Noriaki Kato

Effects of 15-month aldose reductase inhibition with fidarestat on the experimental diabetic neuropathy in rats

We examined the effects of long-term treatment with an aldose reductase inhibitor (ARI) fidarestat on functional, morphological and metabolic changes in the peripheral nerve of 15-month diabetic rats induced by streptozotocin (STZ). Slowed F-wave, motor nerve and sensory nerve conduction velocities were corrected dose-dependently in fidarestat-treated diabetic rats. Morphometric analysis of myelinated fibers demonstrated that frequencies of abnormal fibers such as paranodal demyelination and axonal degeneration were reduced to the extent of normal levels by fidarestat-treatment. Axonal atrophy, distorted axon circularity and reduction of myelin sheath thickness were also inhibited. These effects were associated with normalization of increased levels of sorbitol and fructose and decreased level of myo-inositol in the peripheral nerve by fidarestat. Thus, the results demonstrated that long-term treatment with fidarestat substantially inhibited the functional and structural progression of diabetic neuropathy with inhibition of increased polyol pathway flux in diabetic rats.

Long-term treatment with fidarestat suppresses the development of diabetic retinopathy in STZ-induced diabetic rats.

It is important to suppress retinal vascular changes for prevention of the onset and progression of diabetic retinopathy. In the present study, we investigated the dose-response effect of an aldose reductase (AR) inhibitor, fidarestat, on retinal vascular changes in the retinas of streptozotocin (STZ)-induced diabetic rats. Fidarestat (0.5, 1, and 2 mg/kg) was administered once a day, from 4 days after STZ injection, for 15 months. Microaneurysms and thickness of the basement membrane were frequently observed in the untreated diabetic group as compared to the nondiabetic control group. In addition, the number of pericytes decreased in the untreated diabetic group. Fidarestat diminished the prevalence rate of microaneurysms, basement membrane thickness and decrease in the number of pericytes, and complete suppression was observed at a dose of 2 mg/kg. Fidarestat also dose-dependently inhibited sorbitol accumulation in the retina. Furthermore, a close correlation was observed between the prevalence rate of microaneurysms and the decrease in the number of pericytes, which indicated that damage to pericytes triggers retinal vascular changes. These results suggest that fidarestat, by virtue of its long-term correction of the accelerated polyol pathway, has a potential role in preventing the progression of diabetic retinopathy.

Sorbitol dehydrogenase overexpression potentiates glucose toxicity to cultured retinal pericytes.

The polyol pathway consists of two enzymes, aldose reductase (AR) and sorbitol dehydrogenase (SDH). There is a growing body of evidence to suggest that acceleration of the polyol pathway is implicated in the pathogenesis of diabetic vascular complications. However, a functional role remains to be elucidated for SDH in the development and progression of diabetic retinopathy. In this study, cultured bovine retinal capillary pericytes were used to investigate the effects of SDH overexpression on glucose toxicity. High glucose modestly increased reactive oxygen species (ROS) generation, decreased DNA synthesis, and up-regulated vascular endothelial growth factor (VEGF) mRNA levels in cultured pericytes. SDH overexpression was found to significantly stimulate ROS generation in high glucose-exposed pericytes and subsequently potentiate the cytopathic effects of glucose. Fidarestat, a newly developed AR inhibitor, and N-acetylcysteine, an antioxidant, completely prevented these deleterious effects of SDH overexpression on pericytes. Furthermore, fidarestat administration was found to significantly prevent vascular hyperpermeability, the characteristic changes of the early phase of diabetic retinopathy, in streptozotocin-induced diabetic rats. Our present results suggest that SDH-mediated conversion of sorbitol to fructose and the resultant ROS generation may play an active role in the pathogenesis of diabetic retinopathy. Blockage of sorbitol formation by fidarestat could be a promising therapeutic strategy for the treatment of early phase of diabetic retinopathy.

Neurotrophin-3-induced production of nerve growth factor is suppressed in Schwann cells exposed to high glucose: involvement of the polyol pathway.

Development of hypesthesia, a loss of sensitivity to stimulation, is associated with impaired regeneration of peripheral sensory fibers, in which Schwann cells play a key role by secreting nerve growth factor (NGF). Recent clinical trials indicated that an inhibitor of aldose reductase (AR), the rate‐limiting enzyme in the polyol pathway, significantly improved hypesthesia in diabetic patients. The fact that AR is localized in Schwann cells led us to investigate the role of the polyol pathway in NGF production of isolated Schwann cells. Among various endogenous factors examined, increased production of NGF was demonstrated in the cells treated with neurotrophin‐3 (NT‐3) for 24 h. NT‐3‐induced NGF production was significantly suppressed when cells were cultured in the medium containing high glucose. In these cells, the levels of glutathione (GSH) and cAMP‐response element binding protein (CREB) were reduced, whereas the level of activated nuclear factor‐κB (NF‐κB) was elevated. These changes were abolished when an AR inhibitor fidarestat was included in the medium. NT‐3‐induced NGF production was further attenuated in the cells treated with an inhibitor of GSH synthesis. Together, the enhanced polyol pathway activity under high‐glucose conditions seems to elicit reduced NT‐3‐induced NGF production in Schwann cells. Enhanced oxidative stress linked to the polyol pathway activity may mediate this process.

Continuous Inhibition of Excessive Polyol Pathway Flux in Peripheral Nerves by Aldose Reductase Inhibitor Fidarestat Leads to Improvement of Diabetic Neuropathy

We investigated the effects of three aldose reductase (AR) inhibitors, fidarestat, epalrestat and zenarestat, on the slowing of sensory nerve conduction velocity (SNCV), motor nerve conduction velocity (MNCV), and minimal F-wave latency prolongation in streptozotocin (STZ)-induced diabetic rats. Two weeks after STZ injection, SNCV and MNCV in the diabetic rats were significantly slower than in normal rats. Fidarestat (0.25-2 mg/kg/day), epalrestat (48 to 96 mg/kg/day) or zenarestat (10-40 mg/kg/day) was administered orally for the following 2 weeks, and SNCV, MNCV and F-wave latency were measured 3 h after final administration. Significant prolongation of minimal F-wave latency, as well as slowing of SNCV and MNCV, was found in the untreated diabetic rats 4 weeks after STZ injection. At a dose of 0.5 mg/kg/day or more fidarestat showed significant effects on these nervous dysfunctions, effects that were more potent than those shown by the other inhibitors. Furthermore, following the 2-week administration of fidarestat (1 mg/kg/day), epalrestat (48 mg/kg/day) or zenarestat (20 mg/kg/day), which began 2 weeks after STZ injection, sorbitol content in the sciatic nerve, produced by AR, a rate-limiting enzyme in the polyol pathway, was determined at 3, 8, 12, and 24 h after final administration. At each point in time, sorbitol content in the untreated diabetic rats was much higher than that in the normal control rats. Fidarestat suppressed sorbitol accumulation remarkably and continuously until 24 h after administration. On the other hand, the inhibitory effect by zenarestat declined in a time-dependent manner, and epalrestat did not decrease sorbitol content. Therefore, these results suggest that continuous inhibition of increased polyol pathway flux can improve diabetic neuropathy more potently.

Amelioration of Acute Kidney Injury in Lipopolysaccharide-Induced Systemic Inflammatory Response Syndrome by an Aldose Reductase Inhibitor, Fidarestat

Background Systemic inflammatory response syndrome is a fatal disease because of multiple organ failure. Acute kidney injury is a serious complication of systemic inflammatory response syndrome and its genesis is still unclear posing a difficulty for an effective treatment. Aldose reductase (AR) inhibitor is recently found to suppress lipopolysaccharide (LPS)-induced cardiac failure and its lethality. We studied the effects of AR inhibitor on LPS-induced acute kidney injury and its mechanism. Methods Mice were injected with LPS and the effects of AR inhibitor (Fidarestat 32 mg/kg) before or after LPS injection were examined for the mortality, severity of renal failure and kidney pathology. Serum concentrations of cytokines (interleukin-1β, interleukin-6, monocyte chemotactic protein-1 and tumor necrosis factor-α) and their mRNA expressions in the lung, liver, spleen and kidney were measured. We also evaluated polyol metabolites in the kidney. Results Mortality rate within 72 hours was significantly less in LPS-injected mice treated with AR inhibitor both before (29%) and after LPS injection (40%) than untreated mice (90%). LPS-injected mice showed marked increases in blood urea nitrogen, creatinine and cytokines, and AR inhibitor treatment suppressed the changes. LPS-induced acute kidney injury was associated with vacuolar degeneration and apoptosis of renal tubular cells as well as infiltration of neutrophils and macrophages. With improvement of such pathological findings, AR inhibitor treatment suppressed the elevation of cytokine mRNA levels in multiple organs and renal sorbitol accumulation. Conclusion AR inhibitor treatment ameliorated LPS-induced acute kidney injury, resulting in the lowered mortality.

Serial changes of sensory nerve conduction velocity and minimal F-wave latency in streptozotocin-induced diabetic rats

We studied the serial changes of sensory nerve conduction velocity (SNCV) in the caudal nerve of streptozotocin (STZ)-induced diabetic rats using a new technical method. Minimal F-wave latency was also studied by stimulating the tibial nerve. The SNCV in the diabetic rats was slower than that in the normal rats 2 weeks after STZ injection, and minimal F-wave latency was prolonged compared to normal rats 4 weeks after STZ injection. Treatment of the diabetic rats with insulin for 14 days inhibited SNCV slowing and minimal F-wave latency prolongation. This new method to measure SNCV is useful for various studies, and improvement of diabetic neuropathy with insulin treatment is indicated by recovery from SNCV slowing and minimal F-wave latency prolongation.

The role of the polyol pathway in acute kidney injury caused by hindlimb ischaemia in mice

The polyol pathway, a collateral glycolytic process, previously considered to be active in high glucose milieu, has recently been proposed to play a crucial role in ischaemia/reperfusion tissue injury. In this study, we explored the role of the polyol pathway in acute kidney injury (AKI), a life‐threatening condition, caused by hindlimb ischaemia, and determined if inhibition of the polyol pathway by aldose reductase (AR) inhibitor is beneficial for this serious disorder. Mice 8 weeks of age rendered hindlimb ischaemic for 3 h by the clipping of major supporting arteries revealed marked muscle necrosis with accumulation of sorbitol and fructose in ischaemic muscles. Serum concentrations of blood urea nitrogen (BUN), creatinine phosphokinase (CPK), creatinine, tumour necrosis factor (TNF)‐α as well as interleukin (IL)‐6 were all elevated in these mice. Treatment with AR inhibitor (ARI) effectively suppressed muscle necrosis and accompanying inflammatory reactions and prevented renal failure. Similar to ARI‐treated mice, AR‐deficient mice were protected from severe ischaemic limb injury and renal failure, showing only modest muscle necrosis and significant suppression of serum markers of renal failure and inflammation. Thus, these findings suggest that the polyol pathway is implicated in AKI caused by ischaemic limb injury and that AR may be a potential therapeutic target for this condition. Copyright

Effect of long-term treatment with a new aldose reductase inhibitor, (2S,4S)-6-fluoro-2′,5′-dioxospiro-[chroman-4,4′-imidazolidine]-2-carboxamide (SNK-860), on peripheral neuropathy in streptozotocin-induced diabetic rats

Abstract We studied the long-term effects of a new aldose reductase inhibitor, (2 S ,4 S )-6-fluoro-2′,5′-dioxospiro-[chroman-4,4′-imidazolidine]-2-carboxamide (SNK-860), on functional, biochemical, and structural changes in peripheral nerve of streptozotocin (STZ)-induced diabetic rats. During the experimental period of 26 weeks, the delayed motor-nerve conduction in diabetic rats was significantly prevented by SNK-860 treatment, and elevated sorbitol levels and reduced myo -inositol levels were normalized to 100% and 71% of control levels, respectively. Teased nerve fiber studies demonstrated that the frequency of abnormal fibers was significantly reduced in treated diabetic rats. Morphometric analysis of myelinated fibers also disclosed prevention of axonal atrophy, distorted axonal circularity and preservation of large-sized fibers following SNK-860 treatment. These results suggest that long-term treatment with SNK-860 has a beneficial preventive effect on the development of experimental diabetic neuropathy.

Differential Effects of High Glucose and Methylglyoxal on Viability and Polyol Metabolism in Immortalized Adult Mouse Schwann Cells

Cultured Schwann cells under exposure to high glucose and methylglyoxal (MG) have been individually em- ployed for studying diabetic neuropathy; however, similarities and differences between these two culture models have not been studied. We investigated the effects of high glucose and MG on viability, polyol pathway activity, and expression of oxidative stress markers (4-hydroxy-2-nonenal (4HNE), acrolein (ACR), and hexanoyl lysine (HEL)) in immortalized adult mouse Schwann cells (IMS32 cell line) in culture. Western blot and immunocytochemical analyses revealed that ex- pression of aldose reductase (AR), 4HNE, ACR, and HEL in IMS32 was induced by exposure to both high glucose (30 mM) and MG (0.5 mM) for 48 h. Treatment with MG (0.1, 0.2, and 0.5 mM) induced cell death in a concentration- dependent manner, whereas high glucose environments (30 mM and 56 mM) did not impair cell viability. In contrast, in- tracellular sorbitol and fructose levels were significantly increased by high glucose, but not by MG. Taking these findings together, IMS32 cell line under high glucose conditions appears to be useful for studying oxidative stress in relation to the polyol pathway hyperactivity in diabetes. MG is capable of causing more detrimental damage to IMS32 than high glucose, but MG-induced upregulation of AR is unlikely to accelerate the polyol pathway activity.