As shown in Figure 1, A and B, after 7 days in culture, PTCs from both WT mice and KIM-1mucin mice showed strong antiCKIM-1 Ab staining with an Ab directed against the Ig domain of the molecule. apoptotic cells protects the kidney after acute injury by downregulating innate immunity and inflammation. gene with a promoterCdriven neomycin-resistance cassette on a C57BL/6 genetic background. This mutant mouse generated KIM-1 proteins with the loss of the mucin domain, encoded by exon 3 (KIM-1mucin) (17). KIM-1mucin mice were found to have similar mRNA expression levels of the closest of the TIM genes in the locus (17). We confirmed that other TIMs, TIM-3 and TIM-4, are not differentially regulated in KIM-1mucin tubular cells at the protein level or in B cells compared with KIM-1 WT cells (data not shown). KIM-1mucin interaction with TIM-4 is similar to that in WT KIM-1, indicating that while the KIM-1mucin mutant was deficient in phagocytosis, it retained other KIM-1 functions (17) and did not result in modification of other TIMs tested. To evaluate whether the deletion of the mucin domain in JAK3 this mouse affects KIM-1 expression and function, we incubated fluorescently tagged apoptotic cells with primary cultured PTCs obtained from WT and KIM-1mucin mice and LLC-PK1 cell lines transfected with WT KIM-1 or KIM-1mucin. As shown in Figure 1, A and B, after 7 days in culture, PTCs from both WT mice and KIM-1mucin mice showed strong antiCKIM-1 Ab staining with an Ab directed against the Ig domain of the molecule. The KIM-1mucin PTCs had markedly decreased phagocytosis of apoptotic cells compared with that in WT PTCs (20.2 5.8% SD MK-4827 (Niraparib) vs. 81.3 7.2% of cells containing apoptotic cells, 0.001). A similar reduction in phagocytosis was observed in LLC-PK1 cells transfected with when compared with cells transfected with WT (15.6 5.6% vs. 91.1 10.4%, 0.001). Open in a separate window Figure 1 Decreased phagocytic function of KIM-1mucin in PTCs.(A) AntiCKIM-1 immunostaining (red) in primary PTCs from both WT KIM-1 and KIM-1mucin mice (left panel) and LLC-PK1 cells transfected with empty vector (control), KIM-1 or KIM-1mucin (right panel) exposed to apoptotic lymphocytes (green). Scale bar: 20 m. MK-4827 (Niraparib) (B) The percentage of PTCs that internalized apoptotic lymphocytes was reduced in the KIM-1mucin PTCs (= 3). * 0.01; # 0.001. (C) Representative time course from 5 experiments of the uptake and acidification (red) of apoptotic cells (green) by KIM-1Cexpressing LLC-PK1 cells. Scale bar: 30 m. (D) Representative images of cell outgrowth from coverslips in CHO cells expressing empty vector or KIM-1 (images are representative of 3 experiments). (E) TUNEL+ tubular cells in I/R kidneys from WT KIM-1 and KIM-1mucin mice (= 5 mice/time point/group).* 0.01 vs. sham; # 0.01 vs. WT KIM-1. (F) Colocalization of KIM-1 and TUNEL+ cells (arrows in the left panel show KIM-1Cbinding apoptotic bodies). Scale bar: 50 m. (G) Quantification of TUNEL+ cells in WT KIM-1 and KIM-1mucin mice 24 hours after I/R injury plus vehicle or I/R injury plus Baf (= 3). * 0.05; ** 0.01; *** 0.001. (H) Representative images of apoptotic TUNEL+ cells in WT KIM-1 mice treated with Baf. Scale bar: 100 m. (I) Quantification of mRNA expression in postCI/R injury KIM-1 and KIM-1mucin kidneys (= 3). * 0.05; *** 0.001. (J) Quantification of luminal cellular debris in postCI/R injury WT KIM-1 and KIM-1mucin mice (= 3). ** 0.01. (K) MK-4827 (Niraparib) Representative images of luminal debris.