Peter F. Kador

Autoimmunity in Membranous Nephropathy Targets Aldose Reductase and SOD2

Glomerular targets of autoimmunity in human membranous nephropathy are poorly understood. Here, we used a combined proteomic approach to identify specific antibodies against podocyte proteins in both serum and glomeruli of patients with membranous nephropathy (MN). We detected specific anti-aldose reductase (AR) and anti-manganese superoxide dismutase (SOD2) IgG(4) in sera of patients with MN. We also eluted high titers of anti-AR and anti-SOD2 IgG(4) from microdissected glomeruli of three biopsies of MN kidneys but not from biopsies of other glomerulonephritides characterized by IgG deposition (five lupus nephritis and two membranoproliferative glomerulonephritis). We identified both antigens in MN biopsies but not in other renal pathologies or normal kidney. Confocal and immunoelectron microscopy (IEM) showed co-localization of anti-AR and anti-SOD2 with IgG(4) and C5b-9 in electron-dense podocyte immune deposits. Preliminary in vitro experiments showed an increase of SOD2 expression on podocyte plasma membrane after treatment with hydrogen peroxide. In conclusion, our data support AR and SOD2 as renal antigens of human MN and suggest that oxidative stress may drive glomerular SOD2 expression.

Diabetic cataracts: mechanisms and management†

Diabetes mellitus is associated with a 5‐fold higher prevalence of cataracts, which remains a major cause of blindness in the world. Typical diabetic cataracts contain cortical and/or posterior subcapsular opacities. Adult onset diabetic cataracts also often contain nuclear opacities. Mechanisms of diabetic cataractogenesis have been studied in less detail than those of other diabetic complications. Both animal and human studies support important contribution of increased aldose reductase activity. Surgical extraction is the only cure of diabetic cataract today. An improved understanding of pathogenetic mechanisms, together with finding effective therapeutic agents, remain highest priority for diabetic cataract‐related research and pharmaceutical development. Copyright

Aldose reductase / polyol inhibitors for diabetic retinopathy.

Diabetic retinopathy is the most common microvascular complication of diabetes and the most severe of diabetic ocular complications. This review describes retinal changes at different stages of diabetic retinopathy and risk factors associated with this devastating disease. Special attention is focused on aldose reductase, the first enzyme of the sorbitol pathway of glucose metabolism. The current knowledge on the enzyme localization in the retina, and the role for increased aldose reductase activity in retinal capillary cell loss and formation of acellular capillaries, capillary basement membrane thickening, increased vascular permeability and disruption of blood-retinal barrier, and increased leukocyte adhesion to endothelial cells associated with early diabetic retinopathy, as well as neovascularization associated with advanced (proliferative) diabetic retinopathy, gained through the experimental studies in animal models of diabetes and galactose feeding, is described in detail. The review also analyzes the potential mechanisms underlying aldose reductase involvement in pathogenesis of diabetic retinopathy, and discusses interactions between aldose reductase and other pathogenetic factors such as formation of advanced glycation end-products, oxidative-nitrosative stress, protein kinase C, mitogen-activated protein kinase, and poly(ADP-ribose) polymerase activations, inflammation, and growth factor imbalances. A detailed analysis of clinical diabetic retinopathy trials of aldose reductase inhibitors is also provided.

Poly(ADP-ribose)polymerase inhibition counteracts cataract formation and early retinal changes in streptozotocin-diabetic rats.

PURPOSE This study evaluated the role for poly(ADP-ribose) polymerase (PARP) in diabetes-induced cataractogenesis and early retinal changes. METHODS Control and streptozotocin (STZ)-diabetic rats were treated with or without the PARP inhibitors 1,5-isoquinolinediol (ISO; 3 mg kg(-1) d(-1) intraperitoneally) and 10-(4-methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-1 (GPI-15427, 30 mg kg(-1) d(-1) orally) for 10 weeks after the first 2 weeks without treatment. Lens clarity was evaluated by indirect ophthalmoscopy and slit lamp examination, and retinal changes were evaluated by immunohistochemistry and Western blot analysis. In in vitro studies, cultured human lens epithelial cells and bovine retinal pericytes and endothelial cells were exposed to high glucose or palmitate. RESULTS PARP is expressed in lens, and poly(ADP-ribosyl)ated proteins are primarily localized in the 38- to 87-kDa range of the protein spectrum, with several minor bands at 17 to 38 kDa. The 38- to 87-kDa and the 17- to 38-kDa poly(ADP-ribosyl)ated protein expression increased by 74% and 275%, respectively, after 4 weeks of diabetes and by approximately 65% early after exposure of lens epithelial cells to 30 mM glucose. Both PARP inhibitors delayed, but did not prevent, the formation of diabetic cataract. The number of TUNEL-positive nuclei in flatmounted retinas increased approximately 4-fold in STZ diabetic rats, and this increase was prevented by ISO and GPI-15427. Both PARP inhibitors reduced diabetes-induced retinal oxidative-nitrosative and endoplasmic reticulum stress and glial activation. GPI-15427 (20 microM) prevented oxidative-nitrosative stress and cell death in palmitate-exposed pericytes and endothelial cells. CONCLUSIONS PARP activation is implicated in the formation of diabetic cataract and in early retinal changes. These findings provide a rationale for the development of PARP inhibitors for the prevention of diabetic ocular complications.

Induction of endoplasmic reticulum stress in retinal pericytes by glucose deprivation

Diabetic retinopathy is one of the major microvascular complications associated with diabetes mellitus, and the selective degeneration of retinal capillary pericytes is considered to be a hallmark of early retinopathy. Because glucose fluctuations commonly occur in diabetes, we hypothesized that these fluctuations will increase the endoplasmic reticulum (ER) stress and induce the unfolded protein response (UPR) in retinal pericytes. To study whether ER stress and the UPR can be induced in retinal pericytes, rat retinal capillary pericytes were cultured in different concentrations of glucose. Hypoglycemia but not hyperglycemia was found to activate UPR-specific enzymes in pericytes. Strong UPR activation leading to apoptosis was also observed when pericytes were cultured in glucose concentrations that were reduced from high to low or no glucose. These results indicate that induction of UPR is related not only to absolute concentrations but also to a shifting from higher to lower concentrations of glucose.

Multifunctional antioxidants for the treatment of age-related diseases.

Analogues of N,N-dimethyl-4-(pyrimidin-2-yl)piperazine-1-sulfonamide possessing a free radical scavenger group (FRS), chelating groups (CHL), or both (FRS + CHL) have been synthesized. Electrospray ionization mass spectrometry studies indicate that select members of this series bind ions in the relative order of Cu(1+) = Cu(2+) > Fe(2+) = Fe(3+) > Zn(2+) with no binding of Ca(2+) or Mg(2+) observed. In vitro evaluation of these compounds in human lens epithelial, human retinal pigmented epithelial, and human hippocampal astrocyte cell lines indicates that all analogues possessing the FRS group as well as the water-soluble vitamin E analogue 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid protect these cells against decreased cell viability and glutathione levels induced by hydrogen peroxide. In addition, those compounds possessing CHL groups also protected these cells against hydroxyl radicals generated by the Fenton reaction. These compounds are good candidates for the preventive treatment of cataract, age-related macular degeneration (AMD), and Alzheimers dementia (AD).

Osmotic stress, not aldose reductase activity, directly induces growth factors and MAPK signaling changes during sugar cataract formation.

In sugar cataract formation in rats, aldose reductase (AR) activity is not only linked to lenticular sorbitol (diabetic) or galactitol (galactosemic) formation but also to signal transduction changes, cytotoxic signals and activation of apoptosis. Using both in vitro and in vivo techniques, the interrelationship between AR activity, polyol (sorbitol and galactitol) formation, osmotic stress, growth factor induction, and cell signaling changes have been investigated. For in vitro studies, lenses from Sprague Dawley rats were cultured for up to 48 h in TC-199-bicarbonate media containing either 30 mM fructose (control), or 30 mM glucose or galactose with/without the aldose reductase inhibitors AL1576 or tolrestat, the sorbitol dehydrogenase inhibitor (SDI) CP-470,711, or 15 mM mannitol (osmotic-compensated media). For in vivo studies, lenses were obtained from streptozotocin-induced diabetic Sprague Dawley rats fed diet with/without the ARIs AL1576 or tolrestat for 10 weeks. As expected, lenses cultured in high glucose/galactose media or from untreated diabetic rats all showed a decrease in the GSH pool that was lessened by ARI treatment. Lenses either from diabetic rats or from glucose/galactose culture conditions showed increased expression of basic-FGF, TGF-β, and increased signaling through P-Akt, P-ERK1/2 and P-SAPK/JNK which were also normalized by ARIs to the expression levels observed in non-diabetic controls. Culturing rat lenses in osmotically compensated media containing 30 mM glucose or galactose did not lead to increased growth factor expression or altered signaling. These studies indicate that it is the biophysical response of the lens to osmotic stress that results in an increased intralenticular production of basic-FGF and TGF-β and the altered cytotoxic signaling that is observed during sugar cataract formation.

Orally Active Multi-Functional Antioxidants Delay Cataract Formation in Streptozotocin (Type 1) Diabetic and Gamma-Irradiated Rats

Background Age-related cataract is a worldwide health care problem whose progression has been linked to oxidative stress and the accumulation of redox-active metals. Since there is no specific animal model for human age-related cataract, multiple animal models must be used to evaluate potential therapies that may delay and/or prevent cataract formation. Methods/Principal Findings Proof of concept studies were conducted to evaluate 4-(5-hydroxypyrimidin-2-yl)-N,N-dimethyl-3,5-dioxopiperazine-1-sulfonamide (compound 4) and 4-(5-hydroxy-4,6-dimethoxypyrimidin-2-yl)-N,N-dimethyl-3,5-dioxopiperazine-1-sulfonamide (compound 8), multi-functional antioxidants that can independently chelate redox metals and quench free radicals, on their ability to delay the progression of diabetic “sugar” cataracts and gamma radiation-induced cataracts. Prior to 15 Gy of whole head irradiation, select groups of Long Evans rats received either diet containing compound 4 or 8, or a single i.p. injection of panthethine, a radioprotective agent. Compared to untreated, irradiated rats, treatment with pantethine, 4 and 8 delayed initial lens changes by 4, 47, and 38 days, respectively, and the average formation of posterior subcapsular opacities by 23, 53 and 58 days, respectively. In the second study, select groups of diabetic Sprague Dawley rats were administered chow containing compounds 4, 8 or the aldose reductase inhibitor AL1576. As anticipated, treatment with AL1576 prevented cataract by inhibiting sorbitol formation in the lens. However, compared to untreated rats, compounds 4 and 8 delayed vacuole formation by 20 days and 12 days, respectively, and cortical cataract formation by 8 and 3 days, respectively, without reducing lenticular sorbitol. Using in vitro lens culture in 30 mM xylose to model diabetic “sugar” cataract formation, western blots confirmed that multi-functional antioxidants reduced endoplasmic reticulum stress. Conclusions/Significance Multi-functional antioxidants delayed cataract formation in two diverse rat models. These studies provide a proof of concept that a general cataract treatment focused on reducing oxidative stress instead of a specific mechanism of cataractogenesis can be developed.

Topical KINOSTAT™ ameliorates the clinical development and progression of cataracts in dogs with diabetes mellitus

OBJECTIVE To determine whether topical administration of the aldose reductase inhibitor Kinostat™ can ameliorate the onset or progression of cataracts in dogs with naturally occurring diabetes mellitus (DM). MATERIALS AND METHODS A randomized, prospective, double-masked placebo control pilot study was conducted with 40 dogs newly diagnosed with DM with no or minimal lens changes. Twenty-eight dogs received Kinostat™ and 12 dogs received placebo. PROCEDURES Owners administered the agent into both eyes three times daily for 1 year and compliance was monitored with log sheets. Complete ophthalmic examinations were performed on dilated eyes at the time of enrollment and 1, 2, 3, 6, and 12 months into treatment. Cataract severity was assessed on a scale of 0-3. At 12 months, full bloodwork, including HbA1C and blood Kinostat™ levels were performed. RESULTS After 12 months of treatment, the cataract score in the placebo group significantly increased with seven dogs (14 eyes) developing mature cataracts, two dogs (4 eyes) developing cortical opacities, and one dog (2 eyes) developing equatorial vacuoles with mild punctate cortical opacities. In contrast, the cataract score in the Kinostat™ treated dogs was significantly less with seven developing anterior equatorial vacuoles, two developing incipient anterior cortical cataracts, and four developing mature cataracts. In fact, the cataract scores of the Kinostat™ group at 12 months did not significantly increase from the score at the time of enrollment. The HbA1C values between the two groups after 12 months of treatment were similar, and no blood levels of Kinostat™ were found in any enrolled dog. CONCLUSION The onset and/or progression of cataracts in dogs with DM can be significantly delayed by topical administration of Kinostat™.

Aldose reductase, ocular diabetic complications and the development of topical Kinostat(®).

Diabetes mellitus (DM) is a major health problem with devastating effects on ocular health in both industrialized and developing countries. The control of hyperglycemia is critical to minimizing the impact of DM on ocular tissues because inadequate glycemic control leads to ocular tissue changes that range from a temporary blurring of vision to permanent vision loss. The biochemical mechanisms that promote the development of diabetic complications have been extensively studied. As a result, a number of prominent biochemical pathways have been identified. Among these, the two-step sorbitol pathway has been the most extensively investigated; nevertheless, it remains controversial. To date, long-term pharmacological studies in animal models of diabetes have demonstrated that the onset and development of ocular complications that include keratopathy, retinopathy and cataract can be ameliorated by the control of excess metabolic flux through aldose reductase (AR). Clinically the alleles of AR have been linked to the rapidity of onset and severity of diabetic ocular complications in diabetic patient populations around the globe. In spite of these promising preclinical and human genetic rationales, several clinical trials of varying durations with structurally diverse aldose reductase inhibitors (ARIs) have shown limited success or failure in preventing or arresting diabetic retinopathy. Despite these clinical setbacks, topical ARI Kinostat(®) promises to find a home in clinical veterinary ophthalmology where its anticipated approval by the FDA will present an alternative treatment paradigm to cataract surgery in diabetic dogs. Here, we critically review the role of AR in diabetes mellitus-linked ocular disease and highlight the development of Kinostat(®) for cataract prevention in diabetic dogs. In addition to the veterinary market, we speculate that with further safety and efficacy studies in humans, Kinostat(®) or a closely related product could have a future role in treating diabetic keratopathy.