Abstract

Aims: Activation of the nod-like receptor protein-3 (NLRP3) inflammasome is associated with chronic inflammation across multiple disease states. In diabetic retinopathy, increased connexin43 (Cx43)-hemichannel activity is linked to downstream NLRP3-induced inflammation and fibrosis. In the kidney, this relationship is yet to be reported.
Methods: Transcriptomic analysis of renal datasets on Nephroseq evaluated NLRP3 mRNA expression and correlation to glomerular filtration rate (GFR) in biopsies obtained from people with diabetic nephropathy. Immortalised human kidney (HK2) cells were cultured with high glucose (25mM) conditioned media ± Cx43-hemichannel blocker, Tonabersat (100µM); Caspase-1 inhibitor, YVAD-cmk (10µg/ml); or NLRP3 inhibitor, CY09 (20µM) for 48hr. Immunoblotting assessed protein expression, with carboxyfluorescein and CaspaseGlo-1 assays assessing hemichannel-mediated dye uptake and NLRP3-inflammasome activation, respectively.
Results: Conditioned media upregulated Cx43-hemichannel-mediated dye uptake by 31.7% (P<0.05, n=4) an effect reduced by 50.5±6.9% (P<0.001, n=4) when co-incubated with Tonabersat. Similarly, Tonabersat reduced conditioned media-induced increases in collagen-I and collagen-IV expression by 30.5±6.8% (P<0.001, n=4) and 31.8±6.5% (P<0.01, n=5) respectively. NLRP3 mRNA expression increased by 1.15±0.4 in individuals with diabetic nephropathy (P<0.05, n=10), and correlated with a declining GFR (P<0.01, n=22). Caspase-1 inhibition dampened a conditioned media-induced increase in collagen-IV expression (34.2±9.8%; P<0.05, n=5), whilst inhibition of NLRP3 reduced conditioned media-induced increases in collagen-I expression by 23.8±6.8% (P<0.05, n=5). Lastly, Tonabersat reduced the conditioned media-induced increase in Caspase-1 activity by 24.1±0.6% (P<0.001, n=5).
Conclusion: Tonabersat blocks glucose-induced Cx43-hemichannel-mediated NLRP3-inflammasome activation and downstream extracellular matrix protein changes. Further research is required to determine the therapeutic efficacy of these compounds in vivo.