Chronic Kidney Dysfunction Induced By Jak2V617F-Mediated Mesangiolysis

Cytokine profiles of JAK2-mutated cells depend on STAT signaling and can partially overlap with those of non-malignant ‘bystanders’ or those produced by cells carrying other driver mutations. Secretion of pro-inflammatory cytokines and chemokines causes tissue remodeling and the development of fibrosis. Most recently, analysis from 1420 MPN patients reported up to 29% of MPN patients with severe chronic kidney dysfunction (eGFR <60 mL/min/1.73m²). In the general European population, the prevalence of severe CKD varies between 3.3% in Norway and 17.3% in northeast Germany.

To assess for pathophysiologic correlates of kidney dysfunction, we used established mouse models of Jak2-mutated hematopoiesis and MPN (Jak2VF;Vav-Cre). Jak2VF animals display morphologic kidney alterations with severe glomerular hypertrophy (difference of glomerular tuft area 840.7+/-64.5; p<0.0001) and glomerular capillary dilatation (WT n=6; 1.905 versus VF n=7; 61.22; p=0.0012). Electron microscopy of the glomerular filtration barrier showed an unchanged morphology of glomerular endothelial cells with regular fenestrations, a normal three-layered appearance of the glomerular basement membrane, and a typical podocyte foot process morphology as quantified by unchanged filtration slit density using super-resolution microscopy. Formalin-fixed paraffin embedded kidney sections of Jak2VF and WT animals were immunofluorescence-processed and large tissue areas were acquired with confocal laser scanning microscopy. Glomeruli, single cells, and the mesangium was virtually microdissected using custom-trained Deep-Learning networks. Single-cell expression data was retrieved for every single cell on the slide. In VF animals, the mesangial areas contained significantly more fibronectin-dominant extracellular matrix. Jak2VF animals had significantly fewer mesangial cells (MCs) as indicated by intraglomerular ITGA8-positive cell counts (MCs per 1000um2 glomerular area WT n=34; 4.86 vs. VF n=30; 3.81; p=0.0021). Mesangial cells were activated as demonstrated by Ki67-expression in ITGA8-positive cells indicating MC-proliferation (2.12 vs. 4.16%; p=0.0209) and α-SMA positivity. Cre-mediated induction of the Jak2VF in kidney cells as a potential confounder of the pathologic phenotype was excluded. VF animals showed increased serum creatinine and albuminuria (p=0.04). Pharmacologic Jak-inhibitor treatment with oral Ruxolitinib for 4 weeks resulted in potent reduction of pro-inflammatory cytokines and rescue of the glomerular phenotype and kidney dysfunction.

To assess for inflammatory molecules and pathways contributing to the observed kidney dysfunction, global proteomic analyses on murine glomeruli was performed. Out of 4954 proteins 479 were differentially regulated in VF compared to WT animals. Consistent with the structural data, functional enrichment analysis revealed accumulation of extracellular matrix proteins (p=1.55*10^-8) such as proteins of mesangial or glomerular basement membrane origin like fibronectin, nidogen, and multiple laminins (Laminin alpha 1, 2, 4; beta 2, and gamma 1). In line with the pro-sclerotic phenotype indicating MC activation, highly significant enrichment of actin-cytoskeleton-regulating proteins (p=2.16*10^-46) and pro-inflammatory signaling pathways including necroptosis pathways could be detected. Transcriptome analysis on bulk MCs indicated enrichment of cytokine-related signaling, oxidative phosphorylation and cellular metabolism. Experiments integrating the activated signaling network of MCs as well as analysis on proteomic and transcriptional changes on a single cell level are currently under way.

Together, kidneys of mice with Jak2-mutated hematopoiesis show mesangiolysis and chronic kidney dysfunction (CKD) due to activation of inflammatory cell signaling.

Perner:Syndax: Other: Travel support. Heidel:BMS/Celgene, Novartis, CTI: Research Funding; BMS/Celgene, AOP, Novartis, CTI, Janssen, Abbvie, GSK, Merck, Kartos, Telios: Consultancy.