Hyperoxaluria Requires TNF Receptors to Initiate Crystal Adhesion and Kidney Stone Disease.

Intrarenal crystals trigger inflammation and renal cell necroptosis, processes that involve TNF receptor (TNFR) signaling. Here, we tested the hypothesis that TNFRs also have a direct role in tubular crystal deposition and progression of hyperoxaluria-related CKD. Immunohistochemical analysis revealed upregulated tubular expression of TNFR1 and TNFR2 in human and murine kidneys with calcium oxalate (CaOx) nephrocalcinosis-related CKD compared with controls. Western blot and mRNA expression analyses in mice yielded consistent data. When fed an oxalate-rich diet, wild-type mice developed progressive CKD, whereas Tnfr1-, Tnfr2-, and Tnfr1/2-deficient mice did not. Despite identical levels of hyperoxaluria, Tnfr1-, Tnfr2-, and Tnfr1/2-deficient mice also lacked the intrarenal CaOx deposition and tubular damage observed in wild-type mice. Inhibition of TNFR signaling prevented the induced expression of the crystal adhesion molecules, CD44 and annexin II, in tubular epithelial cells in vitro and in vivo, and treatment with the small molecule TNFR inhibitor R-7050 partially protected hyperoxaluric mice from nephrocalcinosis and CKD. We conclude that TNFR signaling is essential for CaOx crystal adhesion to the luminal membrane of renal tubules as a fundamental initiating mechanism of oxalate nephropathy. Furthermore, therapeutic blockade of TNFR might delay progressive forms of nephrocalcinosis in oxalate nephropathy, such as primary hyperoxaluria.

Oxalate-induced chronic kidney disease with its uremic and cardiovascular complications in C57BL/6 mice.

Chronic kidney disease (CKD) research is limited by the lack of convenient inducible models mimicking human CKD and its complications in experimental animals. We demonstrate that a soluble oxalate-rich diet induces stable stages of CKD in male and female C57BL/6 mice. Renal histology is characterized by tubular damage, remnant atubular glomeruli, interstitial inflammation, and fibrosis, with the extent of tissue involvement depending on the duration of oxalate feeding. Expression profiling of markers and magnetic resonance imaging findings established to reflect inflammation and fibrosis parallel the histological changes. Within 3 wk, the mice reproducibly develop normochromic anemia, metabolic acidosis, hyperkalemia, FGF23 activation, hyperphosphatemia, and hyperparathyroidism. In addition, the model is characterized by profound arterial hypertension as well as cardiac fibrosis that persist following the switch to a control diet. Together, this new model of inducible CKD overcomes a number of previous experimental limitations and should serve useful in research related to CKD and its complications.

Cathepsin S inhibition combines control of systemic and peripheral pathomechanisms of autoimmune tissue injury.

Cathepsin(Cat)-S processing of the invariant chain-MHC-II complex inside antigen presenting cells is a central pathomechanism of autoimmune-diseases. Additionally, Cat-S is released by activated-myeloid cells and was recently described to activate protease-activated-receptor-(PAR)-2 in extracellular compartments. We hypothesized that Cat-S blockade targets both mechanisms and elicits synergistic therapeutic effects on autoimmune tissue injury. MRL-(Fas)lpr mice with spontaneous autoimmune tissue injury were treated with different doses of Cat-S inhibitor RO5459072, mycophenolate mofetil or vehicle. Further, female MRL-(Fas)lpr mice were injected with recombinant Cat-S with/without concomitant Cat-S or PAR-2 blockade. Cat-S blockade dose-dependently reversed aberrant systemic autoimmunity, e.g. plasma cytokines, activation of myeloid cells and hypergammaglobulinemia. Especially IgG autoantibody production was suppressed. Of note (MHC-II-independent) IgM were unaffected by Cat-S blockade while they were suppressed by MMF. Cat-S blockade dose-dependently suppressed immune-complex glomerulonephritis together with a profound and early effect on proteinuria, which was not shared by MMF. In fact, intravenous Cat-S injection induced severe glomerular endothelial injury and albuminuria, which was entirely prevented by Cat-S or PAR-2 blockade. In-vitro studies confirm that Cat-S induces endothelial activation and injury via PAR-2. Therapeutic Cat-S blockade suppresses systemic and peripheral pathomechanisms of autoimmune tissue injury, hence, Cat-S is a promising therapeutic target in lupus nephritis.

Cytotoxicity of crystals involves RIPK3-MLKL-mediated necroptosis.

Crystals cause injury in numerous disorders, and induce inflammation via the NLRP3 inflammasome, however, it remains unclear how crystals induce cell death. Here we report that crystals of calcium oxalate, monosodium urate, calcium pyrophosphate dihydrate and cystine trigger caspase-independent cell death in five different cell types, which is blocked by necrostatin-1. RNA interference for receptor-interacting protein kinase 3 (RIPK3) or mixed lineage kinase domain like (MLKL), two core proteins of the necroptosis pathway, blocks crystal cytotoxicity. Consistent with this, deficiency of RIPK3 or MLKL prevents oxalate crystal-induced acute kidney injury. The related tissue inflammation drives TNF-α-related necroptosis. Also in human oxalate crystal-related acute kidney injury, dying tubular cells stain positive for phosphorylated MLKL. Furthermore, necrostatin-1 and necrosulfonamide, an inhibitor for human MLKL suppress crystal-induced cell death in human renal progenitor cells. Together, TNF-α/TNFR1, RIPK1, RIPK3 and MLKL are molecular targets to limit crystal-induced cytotoxicity, tissue injury and organ failure.