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End-stage renal disease from diabetic nephropathy, mainly due to type II diabetes, is an increasing problem in Western countries. The pathogenesis of diabetic nephropathy is still incompletely understood and much of the experimental insight has been obtained from insulinopenic animal models, resembling type I diabetes. This review therefore aims to describe available rodent models of nephropathy associated with type II diabetes. The review focusses on the metabolic as well as renal functional and structural changes. The usefulness of these rodent models to study renal involvement in type II diabetes is discussed with particular emphasis on confounding factors such as hyperlipidemia, hypertension, immunologic abnormalities, urogenital structural abnormalities and other associated pathological conditions. In addition recent observations on two rat strains, the obese Zucker (fatty) and Goto Kakizaki (GK) rat, are discussed in detail.

Mesangial cell proliferation and matrix accumulation, driven by platelet-derived growth factor (PDGF), contribute to many progressive renal diseases. In a novel approach to antagonize PDGF, we investigated the effects of a nuclease-resistant high-affinity oligonucleotide aptamer in vitro and in vivo. In cultured mesangial cells, the aptamer markedly suppressed PDGF-BB but not epidermal- or fibroblast-growth-factor-2-induced proliferation. In vivo effects of the aptamer were evaluated in a rat mesangioproliferative glomerulonephritis model. Twice-daily intravenous (i.v.) injections from days 3 to 8 after disease induction of 2.2 mg/kg PDGF-B aptamer, coupled to 40-kd polyethylene glycol (PEG), led to 1) a reduction of glomerular mitoses by 64% on day 6 and by 78% on day 9, 2) a reduction of proliferating mesangial cells by 95% on day 9, 3) markedly reduced glomerular expression of endogenous PDGF B-chain, 4) reduced glomerular monocyte/macrophage influx on day 6 after disease induction, and 5) a marked reduction of glomerular extracellular matrix overproduction (as assessed by analysis of fibronectin and type IV collagen) both on the protein and mRNA level. The administration of equivalent amounts of a PEG-coupled aptamer with a scrambled sequence or PEG alone had no beneficial effect on the natural course of the disease. These data show that specific inhibition of growth factors using custom-designed, high-affinity aptamers is feasible and effective.

The second step in mitochondrial fatty acid beta-oxidation is catalyzed by short chain enoyl-CoA hydratase (ECHS1; EC 4.2.1.17). Inherited disorders of this pathway of energy metabolism present clinical and laboratory features resembling sudden infant death syndrome and Reye-like syndrome. To investigate the role of ECHS1 further, the gene structure was analyzed and its chromosomal locus determined. A fragment of rat liver ECHS1 cDNA was employed for isolation and characterization of two overlapping genomic clones encompassing the entire human ECHS1 gene. The gene, approximately 11 kb, is composed of eight exons, with exons I and VIII containing the 5'- and 3'-untranslated regions, respectively. Two major transcription start sites, located 62 and 63 bp upstream of the translation initiation codon, were mapped by primer extension analysis. The immediate 5'-flanking region of the ECHS1 gene is GC-rich and contains several copies of the SP1 binding motive but no typical TATA or CAAT boxes are apparent. Alu repeat elements have been identified within the region -1052/-770 relative to the cap site and in intron 7. The human ECHS1 gene locus was assigned to chromosome 10q26.2-q26.3 by fluorescence in situ hybridization.

Mitochondrial 2-enoyl-CoA hydratase (mECH) and 3,2-trans-enoyl-CoA isomerase (mECI), two enzymes which catalyze totally different reactions in fatty acid beta-oxidation, belong to the low-similarity hydratase/isomerase enzyme superfamily. Their substrates and reaction mechanisms are similar [Müller-Newen, G. & Stoffel, W. (1993) Biochemistry 32, 11,405-11,412]. Glu164 of mECH is the only amino acid with a protic side chain that is conserved in these monofunctional and polyfunctional enzymes with 2-enoyl-CoA hydratase and 3,2-trans-enoyl-CoA isomerase activities. We tested our hypothesis that Glu164 of mECH is the putative active-site amino acid responsible for the base-catalyzed alpha-deprotonation in the hydratase/dehydrase and isomerase reaction. We functionally expressed rat liver mECH wild-type and [E164Q] mutant enzymes in Escherichia coli. Characterization of the purified wild-type and mutant enzymes revealed that the replacement of Glu164 by Gln lowers the kcat value more than 100,000-fold, whereas the Km value is only moderately affected. We have demonstrated in a previous study that Glu165 is indispensable for the 3,2-trans-enoyl-CoA isomerase activity. Taking these results together, we conclude that the conserved glutamic acid is the essential basic group in the active sites of 2-enoyl-CoA hydratase (Glu164) and 3,2-trans-enoyl-CoA isomerase (Glu165), and that these enzymes are not only evolutionarily but also functionally and mechanistically related.

Objective: To investigate the manometrical and clinical long-term results following a tear III., Patients: 27 Primiparae with a tear III following a spontaneous vaginal delivery were retrospectively compared with 22 Primiparae without sphincter injury (follow-up: 27 months)., Methods: Water-Perfusion-Manometry and clinical assessment (modified Kelly-Score). Manometric parameter (at rest and during contraction): sphinctertone and - length, vectorvolume, radial asymmetry. Wilcoxon-Text (p < 0.05)., Results: Tear III patients showed a significant decrease in sphincter length and vector volume both at rest and during contraction. On clinical assessment there was no difference., Conclusion: A tear III weakens the anal sphincter by decreasing sphincter length and vector volume. This weakness can be demonstrated only manometrically but not clinically.

A key enzyme in the mitochondrial beta-oxidation of unsaturated fatty acids is the 3,2-trans-enoyl-CoA isomerase (DCI; EC 5.3.3.8). It catalyzes the transformation of 3-cis and 3-trans intermediates arising during the stepwise degradation of all cis-, mono-, and polyunsaturated fatty acids to the 2-trans-enoyl-CoA intermediates. A genomic clone encoding the human DCI was isolated and characterized by use of the previously cloned human DCI cDNA. The entire gene encompasses approximately 12.5 kb, and the coding sequence is distributed over seven exons. One major and three minor transcription start sites were determined by primer extension analysis. In common with promoters of other housekeeping genes encoding mitochondrial proteins, the GC-rich, immediate 5'-flanking region of the DCI transcription initiation site lacks typical TATA and CAAT boxes; instead, two GC box consensus sequences are present. Introns 2 and 6 contain several Alu repetetive sequences. The human DCI gene locus was assigned to chromosome 16 by use of human-rodent somatic cell hybrids and to chromosome 16p13.3 by chromosomal in situ suppression hybridization studies.

Flumazenil, a new and specific benzodiazepine antagonist that appears to be free of intrinsic pharmacologic action, is extensively metabolized by oxidative processes and represents a high-clearance drug. Consequently, it could be anticipated that hepatic disease affects the elimination and oral bioavailability of flumazenil. Therefore, the pharmacokinetics of flumazenil was evaluated in eight patients who had moderate cirrhosis and in eight age-matched healthy volunteers after a single oral dose (30 mg) and after an intravenous dose (2 mg). The mean half-life (t1/2) was 0.8 versus 1.4 hours (p = 0.003) and total plasma clearance was 1201 versus 705 ml per minute (p = 0.009) for control subjects versus patients with cirrhosis. Bioavailability increased from the normal 28% to 65% (p = 0.001) in patients with hepatic dysfunction. Routine liver tests did not correlate with the elimination of flumazenil in individual patients. It can be concluded that elimination of flumazenil is impaired in patients who have stable alcoholic cirrhosis. Despite the relative wide margin of safety of flumazenil, somewhat lower doses could be effective in such patients if long-term oral use is anticipated.