Losartan reverses fibrotic changes in cortical renal tissue induced by ischemia or ischemia-reperfusion without changes in renal function.

Unilateral renal ischemia for 40 min in rat results in increased fibronectin (FN) expression in proximal tubular cells. This study examines the role of 24 h of blood reperfusion and the role of the renin-angiotensin system (RAS) on these results. Rats were submitted to 40 min of unilateral renal ischemia followed by 24 h of blood reperfusion. Renal function was assayed by clearance measurement in metabolic cages. Intracellular ATP and calcium were determined in proximal tubules. The expression and abundance of FN were investigated by reverse transcription-polymerase chain reaction, ELISA and Western blot either in isolated proximal tubules or cortex homogenates from control, ischemic and ischemic with reperfusion rats. Matrix metalloproteases (MMPs) activity was also measured. Losartan effects on renal function and on the abundance of FN and the MMPs activity in cortical homogenates were also measured. The renal function remained altered after 24 h of reperfusion in untreated and losartan-treated ischemic rats. On the other hand, the abundance of FN is increased after reperfusion both in isolated proximal tubules and total cortex homogenates and the same pattern was observed in the MMPs activity. Twenty-four h of blood reperfusion presented FN-mRNA signals similar to control ones. Losartan pretreated-rats presented diminished FN abundance in homogenates of cortex tissue from ischemic rats with or without reperfusion. Similar results were observed in the MMPs-activity. These results suggest that angiotensin II acting via the AT1 receptor plays a role in the development of tubulointersticial fibrosis after ischemia-reperfusion by activation of intrarenal RAS from the injured kidney.

Trypanosoma brucei oleate desaturase may use a cytochrome b5-like domain in another desaturase as an electron donor.

An open reading frame with fatty acid desaturase similarity was identified in the genome of Trypanosoma brucei. The 1224 bp sequence specifies a protein of 408 amino acids with 59% and 58% similarity to Mortierella alpina and Arabidopsis thaliana Delta12 desaturase, respectively, and 51% with A. thaliana omega3 desaturases. The histidine tracks that compose the iron-binding active centers of the enzyme were more similar to those of the omega3 desaturases. Expression of the trypanosome gene in Saccharomyces cerevisiae resulted in the production of fatty acids that are normally not synthesized in yeast, namely linoleic acid (18:2Delta9,12) and hexadecadienoic acid (16:2Delta9,12), the levels of which were dependent on the culture temperature. At low temperature, the production of bi-unsaturated fatty acids and the 16:2/18:2 ratio were higher. Transformed yeast cultures supplemented with 19:1Delta10 fatty acid yielded 19:2Delta10,13, indicating that the enzyme is able to introduce a double bond at three carbon atoms from a pre-existent olefinic bond. The expression of the gene in a S. cerevisiae mutant defective in cytochrome b5 showed a significant reduction in bi-unsaturated fatty acid production, although it was not totally abolished. Based on the regioselectivity and substrate preferences, we characterized the trypanosome enzyme as a cytochrome b5-dependent oleate desaturase. Expression of the ORF in a double mutant (ole1Delta,cytb5Delta) abolished all oleate desaturase activity completely. OLE1 codes for the endogenous stearoyl-CoA desaturase. Thus, Ole1p has, like Cytb5p, an additional cytochrome b5 function (actually an electron donor function), which is responsible for the activity detected when using the cytb5Delta single mutant.

Fibronectin expression in proximal tubules from ischemic rat kidneys without reperfusion.

The expression of fibronectin (FN), one of the extracellular matrix proteins, was studied in isolated renal proximal tubules in a in vivo rat model of unilateral renal ischemia without reperfusion. FN is involved in cell-extracellular matrix interactions and defective cell-extracellular matrix interactions have been hypothesized to contribute to ischemic renal failure. The expression of FN was investigated by reverse transcription-polymerase chain reaction (RT-PCR), Elisa and Western blot. Isolated proximal tubules from control and post-ischemic rat kidneys were used. ATP, intracellular calcium content, and alkaline phosphatase were also measured to describe the effects associated to 40 min of ischemia. Control tubules expressed FN. Forty minutes of ischemia promoted diminished ATP levels and phosphatase alkaline activity, and increased intracellular calcium in isolated proximal tubules. An increased abundance of FN was observed by ischemic tubules as compared with control tubules. To determine quantitatively the value of FN content, ELISA method was performed. The ischemic tubules also expressed higher amount of FN mRNA. Three amplification products were obtained from both ischemic and control proximal tubular cDNA. The relative amounts of each of the obtained products were the same, strongly suggesting that the augmentation of the FN gene transcription during ischemia is not associated to a modification in the splicing pattern. Moreover, this expression is increased after 40 min of ischemia, not followed by reperfusion.