Interleukin-1 Receptor Activation Aggravates Autosomal Dominant Polycystic Kidney Disease By Modulating Necroptosis

Objective:Autosomal dominant polycystic kidney disease(ADPKD)is the most common inherited kidney disease and is a major cause of end stage kidney disease worldwide.ADPKD is caused by PKD1 or PKD2 mutations,but the mechanisms through which mutations in PKD genes lead to the neoplastic cyst growth in kidneys are still not fully understood.Beyond the well-documented intracellular signaling which contributes to the onset of the disease,the inflammatory microenvironment in the kidney,including recruitment of immune cells and generation of inflammatory cytokines,plays an important role in PKD progression.The interleukin-1 isoforms,IL-1 alpha and IL-1 beta,are the prototypical cytokines from the innate immune system.Both are produced primarily by stimulated monocytes and macrophages,but to a lesser degree by several other cell types,including neutrophils,epithelial and endothelial cells.Previous study reported that in ADPKD patients,urinary excretion of IL-1 receptor antagonist is decreased.However,to our knowledge,the effects of IL-1 signaling on PKD progression have not been directly investigated.Here we use a unique model with deletion of pkd1 restricted to the kidney to establish that activating the receptor for IL-1 worsens the severity of ADPKD,at least in part by attenuating regulated necrosis in the kidney.Methods:1.We established a new IL-1 R;pkd1 double knock out model and analyzed the IL-1 alpha and IL-1 beta level in the double knock out kidneys compared with pkd1 single knock out kidneys.We also extracted the epithelial cells of the kidneys in both group and analyzed the IL-1 alpha and IL-1 beta level after culturing ex vivo for 3 days.2.With the established new IL-1 R;pkd1 double knock out model we analyzed the disease progression.The survival analyses including kidney weight / body weight and cystic index were also performed.3.With the above-mentioned steps,we understood the relationship between IL-1 and ADPKD progression.We further determined the underlying mechenisim.We examined the downstream inflammatory cytokins levels when the IL-1 R were knocked out.4.In the step 3,we revealed that the TNF-alpha level in double knock out group kidneys was upregulated.Since the TNF-alpha is a classic mediator of apoptosis and necroptosis,we further examined the apoptosis and necroptosis markers including RIP3 and MLKL mRNA level and protein phosphorylation level.5.To explore the regulation of necroptosis in ADPKD,multiple ADPKD models were examined.First,we collected human ADPKD surgical specimen from the recipients of kidney transplantation with native nephrectomy patients.Control kidney tissues were from healthy part of renal cell carcinoma surgical specimen.Necroptosis markers including RIP3 and MLKL mRNA level and protein phosphorylation level were analyzed.6.We further explored that whether necroptosis plays a role in cysts progression.We treated the pkd1 flox / flox Ksp Cre + mice with Nec-1 and vehicle and examined the severity of ADPKD at day 7 after birth.The kidney weight body weight ratio and cystic index were tested.Results:1.We employed a mouse model in which pkd1 gene deletion is restricted to the kidney.We measured mRNA expression of the macrophage cytokine MCP-1,known to be important to PKD pathogenesis,and the prototypical macrophage cytokine IL-1 in KPKD1 kidneys.We found that MCP-1 and both isoforms of IL-1 were upregulated in ADPKD tissues.However,when the KPKD1 epithelial cells were isolated and cultured,IL-1 alpha and IL-1 beta levels were downregulated,indicating that the microenvironment in cystic kidneys drives IL-1 upregulation.2.To directly test the role of IL-1 signaling in ADPKD progression,we established KPKD1 mice with genetic deletion of the receptor for IL-1(IL-1R KO KPKD1)and analyzed disease progression in comparison with KPD1 mice expressing normal IL-1R levels in all tissues(IL1R WT KPKD1).Compared with the IL1 R WT KPKD1 cohort,the IL1 R KO KPKD1 animals had a mild prolongation in survival.We also measured kidney weight / body weight ratios at day 8 after birth.The IL1 R KO KPKD1 mice had reduced kidney to body weight ratios and lower cystic indices compared with IL1 R WT controls.3.As IL-1 signaling is important in directing the innate immune response,we posited that IL-1 R activation aggravates PKD progression via effects on inflammatory signals within the kidney.We therefore profiled the expression of several inflammatory cytokines within the IL1 R WT and KO KPDK1 kidneys at day 8.Surprisingly,we could not detect any differences in gene expression for inflammatory cytokines with the exception of TNFalpha.TNF-alpha mRNA levels were significantly higher in the IL1 R KO cohort compared to IL1 R WT controls.This finding was confirmed by quantitating TNF protein levels via Western analysis.Thus,although IL-1 R signaling is typically pro-inflammatory in most diseases,IL-1 R activation in KPKD1 is associated with renal TNF suppression as has been reported in certain contexts.4.We then explored a possible mechanism through which TNF-α could afford protection in our model.As TNF-α is a classic inducer of both apoptosis and necroptosis,we tested markers of apoptosis and regulated necrosis in our experimental animals.We found that mRNA levels for MLKL were increased in IL1 R KO PKD1 kidneys,indicating enhanced susceptibility to regulated necrosis.whereas markers of apoptosis caspase 3 and caspase 8 were not.Thus,by reducing the net survival of proliferating epithelial cells,enhanced regulated necrosis mediated via TNF could contribute to the amelioration in PKD1 seen with IL-1R deficiency.5.We examined markers of regulated necrosis in human ADPKD tissues.To this end,we collected human ADPKD surgical specimens from the recipients of kidney transplantation who underwent native nephrectomy.Control kidney tissues were from healthy parts of renal cell carcinoma surgical specimens.Western analysis indicated that phosphorylation of RIP3 and MLKL is upregulated in human ADPKD tissue consistent with increased necroptosis.Immunohistochemistry on a human ADPKD kidney specimen detected p-MLKL staining in tubular epithelial cells lining the cysts.This pattern recapitulated that which we saw in the KPKD1 mice at day 8 after birth.Compared with non-PKD controls,mRNA levels of RIP3 and MLKL were upregulated in the KPKD1 kidneys,and phosphorylation levels of RIP3 and MLKL were also increased.In primary epithelial cells isolated from KPKD1 mice,RIP3 and MLKL mRNA levels were upregulated by only 1.5 fold compared to controls,rather than the >10 and >2 fold,respectively.seen in vivo,suggesting that the inflammatory milieu in vivo may drive TNF-dependent regulated necrosis in PKD.Indeed,we found that TNF-α mRNA levels were upregulated in KPKD1 tissues and primary KPDK1 tubular epithelial cells.6.To directly assess whether necroptosis plays a role in PKD progression,we treated the KPKD1 mice with an inhibitor of necroptosis or vehicle and examined the severity of ADPKD at day 7 after birth.In the Nec-1-treated animals,the kidney weight body weight ratios were significantly higher than in vehicle-treated controls,and the cystic index trended higher in Nec-1 group.These data suggest that regulated necrosis plays a protective role in PKD by limiting cyst expansion.Conclusions:We find in an ADPKD model that IL1 R activation leads to augmented cystic indices and kidney weight due in part to suppression of regulated necrosis.We posit that,uniquely in PKD,IL1 R stimulation limits necroptosis by suppressing TNF in the kidney.In that case,IL1 R activation aggravates PKD by mitigating rather than augmenting local inflammation with consequent reductions in tubular necrosis.The induction of necroptosis that we detect in multiple ADPKD models may therefore represent a protective counterbalance to increased epithelial cell proliferation.Thus,blunting IL1 R signaling and stimulating necroptosis warrant further testing as novel therapeutic interventions in ADPKD.However,the application of IL-1R antagonists such as anakinra must be approached with caution in human PKD patients given that the downstream effects of TNF on PKD progression appear to be context-specific.