Fig. 4: KIM1 binds DR5 and activates its downstream caspase cascade.

a Co-IP of KIM1 and DR5 in HK-2 cells transfected with the plasmids indicated. The Co-IP assay was performed using anti-HA or respective IgG, while IgG served as the negative control. For IP and lysate groups, HA-tagged KIM1 was detected with anti-HA antibody, and Flag-DR5 was detected using anti-DR5 antibody. b Co-IP of KIM1 and DR5 with/without 24 h cisplatin (Cis) in HK-2 cells. The Co-IP assay used anti-KIM1 or respective IgG. For IP and lysate groups, KIM1 was detected with anti-KIM1 antibody, and DR5 was detected using anti-DR5 antibody. a, b Each experiment was repeated at least three times independently with similar results obtained. c Schematic diagram for FRET assay design. The excitation/emission wavelength of CFP and YFP are 435/485 nm and 485/525 nm respectively. Emission of CFP excites YFP that causes FRET detected at 525 nm; FRET signal is detectable when KIM1-CFP binds DR5-YFP ( < 10 nm). d, e Quantitative FRET signal between KIM1-CFP and DR5-YFP with/without 24 h cisplatin using fluorescence scan (d), and wavelength scan (e). d CFP, pRK-5′Flag-CFP; YFP, pRK-5′Flag-YFP; KIM1-CFP, pRK-5′Flag-KIM1-CFP; DR5-YFP, pRK-5′Flag-KIM1-YFP; n = 3 biological samples per group, each experiment was repeated at least three times independently with similar results obtained. e Each experiment was repeated at least three times independently with similar results obtained. f Representative images of immunohistochemical staining and quantitative results of DR5 on mouse renal sections at Day 3 after cisplatin injury or unilateral renal ischemia-reperfusion injury (uIRI). Scale bar, 50 μm. n = 4 mice per group. g, h The effects of KIM1 overexpression (g) and knockout (h) on activation of DR5 downstream caspase cascade after 24 h cisplatin injury in HK-2 cells. KIM1 KO, lenti-CRISPR/Cas9-based KIM1 knockout. g, h Each experiment was repeated at least three times independently with similar results obtained. i, j Representative images of KIM1 and DR5 staining in HK-2 cells with/without 24 h cisplatin (i) and mouse renal sections at Day 3 after cisplatin injury (j). Scale bars, 50 μm. i n = 3 biological samples per group; j n = 4 mice per group. k–m MTT assay (k), mRNA levels of apoptotic molecules (l), and DR5 downstream caspase cascade (m) in HK-2 cells, with KIM1 overexpression and/or DR5 knockdown, with/without 24 h cisplatin. k n = 5 biological samples per group; l Vec, n = 3 biological samples per group. Each experiment was repeated at least three times independently with similar results obtained. m Each experiment was repeated at least three times independently with similar results obtained. pRK-5′Flag; KIM1, pRK-5′Flag-KIM1; pSuper, pSuper backbone; ShDR5, pSuper-ShDR5; CT, without cisplatin treatment; data shown as mean ± SD. Two-tailed unpaired Student’s t-test was used for two experimental groups, and one-way ANOVA for multiple experimental groups without adjustment. ^*P < 0.05; ^**P < 0.01; ns no significance. Exact P values are provided in Source Data.