IDF21-0092 Influence of the NLRP3 inflammasome in podocytes on the pathogenesis of diabetes dependent hyperfiltration-induced CKD

Abstract

Background: Diabetic nephropathy (DN) is one of the most severe complications of Type 1 diabetes mellitus (T1) that is accounting for about 40% of end-stage renal disease (ESRD). The pathology is seen to correlate with inflammation. Amongst others, pattern recognition receptors are important mediators of disease progression, e.g. nucleotide-binding oligomerization domain receptors, together with inflammasomes. Especially the Nod-like receptor protein 3 (NLRP3) inflammasome multiprotein complex seems to be a key mediator in DN. The assembly of this promotes the maturation of proinflammatory cytokines (canonical pathway) such as IL-1β and IL-18. It is assumed that there are non-canonical pathways, that trigger innate inflammatory response. Whereas the role of NLRP3 in specialized immune cells is well understood, its contribution to processes of disease and homeostasis in renal parenchymal cells is controversially discussed in literature. Aim: With this project we expect to get a better understanding of the role of canonical and non-canonical NLRP3 signaling in podocytes. Furthermore, we aim to investigate if an inhibition of the NLRP3 signaling may lead to an ameliorated or even absent hyperfiltration-induced CKD. Method: To verify, if the NLRP3 inflammasome is involved in canonical, proinflammatory signaling, primary human podocytes derived from renal progenitor cells were stimulated in vitro. By using different concentrations of LPS, ATP, glucose and nigericin, metabolic stress was imitated. IL-1β and IL-18 ELISAs were used to verify canonical pathway activation. We further introduced podocyte-specific NLRP3 inflammasome modulations and Streptozotocin dependent diabetes in mice with a NLRP3 overactive variant (Nlrp3A350V fl/wt) to investigate, if NLRP3 overactivation leads to an aggravated phenotype of hyperfiltration-induced CKD. By using pharmaceuticals such as Oridonine and beta-Hydroxybutyrate we examined, if the inhibition of NLRP3 signaling in C57BL/6J wildtype mice with diabetes leads to an ameliorated outcome. To investigate if it is possible to reverse diabetes dependent hyperfiltration-induced CKD in NLRP3 overactive mice, we treated them with beta-Hydroxybutyrate. The primary endpoint of all in vivo experiments was defined as urinary albumin-creatinine-ratio. Furthermore, relevant histopathological alterations were observed. Results: Our data suggest that human derived podocytes are not susceptible for metabolic stress dependent canonical NLRP3 inflammasome signaling compared with THP-1 + PMA cells. Even though mice presented with severe diabetes and polyuria, we did not observe a significant difference in mice with NLRP3 overactivation regarding urinary albumin excretion and histological changes. So far, also the treatment with NLRP3 inhibitors did not ameliorate hyperfiltration-induced CKD. Discussion: Investigation of the NLRP3 functionality in podocytes provided a deeper insight and better understanding of the pathogenesis of diabetes dependent hyperfiltration-induced CKD. Even though it might have been possible that the specific inhibition of the inflammasome associated proteins should have synergistic effects in inflammation in ESRD and might be a future therapeutic target beyond IL-1β inhibition, our data do not support this hypothesis.