Cyclosporine A Impairs Nucleotide Binding Oligomerization Domain (Nod1)-Mediated Innate Antibacterial Renal Defenses in Mice and Human Transplant Recipients
Figure 4
CsA impairs Tlr4-mediated chemokine production in renal medullary collecting duct cells.
(A) Illustration showing UPEC (in blue) adhering to the luminal surface of cells from collecting duct sections (arrows), identified by the AQP-2 positive staining of the collecting duct principal cells (in dark red). Bar = 20 µm. (B) Identification by reverse transcription PCR of amplified products of Tlr4 (311 bp), Nod1 (525 bp), and Nod2 (507 bp) mRNAs in a confluent culture of medullary collecting ducts (MCDs) dissected from the kidney of a WT mouse. (C) CXCL2 production in cultured WT MCD cells incubated with or without LPS, FK156, or MDP for 6 h at 37°C. (D) Relative levels of Tlr4, Nod1 and Nod2 mRNAs measured by quantitative real time PCR in confluent cultures of WT MCD cells incubated with various concentrations of CsA for 48 h and compared to untreated MCD cells. (E) Immunoblot analysis of Tlr4 and Nod1, and the corresponding β-actin in WT MCD cells incubated with or without various concentrations of CsA for 48 h. (F) CXCL2 production in cultured MCD cells dissected from WT, Tlr4−/−, Nod1−/−, or Nod2−/− kidneys incubated with or without CsA for 48 h, then with or without 104 UPEC (upper panel, n = 3–5 determinations from 3 independent experiments) or 10 ng/ml LPS (lower panel, values from 4 separate cultures of MCD cells dissected from a single mouse in each group) for 3 h or 6 h, respectively. Values are presented as mean ± SE. *, p<0.05 (C, E, Mann-Whitney test; D, Two-tailed, unpaired Student's t test).