Proteomic analysis of the osmotic stress response in the mammalian kidney.

摘要

The papilla of the mammalian kidney must tolerate varying degrees of hyperosmotic stress during urine concentration. Though functionally distinct the papilla is in close physical proximity to the renal cortex, an iso-osmotic region. Proteomic differences between the papilla and cortex of C57BL6 mice were investigated using two-dimensional gel electrophoresis to generate a whole proteome map for each tissue region. Delta2D software was used to quantify (n = 6) two-fold or greater expression changes between the regions leading to detection of 211 cortex spots and 70 papilla spots. MALDI-TOF/TOF mass spectrometry was used to sequence and identify tryptic peptides from the spots identifying 37 different cortex proteins and 16 proteins over-expressed in the papilla. Regional specificity was confirmed by Western Blot and further substantiated by immunohistochemistry for selected proteins. Protein identifications of the renal papilla include alpha B crystallin, Hsp beta-1, Hsp90, 14-3-3 protein, glutathione S-transferase, aldose reductase, actin and tropomyosin which indicates that an array of stress response and structural proteins contribute to offset the hostile environment in the papilla. Proteins over-represented in the cortex were largely related to metabolism. PANTHER analysis of protein identifications revealed an increase in chaperones and protein folding in the papilla compared to cortex. Antidiuretic and diuretic papilla were also examined using a proteomic approach. Two-dimensional gels were analyzed using Delta2D software to quantify (n = 4) 1.25-fold or greater expression changes between the altered hydration state and the control leading to identification of 15 different proteins that change during antidiuresis and 18 different proteins that change during diuresis. Changes were confirmed by Western Blot for selected proteins. Proteins associated with structure (actin, tubulin), signaling (Rho GDP dissociation inhibitor, abhydrolase domain-containing protein 14B), chaperone functioning (Hsp beta-1, alpha B-crystallin, T complex protein-1) and oxidative stress (alpha-enolase, GAPDH and LDH) were altered during either antidiuresis or diuresis.