3-Mercapto-5H-1,2,4-Triazino[5,6-b]Indole-5-Acetic Acid (Cemtirestat) Alleviates Symptoms of Peripheral Diabetic Neuropathy in Zucker Diabetic Fatty (ZDF) Rats: A Role of Aldose Reductase

Peripheral neuropathy is the most prevalent chronic complication of diabetes mellitus. Good glycemic control can delay the appearance of neuropathic symptoms in diabetic patients but it is not sufficient to prevent or cure the disease. Therefore therapeutic approaches should focus on attenuation of pathogenetic mechanisms responsible for the nerve injury. Considering the role of polyol pathway in the etiology of diabetic neuropathy, we evaluated the effect of a novel efficient and selective aldose reductase inhibitor, 3-mercapto-5H-1,2,4-triazino[5,6-b]indole-5-acetic acid (cemtirestat), on symptoms of diabetic peripheral neuropathy in Zucker Diabetic Fatty (ZDF) rats. Since the age of 5 months, male ZDF rats were orally administered cemtirestat, 2.5 and 7.5 mg/kg/day, for two following months. Thermal hypoalgesia was evaluated by tail flick and hot plate tests. Tactile allodynia was determined by a von Frey flexible filament test. Two-month treatment of ZDF rats with cemtirestat (i) did not affect physical and glycemic status of the animals; (ii) partially inhibited sorbitol accumulation in red blood cells and the sciatic nerve; (iii) markedly decreased plasma levels of thiobarbituric acid reactive substances; (iv) normalized symptoms of peripheral neuropathy with high significance. The presented findings indicate that inhibition of aldose reductase by cemtirestat is not solely responsible for the recorded improvement of the behavioral responses. In future studies, potential effects of cemtirestat on consequences of diabetes that are not exclusively dependent on glucose metabolism via polyol pathway should be taken into consideration.

     

Enhanced suicidal death of erythrocytes from gene-targeted mice lacking the Cl-/HCO(3)(-) exchanger AE1

Genetic defects of anion exchanger 1 (AE1) may lead to spherocytic erythrocyte morphology, severe hemolytic anemia, and/or cation leak. In normal erythrocytes, osmotic shock, Cl^– removal, and energy depletion activate Ca²⁺-permeable cation channels with Ca²⁺-induced suicidal erythrocyte death, i.e., surface exposure of phosphatidylserine, cell shrinkage, and membrane blebbing, all features typical for apoptosis of nucleated cells. The present experiments explored whether AE1 deficiency favors suicidal erythrocyte death. Peripheral blood erythrocyte numbers were significantly smaller in gene-targeted mice lacking AE1 (AE1^–/– mice) than in their wild-type littermates (AE1^+/+ mice) despite increased percentages of reticulocytes (AE1^–/–: 49%, AE1^+/+: 2%), an indicator of enhanced erythropoiesis. Annexin binding, reflecting phosphatidylserine exposure, was significantly larger in AE1^–/–erythrocytes/reticulocytes (10%) than in AE1^+/+ erythrocytes (1%). Osmotic shock (addition of 400 mM sucrose), Cl^– removal (replacement with gluconate), or energy depletion (removal of glucose) led to significantly stronger annexin binding in AE1^–/– erythrocytes/reticulocytes than in AE1^+/+ erythrocytes. The increase of annexin binding following exposure to the Ca²⁺ ionophore ionomycin (1 µM) was, however, similar in AE1^–/– and in AE1^+/+ erythrocytes. Fluo3 fluorescence revealed markedly increased cytosolic Ca²⁺ permeability in AE1^–/– erythrocytes/reticulocytes. Clearance of carboxyfluorescein diacetate succinimidyl ester-labeled erythrocytes/reticulocytes from circulating blood was more rapid in AE1^–/– mice than in AE1^+/+ mice and was accelerated by ionomycin treatment in both genotypes. In conclusion, lack of AE1 is associated with enhanced Ca²⁺ entry and subsequent scrambling of cell membrane phospholipids. annexin; cell volume; osmolarity; phosphatidylserine; energy depletion     

Regulation of virulence gene expression in Vibrio cholerae by quorum sensing: HapR functions at the aphA promoter

Quorum sensing negatively influences virulence gene expression in certain toxigenic Vibrio cholerae strains. At high cell densities, the response regulator LuxO fails to reduce the expression of HapR, which, in turn, represses the expression of the virulence cascade. A critical regulatory step in the cascade is activation of tcpPH expression by AphA and AphB. We show here that HapR influences the virulence cascade by directly repressing aphA expression. In strain C6706, aphA expression was increased in a delta hapR mutant and decreased in a delta luxO mutant, indicating a negative and positive influence, respectively, of these gene products on the promoter. Overexpression of HapR also reduced aphA expression in both C6706 and Escherichia coli. DNase I footprinting showed that purified HapR binds to the aphA promoter between -85 and -58. Although it appears that quorum sensing does not influence virulence gene expression in strain O395 solely because of a frameshift in hapR, overproduced HapR did not repress expression from the O395 aphA promoter in either Vibrio or E. coli, nor did the protein bind to the promoter. Two basepair differences from C6706 are present in the O395 HapR binding site at -85 and -77. Introducing the -77 change into C6706 prevented HapR binding and repression of aphA expression. This mutation also eliminated the repression of toxin-co-regulated pilus (TCP) and cholera toxin (CT) that occurs in a delta luxO mutant, indicating that HapR function at aphA is critical for density-dependent regulation of virulence genes.