Serum-induced cytotoxicity of patients with sepsis in HEK cell culture - Preliminary results for the development of a rapid AKI diagnostic test

HUESPE, LUIS; MELANA COLAVITA, JUAN PABLO; BARNES, TAMARA; AGUIRRE, MARÍA VICTORIA; TODARO, JUAN SANTIAGO; AUCHTER, MÓNICA; RODRÍGUEZ, JUAN PABLO

Rio de Janeiro

Congreso; International Sepsis Forum; 2019

Sepsis is a potentially fatal organ dysfunction caused by a dysregulated host response to infection. Acute kidney Injury is the most frequent complication in patients with septic shock and we hypothesize that the damage is due to toxins related to infection, which is why we used in vitro cultures with HEK -293 cell.All patients with Sepsis-3 were included in the study (December 2017-December 2018.)Patients with ARF, we used sepsis as an initial insult and the KDIGO criteria to determine AKI. Blood samples from patients Sepsis.3 (n 15) were obtained with the prior informed consent and the bioethical standards of the Hospital Committee. The biochemical tests were analyzed in quadruplicate and in vitro test using HEK-293 cell line, in a humid atmosphere with 5% C02 and 37°C. Cell monolayer was grown up to 60% confluence using RPMI with 5% fetal bovine serum. Once the monolayers were obtained, culture medium was removed and washed 3 times with 1X PBS. Inverted optical microscopy was used at different magnifications 40-400XFrom all patients (n=385) admitted to ICU, 54 p with Sepsis-3. Average data showed: Age 44.2 years (20-81y), SOFA 7.3 (2-14), p with KDIGO criteria 22 of which 5 patients (18%) required hemodialysis, 18 patients (33.3%) had an Nt-proBNP> 1000 and mortality was 40%. 15 p of them were analyzed in vitro test using HEK-293 cell line and subsequently, cells were treated as the following: A HEK-293 culture with incomplete RPMI medium (control without fetal bovine serum), B HEK-293 cell treated with non-septic serum, C HEK-293 cell treated with septic serum in concentrations (0.5%, 2.5%, 5%, 10% and 15%). Cell cultures incubated for 24 hs, and morphological analysis for monolayers to evaluate changes compatible with cellular death. Control cell grown in incomplete RPMI medium cells treated with non-septic serum did not show significant differences between them and no cytoplasmatic or nuclear damage was observed. However, treated cells showed cell damage in direct relation to the amount of septic serum added to the culture. Acute Kidney Injury and non-recovery at seven days after the initial insult in patients with sepsis and septic shock increases the mortality. This group demonstrate in vitro damage caused by cytoxin from the infectious focus. This result lead us to think that the development of a rapid technique to diagnose AKI with a cell culture laboratory.