This study aimed to characterize the role of female sex in the pathogenesis of diabetic retinopathy. In the retinae of female Ins2Akita-diabetic mice (F-IA), ovariectomized female Ins2Akita-diabetic mice (F-IA/OVX), male Ins2Akita-diabetic mice (M-IA), and female STZ-diabetic mice (F-STZ), the formation of reactive metabolites and post-translational modifications, damage to the neurovascular unit, and expression of cellular stress response genes were analyzed. Compared to the male diabetic retina, the concentrations of the glycation adduct fructosyl-lysine, the Maillard product 3-deoxyglucosone, and the reactive metabolite methylglyoxal were significantly reduced in females. In females, there was also less evidence of diabetic damage to the neurovascular unit, as shown by decreased pericyte loss and reduced microglial activation. In the male diabetic retina, the expression of several members of the crystallin gene family (Cryab, Cryaa, Crybb2, Crybb1, and Cryba4) was increased. Clinical data from type 1 diabetic females showed that premenopausal women had a significantly lower prevalence of diabetic retinopathy compared to postmenopausal women stratified for disease duration and glycemic control. These data emphasize the importance of estradiol in protecting the diabetic retina and highlight the pathogenic relevance of sex in diabetic retinopathy.

The retina is prone to developing pathological neovascularization, a leading cause of blindness in humans. Because excess neovascularization does not affect the entire retina, global inhibition treatment of angiogenesis critically interferes with healthy, unaffected retinal tissue. We therefore established an in vivo photoactivated gene expression paradigm which would allow light-mediated targeting of antiangiogenic genetic treatment only to affected retinal regions. We synthesized a "caged" (i.e., reversibly inhibited) photosensitive 4-hydroxytamoxifen analog. Molecular docking analyses validated its reduced transcriptional activity. Caged 4-hydroxytamoxifen was intravitreally injected into mice harboring the inducible Cre/lox system, with CreERT2 being expressed via the Tie2 promoter in the neurovasculature. Subsequent in vivo irradiation of eyes significantly induced retinal expression of a Cre-dependent transgene in retinal blood vessels. Using GFAP-CreERT2 mice, successful photoactivation was also achieved in eyes and also in ex vivo brain slices for validation of the approach. This highlights the possibility of light-mediated gene therapies specific for the retina, a key first step in personalized medicine.

Background: Diabetic nephropathy (DN) is one of the most severe microvascular complications of diabetes. Interstitial fibrosis/tubular atrophy and renal function decline are typical hallmarks of advanced DN. Pericytes play an important role in kidney vascular function and fibrosis. Murine double minute 2 (MDM2) is an E3 ubiquitin ligase of p53. MDM2 is expressed in glomeruli and involved in podocyte injury. MDM2-inhibition reduces inflammation and fibrosis. However, whether MDM2 affects diabetic renal injury is unclear. This study aims to investigate the effects of pericyte conditional MDM2-KO on renal function and morphology.

Diabetic kidney disease (DKD) is one of the leading causes to develop end-stage kidney disease worldwide. Pericytes are implicated in the development of tissue fibrosis. However, the underlying mechanisms of pericytes in DKD remain largely unknown. We isolated and cultured primary pericytes and rat mesangial cells (HBZY-1). Western blot and qRT-PCR analysis were used to explore the role and regulatory mechanism of Integrin β8/transforming growth factor beta 1 (TGF-β1) pathway. We also constructed pericyte-specific Integrin β8 knock-in mice as the research objects to determine the role of Integrin β8 in vivo. We discovered that reduced Integrin β8 expression was closely associated with pericyte transition in DKD. Overexpressed Integrin β8 in pericytes dramatically suppressed TGF-β1/TGF beta receptor 1 (TGFBR1)/Smad3 signaling pathway and protected glomerular endothelial cells (GECs) in vitro. In vivo, pericyte-specific Integrin β8 knock-in ameliorated pericyte transition, endothelium injury and renal fibrosis in STZ-induced diabetic mice. Mechanistically, Murine double minute 2 (MDM2) was found to increase the degradation of Integrin β8 and caused TGF-β1 release and activation. Knockdown MDM2 could partly reverse the decline of Integrin β8 and suppress pericytes transition. In conclusion, the present findings suggested that upregulated MDM2 expression contributes to the degradation of Integrin β8 and activation of TGF-β1/TGFBR1/Smad3 signaling pathway, which ultimately leads to pericyte transition during DKD progression. These results indicate MDM2/Integrin β8 might be considered as therapeutic targets for DKD.

Diabetic retinopathy (DR) is characterized by vasoregression and glial activation. miRNA-124 (miR-124) reduces retinal microglial activation and alleviates vasoregression in a neurodegenerative rat model. Our aim was to determine whether miR-124 affects vascular and neural damage in the early diabetic retina. Diabetes was induced in 8-week-old Wistar rats by streptozotocin (STZ) injection. At 16 and 20 weeks, the diabetic rats were intravitreally injected with miR-124 mimic, and retinae were analyzed at 24 weeks. Microvascular damage was identified by evaluating pericyte loss and acellular capillary (AC) formation. Müller glial activation was assessed by glial fibrillary acidic protein (GFAP) immunofluorescence staining. Microglial activation was determined by immunofluorescent staining of ionized calcium-binding adaptor molecule 1 (Iba1) in whole mount retinae. The neuroretinal function was assessed by electroretinography. The expression of inflammation-associated genes was evaluated by qRT-PCR. A wound healing assay was performed to quantitate the mobility of microglial cells. The results showed that miR-124 treatment alleviated diabetic vasoregression by reducing AC formation and pericyte loss. miR-124 blunted Müller glial- and microglial activation in diabetic retinae and ameliorated neuroretinal function. The retinal expression of inflammatory factors including Tnf-α, Il-1β, Cd74, Ccl2, Ccl3, Vcam1, Tgf-β1, Arg1, and Il-10 was reduced by miR-124 administration. The elevated mobility of microglia upon high glucose exposure was normalized by miR-124. The expression of the transcription factor PU.1 and lipid raft protein Flot1 was downregulated by miR-124. In rat DR, miR-124 prevents vasoregression and glial activation, improves neuroretinal function, and modulates microglial activation and inflammatory responses.

Background: Zebrafish (Danio rerio) have been established in recent years as a model organism to study Diabetic Retinopathy (DR). Loss of endothelial cells and pericytes is an early hallmark sign of developing DR in the mammalian retina. However, morphology, numbers, ratios, and distributions of different vascular cells in the retinal compartment in zebrafish have not yet been analyzed and compared with the mammalian retina. Methods: The retinal trypsin digest protocol was established on the zebrafish retina. Cell types were identified using the Tg(nflk:EGFP)-reporter line. Cells were quantified using quantitative morphometry. Results: Vascular cells in the zebrafish retina have distinct morphologies and locations. Nuclei of vascular mural cells appear as long and flat nuclei located near the vessel wall. Round nuclei within the vessel walls can be identified as endothelial cells. The vessel diameter decreases from central to peripheral parts of the retina. Additionally, the numbers of vascular cells decrease from central to peripheral parts of the retina. Discussion: The retinal trypsin digest protocol, which can be applied to the zebrafish retina, provides novel insights into the zebrafish retinal vascular architecture. Quantification of the different cell types shows that, in comparison to the mammalian retina, zebrafish have higher numbers of mural cells and an increased mural cell to endothelial cell ratio. This protocol enables to quantify mural cell and endothelial cell numbers, is easily adaptable to different transgenic and mutant zebrafish lines and will enable investigators to compare novel models on a single cell level.

ABSTRACTObjectiveType 2 diabetes mellitus (T2D) is a chronic disease that is influenced by different factors. The extent to which degree adverse childhood events (ACEs) can modify the potential to development of T2D is still not explored and therefore represents one of the central questions of the childhood escape–late life outcome (DRKS00012419) study. In addition, transgenerational effects were considered in the analyses.MethodsThe study analyzed the association of self-reported traumatic experiences and T2D disease of refugees from East Prussia, who were displaced from their former homeland at the end of the World War II. In addition, an independent sample consisting of participants of first-generation offspring of refugees was analyzed.ResultsOf the 242 refugees, all aged between 73 and 93 years, 17.36% reported T2D disease, whereas among the offspring (n= 272), aged between 47 and 73 years, it was 5.5%, meaning reduced T2D prevalence for both generations compared with the German population of comparable age. In the refugee generation, emotional neglect showed a negative association with development of T2D in later life. In women, separation from close caregivers in childhood showed a negative association with later T2D. In contrast, experiencing emotional abuse in childhood showed a positive association with later T2D. The offspring generation showed no associations of adverse childhood events and reported T2D diagnoses in later life.ConclusionsOur results demonstrate that individual trauma in childhood is responded to with different mechanisms that can lead to both increased and decreased reported T2D diagnoses in adulthood and thus should by no means be considered in a generalized manner.

Aims Glucagon-like peptide-1 receptor agonists are effective treatments for type 2 diabetes, effectively lowering glucose without weight gain and with low risk for hypoglycemia. However, their influence on the retinal neurovascular unit remains unclear. In this study, we analyzed the effects of the GLP-1 RA lixisenatide on diabetic retinopathy. Methods Vasculo- and neuroprotective effects were assessed in experimental diabetic retinopathy and high glucose-cultivated C. elegans, respectively. In STZ-diabetic Wistar rats, acellular capillaries and pericytes (quantitative retinal morphometry), neuroretinal function (mfERG), macroglia (GFAP western blot) and microglia (immunohistochemistry) quantification, methylglyoxal (LC–MS/MS) and retinal gene expressions (RNA-sequencing) were determined. The antioxidant properties of lixisenatide were tested in C. elegans. Results Lixisenatide had no effect on glucose metabolism. Lixisenatide preserved the retinal vasculature and neuroretinal function. The macro- and microglial activation was mitigated. Lixisenatide normalized some gene expression changes in diabetic animals to control levels. Ets2 was identified as a regulator of inflammatory genes. In C. elegans, lixisenatide showed the antioxidative property. Conclusions Our data suggest that lixisenatide has a protective effect on the diabetic retina, most likely due to a combination of neuroprotective, anti-inflammatory and antioxidative effects of lixisenatide on the neurovascular unit.

Abstract Background Retinal screening is mandatory to prevent vision loss and blindness due to diabetic retinopathy (DR). The aim of the study was to determine retinopathy screening rates and potential barriers in a German metropolitan diabetes care center. Methods Between May and October 2019, 265 patients with diabetes mellitus (95% type 2 diabetes; age 62±13.2 years; diabetes duration 11.1±8.5 years, HbA1c 7.4±1.0%) were referred to an ophthalmologist (referral form with order “Fundoscopy in diabetes mellitus, findings requested,” completed documentation form “General practitioner’s/diabetologist’s report to the ophthalmologist” and prepared documentation form “Ophthalmologist’s report”). A structured interview was used to assess the level of compliance with the guidelines and to identify potential barriers to retinopathy screening in a real-world setting, including the quantification of extra payments. Results All patients were interviewed at 7.9±2.5 months after the referral for retinopathy screening had been issued. According to patient reporting, fundoscopy was performed in 191 (75%) patients. Ophthalmological reports were obtained from 119/191 (62%) patients (46% of the entire cohort). 10/119 (8%) patients had been previously diagnosed with DR and 6/119 (5%) with new-onset DR. In 158/191 (83%) of patients, the referral had been accepted by the ophthalmology practice, of which 25,1% made a co-payment of 36.2±37.6 €. Discussion Despite a high screening performance in a real-world setting, complete screening in compliance with German guidelines, including written reporting, was found in less than half of the cohort. The prevalence and incidence of DR are high. Even when referred according to the regulations, one-quarter of patients made a co-payment. Efficient solutions to current barriers can emerge with mutual time-saving information prior to examination and feedback about the implementation of findings into treatment.

Diabetic nephropathy (DN) is a major cause of morbidity and mortality in diabetes and is the most common cause of end stage renal disease (ESRD). Renal fibrosis is the final pathological change in DN. It is widely believed that cellular phenotypic switching is the cause of renal fibrosis in diabetic nephropathy. Several types of kidney cells undergo activation and differentiation and become reprogrammed to express markers of mesenchymal cells or podocyte-like cells. However, the development of targeted therapy for DN has not yet been identified. Here, we discussed the pathophysiologic changes of DN and delineated the possible origins that contribute to myofibroblasts and podocytes through phenotypic transitions. We also highlight the molecular signaling pathways involved in the phenotypic transition, which would provide valuable information for the activation of phenotypic switching and designing effective therapies for DN.

Chronic kidney disease (CKD) is a progressive systemic disease, which changes the function and structure of the kidneys irreversibly over months or years. The final common pathological manifestation of chronic kidney disease is renal fibrosis and is characterized by glomerulosclerosis, tubular atrophy, and interstitial fibrosis. In recent years, numerous studies have reported the therapeutic benefits of natural products against modern diseases. Substantial attention has been focused on the biological role of polyphenols, in particular flavonoids, presenting broadly in plants and diets, referring to thousands of plant compounds with a common basic structure. Evidence-based pharmacological data have shown that flavonoids play an important role in preventing and managing CKD and renal fibrosis. These compounds can prevent renal dysfunction and improve renal function by blocking or suppressing deleterious pathways such as oxidative stress and inflammation. In this review, we summarize the function and beneficial properties of common flavonoids for the treatment of CKD and the relative risk factors of CKD.

The pdx1-/- zebrafish mutant was recently established as a novel animal model of diabetic retinopathy. Here, we investigate whether knockout of pdx1 also leads to diabetic kidney disease (DKD). pdx1-/- larvae exhibit several signs of early DKD such as glomerular hypertrophy, impairments in the filtration barrier corresponding to microalbuminuria and glomerular basement membrane (GBM) thickening. Adult pdx1-/- mutants show progressive GBM thickening in comparison to the larval state. Heterozygous pdx1 knockout also leads to glomerular hypertrophy as initial establishment of DKD similar to the pdx1-/- larvae. RNA sequencing (RNA-seq) of adult pdx1+/- kidneys uncovered regulations in multiple expected diabetic pathways related to podocyte disruption and hinting at early vascular dysregulation without obvious morphological alterations. Metabolome analysis and pharmacological intervention experiments revealed the contribution of phosphatidylethanolamine (PtdE) in the early establishment of kidney damage. In conclusion, this study identified the pdx1 mutant as a novel model for the study of DKD showing signs of the early disease progression already in larval stage and several selective features of later DKD in adult mutants.

C. elegans are used to study molecular pathways, linking high glucose levels (HG) to diabetic complications. Persistent exposure of C. elegans to a HG environment induces the mitochondrial formation of reactive oxygen species (ROS) and advanced glycation endproducts (AGEs), leading to neuronal damage and decreased lifespan. Studies suggest that transient high glucose exposure (TGE) exerts different effects than persistent exposure. Thus, the effects of TGE on ROS, AGE-formation and life span were studied in C. elegans. Four-day TGE (400 mM) as compared to controls (0mM) showed a persistent increase of ROS (4-days 286 ± 40 RLUs vs. control 187 ± 23 RLUs) without increased formation of AGEs. TGE increased body motility (1-day 0.14 ± 0.02; 4-days 0.15 ± 0.01; 6-days 0.16 ± 0.02 vs. control 0.10 ± 0.02 in mm/s), and bending angle (1-day 17.7 ± 1.55; 3-days 18.7 ± 1.39; 6-days 20.3 ± 0.61 vs. control 15.3 ± 1.63 in degree/s) as signs of neuronal damage. Lifespan was increased by 27% (21 ± 2.4 days) after one-day TGE, 34% (22 ± 1.2 days) after four-days TGE, and 26% (21 ± 1.4 days) after six-days TGE vs. control (16 ± 1.3 days). These experiments suggest that TGE in C. elegans has positive effects on life span and neuronal function, associated with mildly increased ROS-formation. From the perspective of metabolic memory, hormetic effects outweighed the detrimental effects of a HG environment.

Diabetic kidney disease (DKD) is a major microvascular complication of diabetes mellitus and is one of the leading causes of end-stage kidney disease. Circular RNAs (circRNAs) are a class of endogenous non-coding RNAs that play important roles in various diseases, yet their roles in DKD are poorly understood. CircRNA HIPK3 (circHIPK3), a highly conserved circRNA, is closely related to various cellular functions, including cell proliferation and apoptosis. The association between circHIPK3 and diabetic complications has been well demonstrated in multiple previous studies. However, the role of circHIPK3 in podocyte injury in DKD remains unclear. Herein, we discovered that circHIPK3 expression is markedly elevated in cultured podocytes under high-glucose (HG) conditions and glomeruli of diabetic mice, which is closely associated with podocyte injury in DKD. Functionally, lentivirus-mediated knockdown of circHIPK3 dramatically suppresses HG-induced podocyte apoptosis in vitro. Therapeutically, silencing circHIPK3 by adeno-associated virus-mediated RNA interference ameliorates podocyte injury and albuminuria in STZ-induced diabetic mice. Mechanistically, circHIPK3 facilitates the enrichment of fused in sarcoma (FUS) on the ectodysplasin A2 receptor (EDA2R) promoter, resulting in the upregulation of EDA2R expression and activation of apoptotic signaling. Taken together, these results indicate circHIPK3/FUS/EDA2R axis as a therapeutic target for podocyte injury and DKD progression.

Pericyte dropout, an early sign of retinal diabetic microvascular complications, can be quantified using the retinal trypsin digest protocol. Zebrafish have recently come into focus as a model organism to study diabetic complications, however, the zebrafish retina has not been analysed using the trypsin digest preparation.