Probes for SPP1

Abstract

BACKGROUND/AIMS:

Murine nephrotoxic nephritis (NTN) is a well-established model resembling chronic kidney disease. Investigating gene expression patterns separately in the glomerular and cortical tubulointerstitial structure could provide new knowledge about structure-specific changes in expression of genes in the NTN model.

METHODS:

Glomerular, cortical tubulointerstitial and whole kidney tissues from mice subjected to nephrotoxic serum (NTS) or phosphate buffered saline (PBS) were collected on day 7, 21 and 42 using laser microdissection (LMD). Total RNA was extracted and subjected to nCounter NanoString. Histology, immunohistochemistry, in situ hybridization and/or quantitative real time PCR (qRT PCR) were performed to confirm regulation of selected genes.

RESULTS:

LMD provided detailed information about genes that were regulated differently between structures over time. Some of the fibrotic and inflammatory genes (Col1a1, Col3a1 and Ccl2) were upregulated in both structures, whereas other genes such as Spp1 and Grem1 were differentially regulated suggesting spatial pathogenic mechanisms in the kidney. Downregulation of cortical tubulointerstitium genes involved in iron metabolism was detected along with iron accumulation.

CONCLUSION:

This study demonstrates several regulated genes in pathways important for the pathogenesis of the NTN model and that LMD identifies structure-specific changes in gene expression during disease development. Furthermore, this study shows the benefits of isolating glomeruli and cortical tubulointerstitium in order to identify gene regulation.

Using morphological, histological, and TEM analyses of the cranium, we provide a detailed description of bone and suture growth in zebrafish. Based on expression patterns and localization, we identified osteoblasts at different degrees of maturation. Our data confirm that, unlike in humans, zebrafish cranial sutures maintain lifelong patency to sustain skull growth. The cranial vault develops in a coordinated manner resulting in a structure that protects the brain. The zebrafish cranial roof parallels that of higher vertebrates and contains five major bones: one pair of frontal bones, one pair of parietal bones, and the supraoccipital bone. Parietal and frontal bones are formed by intramembranous ossification within a layer of mesenchyme positioned between the dermal mesenchyme and meninges surrounding the brain. The supraoccipital bone has an endochondral origin. Cranial bones are separated by connective tissue with a distinctive architecture of osteogenic cells and collagen fibrils. Here we show RNA in situ hybridization for col1a1a, col2a1a, col10a1, bglap/osteocalcin, fgfr1a, fgfr1b, fgfr2, fgfr3, foxq1, twist2, twist3, runx2a, runx2b, sp7/osterix, and spp1/ osteopontin, indicating that the expression of genes involved in suture development in mammals is preserved in zebrafish. We also present methods for examining the cranium and its sutures, which permit the study of the mechanisms involved in suture patency as well as their pathological obliteration. The model we develop has implications for the study of human disorders, including craniosynostosis, which affects 1 in 2,500 live births.