Figure 2.

Chrdl1 is expressed in the medullary proximal tubule of the nephron. (A) RT-PCR for Chrdl1 was performed on cDNA from cells representing distal tubule (MDCK), proximal tubule (HK-2), and collecting duct (mIMCD-3). Only the HK-2 proximal tubule cell line expresses Chrdl1. The PCR primers were designed to amplify within a single exon, allowing genomic DNA to serve as a positive control. The template for the negative control was synthesized using DNAsed RNA in a reverse transcription reaction in the absence of reverse transcriptase (No RT). (B) Chrdl1 is shown by in situ hybridization to be expressed in the outer medulla (M) but not in the papilla (P) or the cortex (C) (first panel); boxed region enlarged in the second panel. This expression pattern overlaps with the expression of the proximal tubule marker Lotus Tetragonolobus lectin but not with markers for distal tubule (E-cadherin), collecting duct (Dolichos Bifloris Agglutinin lectin [DBA]), or thick ascending limb (Tamm-Horsfall antigen). (C) Consistent with quantitative PCR data (Figure 1D), in situ hybridization for Chrdl1 shows that expression is reduced 24 h after ischemic injury and returns to approximately normal levels by 20 d after injury. The high-magnification insets show signal in tubular epithelial cells in the sham and at 20 d after ischemia, whereas at 24 h after ischemia, background signal results from nonspecific binding to cellular debris in lumenal protein casts (inset and Supplemental Figure S1). A control tested with a sense probe showed no specific staining (first panel).