in tophaceous or visceral gout. Here, we provide an update on the molecular pathophysiology of these gout manifestations, namely, how MSU crystals can trigger the auto-amplification loop of necroinflammation underlying the crescendo of acute gouty arthritis. We also discuss new findings, such as how aggregating neutrophil extracellular traps (NETs) might drive the resolution of arthritis and how these structures, together with granuloma formation, might support immune anergy, but yet promote tissue damage and remodeling during tophaceous gout.

Phagocytosis of environmental or metabolic crystalline particles induces cytotoxicity by triggering necroptosis across a broad range of particle size and shape

In crystallopathies, crystals or crystalline particles of environmental and metabolic origin deposit within tissues, induce inflammation, injury and cell death and eventually lead to organ-failure. The NLRP3-inflammasome is involved in mediating crystalline particles-induced inflammation, but pathways leading to cell death are still unknown. Here, we have used broad range of intrinsic and extrinsic crystal- or crystalline particle-sizes and shapes, e.g. calcium phosphate, silica, titanium dioxide, cholesterol, calcium oxalate, and monosodium urate. As kidney is commonly affected by crystallopathies, we used human and murine renal tubular cells as a model system. We showed that all of the analysed crystalline particles induce caspase-independent cell death. Deficiency of MLKL, siRNA knockdown of RIPK3, or inhibitors of necroptosis signaling e.g. RIPK-1 inhibitor necrostatin-1s, RIPK3 inhibitor dabrafenib, and MLKL inhibitor necrosulfonamide, partially protected tubular cells from crystalline particles cytotoxicity. Furthermore, we identify phagocytosis of crystalline particles as an upstream event in their cytotoxicity since a phagocytosis inhibitor, cytochalasin D, prevented their cytotoxicity. Taken together, our data confirmed the involvement of necroptosis as one of the pathways leading to cell death in crystallopathies. Our data identified RIPK-1, RIPK3, and MLKL as molecular targets to limit tissue injury and organ failure in crystallopathies.

Anti-Transforming Growth Factor β IgG Elicits a Dual Effect on Calcium Oxalate Crystallization and Progressive Nephrocalcinosis-Related Chronic Kidney Disease

Crystallopathies are a heterogeneous group of diseases caused by intrinsic or environmental microparticles or crystals, promoting tissue inflammation and scarring. Certain proteins interfere with crystal formation and growth, e.g., with intrarenal calcium oxalate (CaOx) crystal formation, a common cause of kidney stone disease or nephrocalcinosis-related chronic kidney disease (CKD). We hypothesized that immunoglobulins can modulate CaOx microcrystal formation and crystal growth and that therefore, biological IgG-based drugs designed to specifically target disease modifying proteins would elicit a dual effect on the outcome of CaOx-related crystallopathies. Indeed, both the anti-transforming growth factor (TGF)β IgG and control IgG1 antibody impaired CaOx crystallization in vitro, and decreased intrarenal CaOx crystal deposition and subsequent CKD in mice on an oxalate-rich diet compared to oxalate-fed control mice. However, the TGFβ-specific IgG antibody showed nephroprotective effects beyond those of control IgG1 and substantially reduced interstitial fibrosis as indicated by magnetic resonance imaging, silver and α-smooth muscle actin staining, RT-qPCR, and flow cytometry for pro-fibrotic macrophages. Suppressing interstitial fibrosis slowed the decline of glomerular filtration rate (GFR) compared to treatment with control IgG1 [slope of m = −8.9 vs. m = −14.5 μl/min/100 g body weight (BW)/day, Δ = 38.3%], an increased GFR at the end of the study (120.4 vs. 42.6 μl/min/100 g BW, Δ = 64.6%), and prolonged end stage renal disease (ESRD)-free renal survival by 10 days (Δ = 38.5%). Delayed onset of anti-TGFβ IgG from day 7 was no longer effective. Our results suggest that biological drugs can elicit dual therapeutic effects on intrinsic crystallopathies, such as anti-TGFβ IgG antibody treatment inhibits CaOx crystallization as well as interstitial fibrosis in nephrocalcinosis-related CKD.

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.

Aristolochic acid I determine the phenotype and activation of macrophages in acute and chronic kidney disease

Acute and chronic kidney injuries are multifactorial traits that involve various risk factors. Experimental animal models are crucial to unravel important aspects of injury and its pathophysiological mechanisms. Translating knowledge obtained from experimental approaches into clinically useful information is difficult; therefore, significant attention needs to be paid to experimental procedures that mimic human disease. Herein, we compared aristolochic acid I (AAI) acute and chronic kidney injury model with unilateral ischemic-reperfusion injury (uIRI), cisplatin (CP)- or folic acid (FA)-induced renal damage. The administration of AAI showed significant changes in serum creatinine and BUN upon CKD. The number of neutrophils and macrophages were highly increased as well as AAI-induced CKD characterized by loss of tubular epithelial cells and fibrosis. The in vitro and in vivo data indicated that macrophages play an important role in the pathogenesis of AA-induced nephropathy (AAN) associated with an excessive macrophage accumulation and an alternative activated macrophage phenotype. Taken together, we conclude that AA-induced injury represents a suitable and relatively easy model to induce acute and chronic kidney injury. Moreover, our data indicate that this model is appropriate and superior to study detailed questions associated with renal macrophage phenotypes.

Author Correction: Particles of different sizes and shapes induce neutrophil necroptosis followed by the release of neutrophil extracellular trap-like chromatin

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

The Long Pentraxin PTX3 Is an Endogenous Inhibitor of Hyperoxaluria-Related Nephrocalcinosis and Chronic Kidney Disease

The long pentraxin 3 (PTX3) exerts a variety of regulatory functions in acute and chronic tissue inflammation. In particular, PTX3 acts as an opsonin for a variety of pathogens and endogenous particles. We hypothesized that PTX3 would exhibit opsonin-like functions toward calcium oxalate crystals, too, and inhibit crystal growth. This process is fundamental in kidney stone disease as well as in hyperoxaluria-related nephrocalcinosis, the paradigmatic cause of chronic kidney disease (CKD) in children with primary hyperoxaluria type I due to genetic defects in oxalate metabolism. Direct effects of PTX3 on calcium oxalate crystals were investigated in chemico by adding recombinant PTX3 to supersaturated calcium and oxalate solutions. PTX3, but not isomolar concentrations of albumin, dose-dependently inhibited crystal growth. In vivo, the PTX3 protein was undetectable in tubular epithelial cells and urine of wild-type mice under physiological conditions. However, its levels increased within 3 weeks of feeding an oxalate-rich diet, an exposure inducing hyperoxaluria-related nephrocalcinosis and CKD in selected mouse strains (male and female C57BL/6N and male Balb/c mice) but not in others (male and female 129SV and CD-1, male and female Balb/c mice). Genetic ablation of ptx3 in nephrocalcinosis un-susceptible B6;129 mice was sufficient to raise the oxalate nephropathy phenotype observed in susceptible strains. We conclude that PTX3 is an endogenous inhibitor of calcium oxalate crystal growth. This mechanism limits hyperoxaluria-related nephrocalcinosis, e.g., in primary or secondary hyperoxaluria, and potentially also in the more prevalent kidney stone disease.