Ischemic time impacts biological integrity of phospho-proteins in PI3K/Akt, Erk/MAPK, and p38 MAPK signaling networks.

Post-translational modifications of proteins, such as phosphorylation, are labile events dynamically regulated by opposing kinase and phosphatase activities. Preanalytical factors, such as ischemic time before fixation, affect these activities and can have a significant impact on the ability to elucidate signaling pathways in tissue. Immunohistochemical analysis of phosphorylated proteins involved in PI3K/Akt, Erk/MAPK, and p38 MAPK signaling networks was performed in human cell line xenografts from lung, brain, ovary, and prostate tumors. In order to replicate real-world practices, the tissues were subjected to ischemic times of 0 (baseline), 1, 4, and 24 hours before fixation in formalin. Two key concepts emerge from this analysis: (1) the stability of different phospho-epitopes within a given tumor type is variable (e.g. phospho-PRAS40 is more labile than phospho-S6 ribosomal protein) and (2) the stability of a given phospho-epitope (e.g. phospho-MAPKAPK2) varies significantly across different tumor types. These results highlight the importance of proper tissue acquisition and rapid fixation to preserve the biological integrity of signal transduction pathways that may guide therapeutic decision making.

Acute hyperglycemia induced by ketamine/xylazine anesthesia in rats: mechanisms and implications for preclinical models.

The effects of anesthetic agents, commonly used in animal models, on blood glucose levels in fed and fasted rats were investigated. In fed Sprague-Dawley rats, ketamine (100 mg/kg)/xylazine (10 mg/kg) (KX) produced acute hyperglycemia (blood glucose 178.4 +/- 8.0 mg/dl) within 20 min. The baseline blood glucose levels (104.8 +/- 5.7 mg/dl) reached maximum levels (291.7 +/- 23.8 mg/dl) at 120 min. Ketamine alone did not elevate glucose levels in fed rats. Isoflurane also produced acute hyperglycemia similar to KX. Administration of pentobarbital sodium did not produce hyperglycemia in fed rats. In contrast, none of these anesthetic agents produced hyperglycemia in fasted rats. The acute hyperglycemic effect of KX in fed rats was associated with decreased plasma levels of insulin, adrenocorticotropic hormone (ACTH), and corticosterone and increased levels of glucagon and growth hormone (GH). The acute hyperglycemic response to KX was dose-dependently inhibited by the specific alpha2-adrenergic receptor antagonist yohimbine (1-4 mg/kg). KX-induced changes of glucoregulatory hormone levels such as insulin, GH, ACTH, and corticosterone were significantly altered by yohimbine, whereas the glucagon levels remained unaffected. In conclusion, the present study indicates that both KX and isoflurane produce acute hyperglycemia in fed rats. The effect of KX is mediated by modulation of the glucoregulatory hormones through stimulation of alpha2-adrenergic receptors. Pentobarbital sodium did not produce hyperglycemia in either fed or fasted rats. Based on these findings, it is suggested that caution needs to be taken when selecting anesthetic agents, and fed or fasted state of animals in studies of diabetic disease or other models where glucose and/or glucoregulatory hormone levels may influence outcome and thus interpretation. However, fed animals are of value when exploring the hyperglycemic response to anesthetic agents.

Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis.

Connective tissue growth factor (CTGF) is a peptide secreted by cultured endothelial cells and fibroblasts when stimulated by transforming growth factor-beta (TGF-beta), and is overexpressed during fibrotic processes in coronary arteries and in skin. To determine whether CTGF is implicated in the pathogenesis of diabetic glomerulosclerosis, cultured rat mesangial cells (MC) as well as kidney cortex and microdissected glomeruli were examined from obese, diabetic db/db mice and their normal counterparts. Exposure of MC to recombinant human CTGF significantly increased fibronectin and collagen type I production. Furthermore, unstimulated MC expressed low levels of CTGF message and secreted minimal amounts of CTGF protein (36 to 38 kD) into the media. However, sodium heparin treatment resulted in a greater than fourfold increase in media-associated CTGF, suggesting that the majority of CTGF produced was cell- or matrix-bound. Exposure of MC to TGF-beta, increased glucose concentrations, or cyclic mechanical strain, all causal factors in diabetic glomerulosclerosis, markedly induced the expression of CTGF transcripts, while recombinant human CTGF was able to autoinduce its own expression. TGF-, and high glucose, but not mechanical strain, stimulated the concomitant secretion of CTGF protein, the former also inducing abundant quantities of a small molecular weight form of CTGF (18 kD) containing the heparin-binding domain. The induction of CTGF protein by a high glucose concentration was mediated by TGF-beta, since a TGF-beta-neutralizing antibody blocked this stimulation. In vivo studies using quantitative reverse transcription-PCR demonstrated that although CTGF transcripts were low in the glomeruli of control mice, expression was increased 28-fold after approximately 3.5 mo of diabetes. This change occurred early in the course of diabetic nephropathy when mesangial expansion was mild, and interstitial disease and proteinuria were absent. A substantially reduced elevation of CTGF mRNA (twofold) observed in whole kidney cortices indicated that the primary alteration of CTGF expression was in the glomerulus. These results suggest that CTGF upregulation is an important factor in the pathogenesis of mesangial matrix accumulation and progressive glomerulosclerosis, acting downstream of TGF-beta.