Identification and characterization of a mitochondrial thioredoxin system in plants.

Plants possess two well described thioredoxin systems: a cytoplasmic system including several thioredoxins and an NADPH-dependent thioredoxin reductase and a specific chloroplastic system characterized by a ferredoxin-dependent thioredoxin reductase. On the basis of biochemical activities, plants also are supposed to have a mitochondrial thioredoxin system as described in yeast and mammals, but no gene encoding plant mitochondrial thioredoxin or thioredoxin reductase has been identified yet. We report the characterization of a plant thioredoxin system located in mitochondria. Arabidopsis thaliana genome sequencing has revealed numerous thioredoxin genes among which we have identified AtTRX-o1, a gene encoding a thioredoxin with a potential mitochondrial transit peptide. AtTRX-o1 and a second gene, AtTRX-o2, define, on the basis of the sequence and intron positions, a new thioredoxin type up to now specific to plants. We also have characterized AtNTRA, a gene encoding a protein highly similar to the previously described cytosolic NADPH-dependent thioredoxin reductase AtNTRB but with a putative presequence for import into mitochondria. Western blot analysis of A. thaliana subcellular and submitochondrial fractions and in vitro import experiments show that AtTRX-o1 and AtNTRA are targeted to the mitochondrial matrix through their cleavable N-terminal signal. The two proteins truncated to the estimated mature forms were produced in Escherichia coli; AtTRX-o1 efficiently reduces insulin in the presence of DTT and is reduced efficiently by AtNTRA and NADPH. Therefore, the thioredoxin and the NADPH-dependent thioredoxin reductase described here are proposed to constitute a functional plant mitochondrial thioredoxin system.

Accumulation of advanced glycation endproducts in the rat nephron: link with circulating AGEs during aging.

The accumulation of advanced glycosylation end products (AGEs) is believed to be a factor in the development of aging nephropathy. We have attempted to establish a link between the formation of AGEs and the onset of renal impairment with aging, indicated by albuminuria, using a fluorescence assay and immunohistochemical detection of AGEs in the renal extracellular matrix in rats. The fluorescence of collagenase-digested Type IV collagen from GBM increased with age, from 1.65 +/- 0.05 AU/mM OHPro (3 months) and 1.58 +/- 0.04 (10 months) to 2.16 +/- 0.06 (26 months) (p < 0.001) and 2.53 +/- 0.18 (30 months) (p < 0.001). In contrast, the extent of early glycation products significantly decreased from 5.35 +/- 0.25 nmol HCHO/nmol OHPro at 3 months to 3.14 +/- 0.19 at 10 months (p < 0.001), 3.42 +/- 0.38 at 26 months, and 0.74 +/- 0.08 at 30 months (p < 0.001). The urinary fluorescence of circulating AGE rose from 2.42 +/- 0.15 AU/mg protein (3 months), 1.69 +/- 0.07 (10 months), to 4.63 +/- 0.35 (26 months) (p < 0.01) and 4.73 +/- 0.72 (30 months), while the serum fluorescence increased from 0.39 +/- 0.02 AU/mg protein at 3 months and 0.43 +/- 0.02 at 10 months to 0.59 +/- 0.04 at 26 months (p < 0.001) and 0.54 +/- 0.03 at 30 months (p < 0.04). Polyclonal antibodies raised against AGE RNase showed faint areas of AGE immunoreactivity in mesangial areas in the nephrons of young rats. The immunolabeling of Bowman's capsule, the mesangial matrices, and the peripheral loops of glomerular and tubule basement membranes increased with rat age. The increase in circulating AGE peptides parallels the accumulation of AGEs in the nephron, and this parallels the pattern of extracellular matrix deposition, suggesting a close link between AGE accumulation and renal impairment in aging rats.

Enhancement of glomerular permeability to anionic ferritin induced by kidney perfusion with collagenase.

The role of collagen in ultrafiltration properties of the glomerular basement membrane (GBM) was tested after a single administration of bacterial collagenase, using native ferritin as a tracer which does not pass through the GBM under physiological conditions. Experiments were performed both in situ and with isolated kidneys. Increased permeability to ferritin occurs 6 hr following enzyme perfusion and becomes patent after 30 hr, numerous tracer molecules appearing in urinary space, without any readily observable changes either in distribution of fixed negative charges (as revealed by colloidal iron and polyethyleneimine) or in structural organization of the glomerulus. Selective permeability of the GBM is progressively restored so that ferritin is almost confined to capillary lumen one month after enzyme injection. We conclude that collagen plays an important part in restricting plasma protein filtration.

[Effect of collagenase on the permeability of the glomerular basement membrane in the rat kidney]. / Action de la collagénase sur la perméabilite de la membrane basale glomérulaire du rein de rat.

Recently, several authors have emphasized the role of negative sites located in th laminae rarae of the glomerular basement membrane (GBM), in restricting glomerular permeability to anionic macromolecules. In this work, we point out that ultrafiltration properties involve integrity of the GBM. Indeed after intravenous perfusion of bacterian collagenase, anionic ferritin permeates the GBM though negative site distribution (as shown by fixation of colloidal iron) is unaffected.