Differential expression of pancreatic protein and chemosensing receptor mRNAs in NKCC1-null intestine

Abstract

Aim: To investigate the intestinal functions of the NKCC1 Na(+)-K(+)-2Cl cotransporter (SLC12a2 gene), differential mRNA expression changes in NKCC1-null intestine were analyzed.

Methods: Microarray analysis of mRNA from intestines of adult wild-type mice and gene-targeted NKCC1-null mice (n = 6 of each genotype) was performed to identify patterns of differential gene expression changes. Differential expression patterns were further examined by Gene Ontology analysis using the online Gorilla program, and expression changes of selected genes were verified using northern blot analysis and quantitative real time-polymerase chain reaction. Histological staining and immunofluorescence were performed to identify cell types in which upregulated pancreatic digestive enzymes were expressed.

Results: Genes typically associated with pancreatic function were upregulated. These included lipase, amylase, elastase, and serine proteases indicative of pancreatic exocrine function, as well as insulin and regenerating islet genes, representative of endocrine function. Northern blot analysis and immunohistochemistry showed that differential expression of exocrine pancreas mRNAs was specific to the duodenum and localized to a subset of goblet cells. In addition, a major pattern of changes involving differential expression of olfactory receptors that function in chemical sensing, as well as other chemosensing G-protein coupled receptors, was observed. These changes in chemosensory receptor expression may be related to the failure of intestinal function and dependency on parenteral nutrition observed in humans with SLC12a2 mutations.

Conclusion: The results suggest that loss of NKCC1 affects not only secretion, but also goblet cell function and chemosensing of intestinal contents via G-protein coupled chemosensory receptors.

Keywords: Chemosensitivity; Chemosensory; Dyspepsia; Gastrointestinal; SLC12a2.

Figure 1

Northern blot and polymerase chain reaction analysis of duodenal gene expression. A: Northern blots show upregulation of Ela, Gp2, and Try primarily in the duodenum of Nkcc1^-/- (KO) mice relative to WT; n = 3 WT and 3 KO for each segment; B: Duodenal tissue from a separate cohort of mice confirms upregulation of elastase (4.7-fold, P = 0.002), glycoprotein 2 (3.5-fold, P = 0.02) and trypsin (6.0-fold, P = 0.002); n = 4 WT and 4 KO; C: Quantitative real-time polymerase chain reaction analysis of dx1, Ins1, and Glut2 shows increased expression of these genes in Nkcc1^-/- duodenum. Expression levels were normalized using the L32 ribosomal subunit mRNA; n = 4 WT (black bars) and 4 KO (grey bars). ^aP < 0.01 vs WT, ^bP < 0.002 vs WT. Gp2: Glycoprotein 2; Ela: Elastase; Try: Trypsin; WT: Wild-type; KO: Nkcc1^-/-; Pdx1: Pancreatic duodenal homeobox 1; Ins1: Insulin1; Glut2: Glucose transporter type 2.

Figure 2

Colocalization of peptidases in goblet cells of the duodenum. DAPI (blue) identifies nuclei and TFF3 (intestinal trefoil factor, green fluorescence) identifies goblet cells. Elastase, amylase and trypsin are identified by red fluorescence. The merged images show colocalization of each of these proteases and TFF3. Staining patterns were assessed in 4 wild-type and 3 Nkcc1^-/- mice. The images shown are for NKCC1-null mice. Control sections with only primary or only secondary antibodies do not identify goblet cells (not shown).

Figure 3

Amylase is expressed in a subset of goblet cells. Immunofluorescence of amylase (top, red) identifies many, but not all, goblet cells that are clearly stained with AB/PAS (bottom). The same sections (slides) from NKCC1-null intestine was used for immunofluorescence, imaged, and then stained with AB/PAS. Arrows indicate the same cell in both images. AB/PAS: Alcian Blue/Periodic Acid-Schiff.