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The cardioprotective effects of sodium glucose cotrasponter 2 (SGLT2) inhibitors seem to be independent from the effects on glycemic control, through little-known mechanisms. In this study, we investigate whether the cardioprotective effects of empagliflozin, a SGLT2 inhibitor, may be associated with myocardial sympathetic activity and inflammatory cell infiltration in an experimental model of angiotensin II-dependent hypertension. Angiotensin II (Ang II), Ang II plus Empagliflozin, physiological saline, or physiological saline plus empagliflozin were administered to Sprague Dawley rats for two weeks. Blood pressure was measured by plethysmographic method. Myocardial hypertrophy and fibrosis were analysed by histomorphometry, and inflammatory cell infiltration and tyrosine hydroxylase expression, implemented as a marker of sympathetic activity, were evaluated by immunohistochemistry. Ang II increased blood pressure, myocardial hypertrophy, fibrosis, inflammatory infiltrates and tyrosine hydroxylase expression, as compared to the control group. Empagliflozin administration prevented the development of myocardial hypertrophy, fibrosis, inflammatory infiltrates and tyrosine hydroxylase overexpression in Ang II-treated rats, without affecting blood glucose and the Ang II-dependent increase in blood pressure. These data demonstrate that the cardioprotective effects of SGLT2 inhibition in Ang II-dependent hypertension may result from the myocardial reduction of sympathetic activity and inflammation and are independent of the modulation of blood pressure and blood glucose levels.

Purpose There is no valid medical treatment for diabetic retinopathy mostly because its pathogenesis remains largely unknown. Early stages of diabetic retinopathy, just like glaucoma, are characterized by the loss of retinal ganglion cells. Whether the two diseases may share a similar pathogenic background is unknown. Methods To clarify this issue the thickness of retinal nerve fiber layer was studied in vivo by optical coherence tomography in 10 Ins2Akita (diabetic) and 10 C57BL/6J (control) mice. The number of retinal ganglion cells and retina's surface covered by neurofilaments were quantified ex vivo in 12 normoglycemic DBA/2J (glaucoma) and 11 diabetic (alloxan-induced) DBA/2J mice (glaucoma + diabetes). Results At 16 weeks of age retinal nerve fiber layer was significantly thinner in Ins2Akita mice confirming the neurodegenerative impact of diabetes. Number of retinal ganglion cells and retina's surface covered by neurofilaments were similar in normoglycemic and diabetic DBA/2J mice with the exception of the superior quadrant where the number of retinal ganglion cells was increased in animals with glaucoma + diabetes. Conclusions In presence of glaucoma, diabetes is unable to induce further retinal ganglion cells loss. The hypothesis that the mechanism leading to retinal ganglion cells loss may be shared by the two diseases cannot be ruled out. Whether early diabetes-driven retinal neurodegeneration could be prevented by neuroprotective treatment proven to be effective in case of glaucoma, remains to be clarified.

Aims Sodium-glucose cotransporter 2 (SGLT2) inhibitors, a new class of antidiabetic drugs, are nephroprotective in case of diabetes, but whether a similar beneficial effect may be detectable also in case of chronic non-diabetic kidney diseases remains still unknown. The aim of this study was to evaluate the effects of empagliflozin, a SGLT-2 inhibitor, on the progression of cyclosporine nephropathy, in the absence of diabetes. Methods Sprague Dawley rats (n = 27) have been fed with low-salt diet starting 10 days before the beginning and finished at the end of the experimental period. Cyclosporine-A (CsA, 15 mg/kg/day, intraperitoneal injection, n = 8) and CsA plus empagliflozin (Empa, 10 mg/kg/day, per os, n = 7) were administered for 4 weeks. The control groups were treated with placebo (Control, n = 7) or empagliflozin (Control + Empa, n = 5). Blood pressure (plethysmographic method) was measured at the beginning and at the end of the experimental period. At the end of the experimental protocol, the kidneys were excised for histomorphometric analysis of renal fibrosis and for immunohistochemical evaluation of inflammatory infiltrates (monocytes/macrophages), type I and type IV collagen expression, and tyrosine hydroxylase expression, used as marker of sympathetic nerve activity. Results CsA-treated rats showed a significant increase (p p p p p p p p p p p Conclusion Empagliflozin administration caused a reduction in blood pressure in CsA-treated rats and showed a protective effect on CsA nephropathy by decreasing renal fibrosis, type I and type IV collagen expression, macrophage infiltration and tyrosine hydroxylase expression. These data suggest that empagliflozin promotes nephroprotection also in non-diabetic kidney disease.

Background: Clinical trials have shown that empagliflozin (Empa), a sodium-glucose cotransporter 2 (SGLT2) inhibitor, promotes nephroprotective effects in diabetic patients. The mechanisms underlying nephroprotection are not completely known and it is not known whether the renal beneficial action is present even in non-diabetic kidney disease. The aim of this study was to evaluate the effect of Empa administration on the development of renal fibrosis in an experimental model of angiotensin II (Ang II)-dependent hypertension. Methods: Sprague Dawley rats (n = 31) were divided into 4 experimental groups. Ang II (200 ng/kg/min, osmotic minipumps, s.c., n = 9) or Ang II + Empa (10 mg/kg/day, per os, n = 10) were administered for 2 weeks. Control rats were treated with placebo (physiological saline, n = 6), and another group was treated with placebo plus Empa (n = 6) for the same period. Blood pressure (plethysmographic method) was measured at the beginning and at the end of the experimental protocol. After 2 weeks, the rats were euthanized and the kidneys were excised for histomorphometric evaluation of glomerular and tubulo-interstitial fibrosis and for the immunohistochemical evaluation of inflammatory infiltrates (monocytes/macrophages) and types I and IV collagen expression. Results: The administration of Ang II resulted in an increase in blood pressure (p < 0.01), glomerular (p < 0.05) and tubulo-interstitial (p < 0.01) fibrosis, renal inflammatory infiltrates (p < 0.01) and type I (p < 0.01) and type IV collagen expression (p < 0.05) compared to the control group. Treatment with Empa did not significantly modify the increase in blood pressure due to Ang II, but prevented the development of renal glomerular and tubulo-interstitial fibrosis, and the increase in inflammatory infiltrates and types I and IV collagen expression in Ang II-treated rats (p < 0.01). Conclusions: These data demonstrate that the treatment with Empa prevents the development of renal fibrosis in Ang II-dependent hypertension. In Ang II-dependent hypertension, the anti-fibrotic effect due to SGLT2 inhibition is caused by the reduction of inflammatory infiltrates and it is independent on the modulation of blood pressure increase.

Background Pre-eclampsia has a major impact on renal function as shown by the development of proteinuria and podocyturia. How the systemic, soluble Fms-like tyrosine kinase-1 (sFlt-1)-driven inhibition of vascular endothelial growth factor (VEGF) activity detected in pre-eclampsia directly affects renal function remains unknown. The aim of the study was to clarify whether a non-canonical, renal-centred escape from VEGF inhibition in the case of pre-eclamptic pregnancy might have a direct impact on renal function. Methods We evaluated plasma and urinary VEGF and placental growth factor (PlGF), plasma sFlt-1 and carbonic anhydrase IX (CAIX), albuminuria and podocyturia in 18 women with uncomplicated pregnancy, 21 with pre-eclampsia and 18 non-pregnant. The three groups were matched for age and the pregnant groups also for gestational age at enrolment. Results Plasma VEGF was reduced in uncomplicated (P = 0.001) and pre-eclamptic (P = 0.0003) pregnancies when compared with controls. In uncomplicated pregnancy, the dysfunction was balanced by an increase (P = 0.009) of plasma PlGF. Increased (P = 0.0001) plasma CAIX in pre-eclampsia was in line with hypoxia. Pre-eclampsia resulted in a paradoxical increase (P = 0.0004) of urinary excretion of VEGF. Urinary concentrations of VEGF and podocytes were correlated to each other (r2 = 0.48, P Conclusions In the case of pre-eclampsia, the systemic VEGF inhibition leads the kidney, possibly the podocyte, to increase the VEGF synthesis. The mechanisms leading to local VEGF overproduction or the overproduced VEGF itself are reasonably involved in the pathogenesis of podocyturia and, as a consequence, renal dysfunction in pre-eclampsia.

Despite research progresses, the chance to accurately predict the risk for diabetic nephropathy (DN) is still poor. So far, the first evidence of DN is micro-albuminuria, which is detected only 10–20 years after the onset of diabetes. Our goal is to develop new predictive tools of nephropathy starting from urine, which can be easily obtained using noninvasive procedures and it is directly related to kidney. Since it is reasonable to suppose that, in predisposed patients, the mechanisms leading to nephropathy start acting since the diabetes onset, urine from children with recent diagnosis of type 1 diabetes was subjected to proteomic analysis in comparison to age-matched controls. Targeted confirmation was performed on children with a longer history of diabetes using Western Blotting and applying a urinary lipidomic approach. To definitively understand whether the observed alterations could be related to diabetic nephropathy, urine from diabetic adults with or without albuminuria was also examined. For the first time, lipid metabolisms of prostaglandin and ceramide, which are significantly and specifically modified in association with DN, are shown to be already altered in children with a recent diabetes diagnosis. Future studies on larger cohorts are needed to improve the validity and generalizability of these findings. Data are available via ProteomeXchange with identifier PXD011183 Submission details: Project Name: Urinary proteomics by 2DE and LC-MS/MS. Project accession: PXD011183 Project DOI: https://doi.org/10.6019/PXD011183 Significance Nephropathy is a very common diabetic complication. Once established, its progression can only be slowed down but full control or remission is achieved in very few cases, thus posing a large burden on worldwide health. The first evidence of diabetic nephropathy (DN) is micro-albuminuria, but only 30% of patients with micro-albuminuria progress to proteinuria, while in some patients it spontaneously reverts to normo-albuminuria. Thus, there is clear need for biomarkers that can accurately predict the risk to develop DN. Herein, by applying proteomic and lipidomic approaches on urine samples, we show that alteration of prostaglandin and ceramide metabolisms specifically occurs in association with DN. Interestingly, we demonstrate that the modification of these metabolic pathways is an early event in diabetic patients, suggesting the identified changed proteins as possible predictive biomarkers of diabetes-induced renal function decline.

Background and Aims Sodium glucose cotransporter 2 (SGLT2) inhibitors, a new class of antidiabetic drugs, showed nephroprotection in type 2 diabetic patients. The mechanisms underlying nephroprotection are not completely known and it is unclear whether the nephroprotective effects are present also in non-diabetic nephropathy. The aim of this study was to evaluate the effects of empagliflozin, a SGLT-2 inhibitor, in cyclosporine nephropathy in the absence of diabetes. Method Ten days before the beginning and then during the entire experimental periods, low-salt diet (Teklad 7034) was administered to Sprague Dawley rats. Cyclosporine-A (CsA, 15 mg/kg/day, intraperitoneal injection; n=6) and CsA plus empagliflozin (Empa, 10 mg/kg /day, per os; n=6) were administered for 4 weeks. Control group was treated with placebo (n=6). Blood pressure was measured by plethysmographic method at the beginning and at the end of the experimental period. At the end of the protocol, the kidneys were excised for histomorphometric analysis of renal fibrosis and for immunohistochemical evaluation of inflammatory infiltrates and tyrosine hydroxylase expression, used as marker of symphatetic nerve activity. Results The rats treated with CsA showed a significant increase (p Conclusion Empagliflozin administration showed protective effects on cyclosporine nephropathy, decreasing renal fibrosis, macrophage infiltration and tyrosine hydroxylase expression. These data suggest that the nephroprotective role of empagliflozin could not be restricted only to diabetic nephropathy.

The increased mortality reported with intensive glycaemic control has been attributed to an increased risk of treatment-related hypoglycaemia. This study investigated the relationships of haemoglobin (Hb) A1c, anti-hyperglycaemic treatment, and potential risks of adverse effects with all-cause mortality in patients with type 2 diabetes. Patients (n = 15,773) were stratified into four categories according to baseline HbA1c and then assigned to three target categories, based on whether HbA1c was &le, 0.5% below or above (on-target), &gt, 0.5% below (below-target) or &gt, 0.5% above (above-target) their HbA1c goal, personalized according to the number of potential risks among age &gt, 70 years, diabetes duration &gt, 10 years, advanced complication(s), and severe comorbidity (ies). The vital status was retrieved for 15,656 patients (99.26%). Over a 7.4-year follow-up, mortality risk was increased among patients in the highest HbA1c category (&ge, 8.5%) (adjusted hazard ratio, 1.34 (95% confidence interval, 1.22&ndash, 1.47), p &lt, 0.001) and those above-target (1.42 (1.29&ndash, 1.57), p &lt, 0.001). Risk was increased among individuals in the lowest HbA1c category (&lt, 6.5%) and those below-target only if treated with agents causing hypoglycaemia (1.16 (1.03&ndash, 1.29), p = 0.01 and 1.10 (1.01&ndash, 1.22), p = 0.04, respectively). These data suggest the importance of setting both upper and lower personalized HbA1c goals to avoid overtreatment in high-risk individuals with type 2 diabetes treated with agents causing hypoglycaemia.

Purpose: The aim of this study was to characterize the response to aflibercept in a mouse model of type 3 neovascularization, the neoretinal vascularization (NRV) 2 mouse line. Methods: Twelve NRV2 mice were assigned to one of the following groups: (1) 6 mice were injected with aflibercept 3 µg/g at postnatal day (P) 15 (“aflibercept” group), and (2) the remaining 6 mice did not receive any treatment (“placebo” group). The mice were examined at P30 and P44. Results: The NRV mice’s retinas were characterized by regions of depigmentation that were topographically associated with hyperfluorescent lesions seen on fluorescein angiography (FA) images. On optical coherence tomography images, intraretinal neovascularizations were visualized as hyperreflective lesions mainly localized within the outer plexiform and outer nuclear layers. The average number of intraretinal neovascular lesions visualized on FA at P30 was 5.0 ± 2.2 in the aflibercept group and 20.7 ± 2.4 in the placebo group (p < 0.0001). At P44, the average number of intraretinal lesions was still lower in the aflibercept group, although this difference was not statistically significant (p = 0.088). Conclusion: Aflibercept therapy was effective in inhibiting pathologic angiogenesis in the NRV2 mouse model. However, the successive treatment washout resulted in an increase in the number of lesions.

There is a lack of studies evaluating the sub-clinical retinal changes in patients with long-term type 1 diabetes mellitus (T1DM) and without history of systemic/ocular complications. The aim of this cross-sectional study was to investigate sub-clinical structural and/or vascular retinal changes in patients with long-term (≥30 years) T1DM and without systemic/ocular complications (“happy few” patients) using structural optical coherence tomography (OCT), OCT-angiography and microperimetry. Twelve eyes of 12 consecutive T1DM patients (mean age 52 ± 12 years, mean duration of disease 35 ± 3 years, mean HbA1c level 7.3 ± 2.8%), without micro/macrovascular complications associated with long-standing T1DM, and twelve healthy subjects were consecutively included. No statistically significant differences were disclosed comparing patients and controls for age, sex, best-corrected visual acuity, central macular thickness, and choroidal thickness. Using OCT-angiography, we did not find any significant difference in foveal avascular zone area, perfusion density, vessel length density, and tortuosity. Moreover, no significant differences were disclosed in retinal nerve fiber layer and ganglion cell complex thickness using structural OCT. No differences were disclosed in retinal sensitivity by microperimetry. New diagnostic tools are able to confirm the presence of a particular population of patients with type 1 diabetes who have been completely spared from diabetic retinal complications. The finding of these “happy few” patients could help us to better understand and target future treatments for diabetes.

BACKGROUND: Although diabetic kidney disease demonstrates both familial clustering and single nucleotide polymorphism heritability, the specific genetic factors influencing risk remain largely unknown.METHODS: To identify genetic variants predisposing to diabetic kidney disease, we performed genome-wide association study (GWAS) analyses. Through collaboration with the Diabetes Nephropathy Collaborative Research Initiative, we assembled a large collection of type 1 diabetes cohorts with harmonized diabetic kidney disease phenotypes. We used a spectrum of ten diabetic kidney disease definitions based on albuminuria and renal function.RESULTS: Our GWAS meta-analysis included association results for up to 19,406 individuals of European descent with type 1 diabetes. We identified 16 genome-wide significant risk loci. The variant with the strongest association (rs55703767) is a common missense mutation in the collagen type IV alpha 3 chain (COL4A3) gene, which encodes a major structural component of the glomerular basement membrane (GBM). Mutations in COL4A3 are implicated in heritable nephropathies, including the progressive inherited nephropathy Alport syndrome. The rs55703767 minor allele (Asp326Tyr) is protective against several definitions of diabetic kidney disease, including albuminuria and ESKD, and demonstrated a significant association with GBM width; protective allele carriers had thinner GBM before any signs of kidney disease, and its effect was dependent on glycemia. Three other loci are in or near genes with known or suggestive involvement in this condition (BMP7) or renal biology (COLEC11 and DDR1).CONCLUSIONS: The 16 diabetic kidney disease-associated loci may provide novel insights into the pathogenesis of this condition and help identify potential biologic targets for prevention and treatment.

Diabetic kidney disease (DKD) is a heritable but poorly understood complication of diabetes. To identify genetic variants predisposing to DKD, we performed genome-wide association analyses in 19,406 individuals with type 1 diabetes (T1D) using a spectrum of DKD definitions basedon albuminuria and renal function. We identified 16 genome-wide significant loci. The variant with the strongest association (rs55703767) is a common missense mutation in the collagen type IV alpha 3 chain(COL4A3)gene, which encodes a major structural component of the glomerular basement membrane (GBM) implicated in heritable nephropathies. The rs55703767 minor allele (Asp326Tyr) is protective against several definitions of DKD, including albuminuria and end-stage renal disease. Three other loci are in or near genes with known or suggestive involvement in DKD(BMP7)or renal biology (COLEC11andDDR1). The 16 DKD-associated loci provide novel insights into the pathogenesis of DKD, identifying potential biological targets for prevention and treatment.

To evaluate the plasma concentration of the soluble form of the urokinase-type plasminogen activator receptor ((s)uPAR), an established biomarker of chronic inflammation, in patients affected by neovascular age-related macular degeneration. Forty consecutive patients affected by age-related macular degeneration and 52 subjects with no history of the disease were included in this case–control study. The two groups of individuals considered for the study were matched for age, sex, and class of medications taken. Plasma concentration of suPAR was measured using a specific ELISA assay (suPARnostic, Birkeroed, Denmark). The case and control groups were similar for age, gender distribution, weight, height, and systolic and diastolic blood pressure, as well as for dyslipidemia and high blood pressure medication (P > 0.28). The plasma concentrations of suPAR were significantly increased in patients with neovascular age-related macular degeneration when compared to controls (6.19 ± 2.2 ng/ml, vs 5.21 ± 1.5, respectively, mean ± SD P = 0.01). Patients with neovascular age-related macular degeneration display increased plasma levels of suPAR, suggesting that chronic inflammation may be involved in the pathogenesis of the disease.

It usually takes several years (in some cases, decades) for predisposed individuals to move from the onset of type 1 or type 2 diabetes to the development of microalbuminuria, the first sign of diabetic nephropathy. This long, complication-free, period represents the best possible moment to start a successful preventive strategy (primary prevention) aimed to avoid or at least to postpone the increase of albumin excretion rate. Prevention is based on understanding and counteracting the initial mechanisms leading to the development of the disease and unfortunately, in case of diabetic nephropathy, most of them remain unclear. Little is also known about which, among endothelial cells and podocytes, represent the first glomerular target of the complication. Selective damage of the endothelium or of the podocyte results, as a common consequence, in an increase of albumin excretion rate. Albuminuria by itself cannot therefore be of help to solve the case. Endothelium and podocytes are involved in a continuous cross-talk and by studying the impact of diabetes on this "communication" process it should be possible to obtain some information regarding the weak component of the glomerular filter. Finally, the careful investigation of the mechanisms leading to the development podocyturia, a recently identified glomerular dysfunction associated to the pathogenesis of diabetic nephropathy, could contribute to shed some more light on the very early stages of this complication.

Aims: Compound 21 (C21), selective AT2 receptor agonist, has cardioprotective effects in experimental models of hypertension and myocardial infarction. The aims of the study was to evaluate the effect of C21, losartan, or both in Zucker diabetic fatty (ZDF) rats (type 2 diabetes) on (1) the prevention of myocardial hypertrophy; (2) myocardial expression of phosphatase and tensin homolog (PTEN), a target gene of miR-30a-3p, involved in myocardial remodelling. Methods: Experiments were performed in ZDF (n = 33) and in control Lean (8) rats. From the 6th to the 20th week of age, we administered C21 (0.3 mg/kg/day) to 8 ZDF rats. 8 ZDF rats were treated with losartan (10 mg/kg/day), 8 rats underwent combination treatment, C21+ losartan, and 9 ZDF rats were left untreated. Blood glucose and blood pressure were measured every 4 weeks. At the end of the study the hearts were removed, the apex was cut for the quantification of PTEN mRNA and miR-30a-3p expression (realtime-PCR). Myocardial hypertrophy was evaluated by histomorphometric analysis, and nitrotyrosine expression (as marker of oxidative stress) by immunohistochemistry. Results: ZDF rats had higher blood glucose (p < 0.0001) with respect to control Lean rats, while blood pressure did not change. Both parameters were not modified by C21 treatment, while losartan and losartan + C21 reduced blood pressure in ZDF rats (p < 0.05). miR-30a-3p expression was increased in ZDF rats (p < 0.01) and PTEN mRNA expression was decreased (p < 0.05). ZDF rats developed myocardial hypertrophy (p < 0.01) and increased oxidative stress (p < 0.01), both were prevented by C21 or losartan, or combination treatment. C21 or losartan normalized the expression of miR-30a-3p and PTEN. Conclusions: Activation of AT2 receptors or AT1 receptor blockade prevents the development of myocardial hypertrophy in ZDF rats. This occurs through the modulation of the miR-30a-3p/PTEN interaction.

Aims: To define the contribution of chronic kidney disease (CKD) to excess mortality in patients with type 2 diabetes and identify the baseline variables associated with all-cause death in those with and without CKD using the RECursive Partitioning and Amalgamation (RECPAM) method. Methods: This observational, longitudinal, cohort study enrolled 15,773 consecutive non-dialytic patients with type 2 diabetes in 19 Diabetes Clinics throughout Italy in 2006–2008. Based on the presence of albuminuria ≥ 30 mg day−1 and/or estimated glomerular filtration rate (eGFR) < 60 mL min−1·1.73 m−2 at baseline, patients were classified as having or not CKD. Vital status was verified on October 31, 2015 for 99.26% of patients. Results: Mortality increased with increasing albuminuria and eGFR category. Excess risk versus the general population was maximal in patients aged < 55 years in the worse albuminuria or eGFR category. Conversely, in subjects aged ≥ 75 years with albuminuria < 10 mg day−1 or eGFR ≥ 75 mL min−1·1.73 m−2, excess mortality was no longer detectable. At RECPAM analysis, the main correlates of death in the whole cohort were albuminuria > 44 mg day−1, prevalent CVD, and eGFR < ~ 75 mL min−1·1.73 m−2; gender, prevalent CVD, and higher albuminuria in the normoalbuminuric range, in patients without CKD; and CVD, eGFR ~ < 50 mL min−1·1.73 m−2, and albuminuria > 53 mg day−1, in those with CKD. Conclusions: CKD is a major contributor to excess mortality in type 2 diabetes, conferring a very high risk in younger patients and fully accounting for excess risk in the older ones. Higher albuminuria and lower eGFR, even in the normal range, identify individuals with increased mortality risk. Trial registration ClinicalTrials.gov (NCT00715481; https://clinicaltrials.gov/ct2/show/NCT00715481).

Recent evidences indicate early, diabetes-driven, retinal neurodegeneration as the origin of diabetic retinopathy. To verify the possibility to prevent the disease, we investigated in a mouse model of type 1 diabetes the effect of long-lasting hyperglycemia on retinal and choroidal structures and, in parallel, we tested the effect of topical treatment with the neuroprotective agent citicoline. Forty wild-type C57B6 mice were included in this study. Diabetes was induced by a single intravenous injection of alloxan. Five animals were considered as sham-treated controls, 15 animals as sham-treated diabetic mice, 5 animals as citicoline-treated controls and 15 animals as citicoline-treated diabetic mice. After eight months of diabetes in vivo analysis of the retina was performed using the Spectralis HRA (Heidelberg Retinal Angiography) + OCT. Neuroretinal abnormalities, in particular a significant narrowing of Retinal Nerve Fiber Layer (19.3 ± 2.2 vs 23.3 ± 2.4, ?m ± SD, p=0.01), Ganglion Cells/Inner Plexiform Layer (54.3 ± 5.1 vs 62.6 ± 4.0, p=0.03), Ganglion Cells Complex (73.9 ± 4.8 vs 83.8 ± 3.4, p=0.003) and Retinal thickness (223.8 ± 3.9 vs 236.7 ± 5.8, p=0.0004) were detected in the diabetic mouse that showed also a significant reduction of Choroidal thickness (67.4 ± 3.3 vs 84.7 ± 1.9, p=0.0001). In line with the hypothesis that neuroprotection might help preventing diabetic retinopathy, neuroretinal but not choroidal (choroid lacks a neuronal component) dysfunctions were prevented by citicoline. Altogether these findings demonstrate that diabetes-driven neuroretinal dysfunctions can be monitored in vivo by OCT in the mouse. Retinal neuroprotection as obtained by topical citicoline protects from these abnormalities suggesting this approach as a possible way to prevent diabetic retinopathy.

Endothelial progenitor cells (EPCs) enter the systemic circulation in response to cues related to vascular damage and need for neovascularization. Thus, EPCs could become readily accessible informers of vascular status and enable the survey of vascular pathologies during preclinical stages. To identify EPC changes with biomarker potential, we investigated whether discrete EPC abnormalities were associated with early nonproliferative diabetic retinopathy (NPDR). Two EPC subtypes with different functions have been characterized to date—one solely committed to the endothelial lineage and the other carrying both endothelial and monocytic markers. We found that only the latter, colony-forming units (CFU)-Hill cells, manifested abnormalities in type 1 diabetic patients with NPDR compared with control subjects. The abnormalities consisted in an increased number of colonies formed in vitro and downregulation of the molecules that facilitate homing at sites of vascular injury. The abnormalities were absent in type 1 diabetic patients free of retinopathy and other complications, despite long diabetes duration, but were detected in some of the patients without clinical retinopathy after short diabetes duration. CFU-Hill cells are potential informers of diabetic microangiopathy but may be preempted from carrying out reparative functions if the molecular abnormalities compromise interactions with the damaged vascular wall.

Although microvascular complications of diabetes (retinopathy, neuropathy, and nephropathy) affect different organs, they are strongly correlated to each other. Based on recent data, their onset and progression could be directly monitored, focusing our attention only on the eye. When confirmed and standardized, this approach could allow one to simplify the way in which we follow the progression of different diabetic complications, and thus establish new strategies aimed at preventing, treating and, hopefully, inducing the remission of microvascular complications of diabetes.

Aims: To analyze retinal vascular plexuses and choriocapillaris by optical coherence tomography angiography (OCT-A) and retinal nerve fiber layer and ganglion cell layer (GCL) by structural optical coherence tomography (OCT) in patients with type 1 diabetes mellitus (T1DM) without diabetic retinopathy (DR). Methods: A total of 25 eyes of 25 consecutive T1DM patients without signs of DR were prospectively recruited and compared to 25 healthy subjects (control eyes). All patients underwent OCT-A (CIRRUS HD-OCT model 5000, Carl Zeiss Meditec, Dublin, CA) and structural OCT. Qualitative and quantitative analyses with vessel density were performed on OCT-A images in the superficial capillary plexus (SCP), deep capillary plexus (DCP) and choriocapillaris for all patients. Results: By means of OCT-A, a rarefaction of the perifoveal capillary network in SCP was detected in 7 out of 25 eyes. No significant difference was found in FAZ area of both SCP and DCP comparing diabetic and control groups. By analyzing the DCP, diabetic eyes revealed a significant decreased vessel density compared to control eyes [0.464±0.016 and 0.477±0.014, respectively (p=0.005)]. Instead, no significant difference was found in the vessel density of all-retina plexus, SCP and choriocapillaris. By RFNL and GCL thickness analysis, no significant differences were disclosed between diabetics and healthy subjects. Conclusions: We demonstrated the ability of OCT-A to disclose early vascular alterations in patients with T1DM diagnosed as without any signs of DR on the basis of fundus biomicroscopy. Our results also suggest that microvascular changes could precede detectable damage of diabetic neuroretinopathy.

Although microvascular complications of diabetes (retinopathy, neuropathy, and nephropathy) affect different organs, they are strongly correlated to each other. Based on recent data, their onset and progression could be directly monitored, focusing our attention only on the eye. When confirmed and standardized, this approach could allow one to simplify the way in which we follow the progression of different diabetic complications, and thus establish new strategies aimed at preventing, treating and, hopefully, inducing the remission of microvascular complications of diabetes.

The aim of this study was to evaluate the effect of compound 21 (C21), a selective AT2 receptor agonist, on diabetic nephropathy and the potential additive effect of C21, when associated with losartan treatment, on the development of albuminuria and renal fibrosis in Zucker diabetic fatty (ZDF) rats. The experiments lasted 15 wk (from 5 to 20 wk of age) and were performed in 40 ZDF rats and 12 control lean rats. ZDF rats were divided into 4 groups: 1) 9 rats were treated with losartan; 2) 10 rats were treated with C21; 3) 9 rats were treated with losartan plus C21; and 4) 12 rats were maintained without any treatment. ZDF rats showed an increase in blood glucose level, albuminuria, renal fibrosis, macrophage infiltration, and TNF-α expression and a reduction of glomerular nephrin expression compared with control lean rats. C21 treatment reduced renal glomerular, tubulointerstitial, and perivascular fibrosis, and macrophage infiltration and TNF-α expression in ZDF rats. C21 treatment caused a decrease in albuminuria in ZDF rats up to 11 wk of age. Losartan decreased macrophage infiltration, TNF-α expression, and renal glomerular and perivascular fibrosis, restored glomerular nephrin expression, but did not affect tubulointerstitial fibrosis. Losartan treatment caused a decrease in albuminuria in ZDF rats up to 15 wk of age. At the end of the protocol, only the combination of C21 plus losartan significantly reduced albuminuria in ZDF rats. These data demonstrate that C21 has beneficial effects on diabetic nephropathy, suggesting the combination of C21 and losartan as a novel pharmacological tool to slow the progression of nephropathy in type II diabetes.

AIMS/HYPOTHESIS: An abnormal urinary albumin excretion rate (AER) is often the first clinically detectable manifestation of diabetic nephropathy. Our aim was to estimate the heritability and to detect genetic variation associated with elevated AER in patients with type 1 diabetes.METHODS: The discovery phase genome-wide association study (GWAS) included 1,925 patients with type 1 diabetes and with data on 24 h AER. AER was analysed as a continuous trait and the analysis was stratified by the use of antihypertensive medication. Signals with a p value RESULTS: The narrow-sense heritability, captured with our genotyping platform, was estimated to explain 27.3% of the total AER variability, and 37.6% after adjustment for covariates. In the discovery stage, five single nucleotide polymorphisms in the GLRA3 gene were strongly associated with albuminuria (p CONCLUSIONS/INTERPRETATION: This study suggests novel pathways and molecular mechanisms for the pathogenesis of albuminuria in type 1 diabetes.

There is still an unmet clinical need for small-caliber artery substitution. Decellularized scaffolds in tissue engineering represent a promising solution. We have developed an innovative system for the automatic decellularization of blood vessels, used to process pig arteries. The system is able to automatically drive a decellularization process in a safe and reliable environment, with complex time patterns, using up to three different decellularization solutions, and providing at the same time a physical stress to improve the decellularization. The decellularization of pig arteries was evaluated by means of histology, DNA quantification and mechanical testing. Outcomes showed scaffolds with no cellular or nuclear remnants and a well-preserved tissue structure, corroborated by mechanical properties similar to native tissue. Decellularized scaffolds were seeded on the inner layer with human endothelial cells and implanted as iliac artery replacement in 4 pharmacologically immune-compromised pigs. This chimeric model was performed as a very preliminary evaluation to investigate the performances of these scaffolds in vivo, and to investigate the fate of seeded cells. Recipients were sacrificed on day 14 and day 70 after surgery, and vessels were found to be patent and with no evidence of thrombi formation. The inner layer was covered by endothelial cells, and the migration of cells positive for α-smooth-muscle actin was observed from the outer layer towards the tunica media. Intriguingly, the endothelial cells on explanted vessels were entirely derived from the host while the seeded cells were lost. In conclusion, this work presents a novel tool for a safe and controlled production of arterial scaffolds, with good decellularization outcomes and a good performance in a short-term, large-animal implantation.

The aim of this cross-sectional case-control study is to investigate the possible presence of vascular/neurodegenerative alterations in the retina of type 1 diabetes mellitus (T1DM) patients without diabetic retinopathy (DR). Thirty-four eyes of 34 consecutive T1DM without DR (mean age 21 &plusmn, 2 years) were included. Another cohort of 27 eyes (27 healthy control subjects matched with age and sex) was also recruited. All patients underwent multimodal imaging evaluation using structural optical coherence tomography (OCT), OCT-angiography (OCT-A), dynamic vessel analyzer (DVA) and microperimetry. No significant differences were disclosed comparing diabetics and controls for visual acuity, central macular thickness, and subfoveal choroidal thickness. On retinal nerve fiber layer and ganglion cell complex thickness, no significant differences were disclosed comparing each 3-mm-diameter macular and peripapillary subfield between two groups. Using OCT-A, deep capillary plexus perfusion density (PD) of diabetics was significantly lower compared to control group, whereas PD of other retinal/choriocapillaris plexuses and foveal avascular zone area did not show any significant difference. Using DVA, diabetic eyes revealed a significantly decreased vessel response to flicker light in comparison to controls. No differences were disclosed using microperimetry analysis. Taken together, these results suggest that vascular alterations could be the first detectable retinal change in the development of DR.

Sex and genetic variation influence the risk of developing diabetic nephropathy and ESRD in patients with type 1 diabetes. We performed a genome-wide association study in a cohort of 3652 patients from the Finnish Diabetic Nephropathy (FinnDiane) Study with type 1 diabetes to determine whether sex-specific genetic risk factors for ESRD exist. A common variant, rs4972593 on chromosome 2q31.1, was associated with ESRD in women (P5×10(-8)) but not in men (P=0.77). This association was replicated in the meta-analysis of three independent type 1 diabetes cohorts (P=0.02) and remained significant for women (P5×10(-8); odds ratio, 1.81 [95% confidence interval, 1.47 to 2.24]) upon combined meta-analysis of the discovery and replication cohorts. rs4972593 is located between the genes that code for the Sp3 transcription factor, which interacts directly with estrogen receptor α and regulates the expression of genes linked to glomerular function and the pathogenesis of nephropathy, and the CDCA7 transcription factor, which regulates cell proliferation. Further examination revealed potential transcription factor-binding sites within rs4972593 and predicted eight estrogen-responsive elements within 5 kb of this locus. Moreover, we found sex-specific differences in the glomerular expression levels of SP3 (P=0.004). Overall, these results suggest that rs4972593 is a sex-specific genetic variant associated with ESRD in patients with type 1 diabetes and may underlie the sex-specific protection against ESRD.

Despite the evidence that both type 1 and type 2 diabetes are presently treated with up-to-date therapeutic protocols, complete normalization of glycemic control for both diseases appears to be still out of reach. The gap existing between the “ideal” cure still to come and the present pharmacological treatment of diabetes is the cause of the development of microvascular diabetic complications such as nephropathy and retinopathy.

Summary: The aim of this study was to evaluate the role of the angiotensin II (Ang II) induced-differential miRNA expression in renal glomerular and tubulo-interstitial fibrosis in an experimental model of Ang II-dependent hypertension. To clarify this issue, Sprague Dawley rats were treated with Ang II (200 ng/kg per minute, n = 15) or physiological saline (n = 14) for 4 weeks. Systolic blood pressure and albuminuria were measured every 2 weeks. At the end of the experimental period, renal glomerular and tubulo-interstitial fibrosis was evaluated by histomorphometric analysis, after Sirius-Red and Masson's trichrome staining. Ang II increased systolic blood pressure (P < 0.0001), albuminuria (P < 0.01) and both glomerular and tubulo-interstitial fibrosis (P < 0.01). Using laser capture microdissection and miRNA microarray analysis this study showed that miR-29a-3p was down-regulated in renal tubules and up-regulated in glomeruli. Real-time polymerase chain reaction (PCR) experiments confirmed in Ang II-treated rats a down-regulation of miR-29a-3p in tubules (P < 0.01), while no significant changes were observed in glomeruli. Matrix metalloproteinase-2 (MMP-2) was identified as putative miR-29a-3p target (by TargetScan, miRanda, Tarbase software) and functionally confirmed by luciferase activity assay. These data demonstrate that the effects of Ang II on miR-29a-3p expression in renal tubules is different from the one exerted in the glomeruli and that miR-29a-3p targets MMP-2. These results suggest that the development of renal fibrosis at glomerular and tubulo-interstitial level depends on different molecular mechanisms.

Compound 21 (C21), selective agonist of angiotensin type-2 (AT-2) receptors, shows anti-inflammatory effects in experimental models of hypertension and nephroprotection in diabetes. The aim of the present study was to evaluate the effects of C21 in cyclosporine nephropathy, which is characterized mainly by tubulo-interstitial fibrosis. Ten days before and during the experimental periods, low-salt diet was administered to Sprague–Dawley rats. Cyclosporine-A (CsA; 15 mg/kg per day, intraperitoneal injection) and CsA plus C21 (0.3 mg/kg per day, intraperitoneal injection) were administered for 1 and 4 weeks. Control groups were left without any treatment. Blood pressure (plethysmographic method) and 24 h urinary albumin excretion were measured once a week. At the end of the experimental protocols, the kidneys were excised for histomorphometric analysis of renal fibrosis and for immunohistochemical evaluation of inflammatory infiltrates and type I and type IV collagen expression. After 1 and 4 weeks, the rats treated with CsA showed a significant increase (P

OBJECTIVE To evaluate the rate and determinants of concordance between advanced diabetic retinopathy (DR) and chronic kidney disease (CKD), as assessed by both albuminuria and estimated glomerular filtration rate (eGFR), in the large cohort of the Renal Insufficiency And Cardiovascular Events (RIACE) Italian multicenter study. RESEARCH DESIGN AND METHODS Patients with type 2 diabetes (n = 15,773) visiting consecutively 19 hospital–based diabetes clinics in years 2007 and 2008 were examined. DR was assessed by dilated fundoscopy. CKD was defined based on albuminuria and eGFR. RESULTS CKD was present in 58.64% of subjects with advanced DR, whereas advanced DR was detectable only in 15.28% of individuals with any CKD and correlated with the albuminuric CKD phenotypes more than with the nonalbuminuric phenotype. Age, male sex, diabetes duration, hemoglobin A1c, hypertension, triglycerides, previous cardiovascular disease, and, inversely, HDL-cholesterol correlated independently with the presence of any CKD in individuals with advanced DR; correlates differed according to the presence of albuminuria, reduced eGFR, or both. Conversely, factors associated with the presence of advanced DR in subjects with any CKD were diabetes treatment, previous cardiovascular disease, albuminuria, and, inversely, smoking, eGFR, and age at diagnosis. CONCLUSIONS Concordance of CKD with advanced DR is low in subjects with type 2 diabetes, and CKD without advanced DR is more frequent than isolated advanced DR, at variance with type 1 diabetes. Factors independently associated with the presence of any CKD in individuals with advanced DR differ, at least in part, from those correlating with the presence of advanced DR in subjects with any CKD and by CKD phenotype.

Nuclear envelope-related muscular dystrophies, in particular those referred to as laminopathies, are relatively novel and unclear diseases, also considering the increasing number of mutations identified so far in genes of the nuclear envelope. As regard LMNA gene, only tentative relations between phenotype, type and localization of the mutations have been established in striated muscle diseases, while laminopathies affecting adipose tissue, peripheral nerves or progerioid syndromes could be linked to specific genetic variants. This study describes the biochemical phenotype of neuromuscular laminopathies in samples derived from LMNA mutant patients. Since it has been reported that nuclear alterations, due to LMNA defects, are present also in fibroblasts from Emery–Dreifuss muscular dystrophy and familial partial lipodystrophy patients, we analyzed 2D-maps of skin fibroblasts of patients carrying 12 different LMNA mutations spread along the entire gene. To recognize distinctive proteins underlying affected biochemical pathways, we compared them with fibroblasts from healthy controls and, more importantly, fibroblasts from patients with non-lamin related neuromuscular disorders. We found less abundance of cytoskeletal/structural proteins, confirming a dominant role for Lamin A/C in structural support of nuclear architecture. Interestingly, we also established significant changes in the expression of proteins involved in cellular energy production and oxidative stress response. To our knowledge, this is the first report where proteomics was applied to characterize ex-vivo cells from LMNA patients, suggesting that this may represent a new approach to better understand the molecular mechanisms of these rare diseases and facilitate the development of novel therapeutic treatments.

Objectives- Circulating angiogenic cells (CACs) are vascular-committed bone-marrow derived cells expressing endothelial and stem cell markers that are dysfunctional in type 1 diabetes (T1D). We studied if restoration of endogenous beta cells function with islet transplantation is associated with better CACs function. Research design and methods- 18 T1D patients, 14 insulin independent islet-transplanted patients (ITA) and 14 healthy controls (C) were studied cross-sectionally. In vivo (CACs percentage/apoptosis and migratory/differentiation assay) and in vitro studies [colony forming unit endothelial cells (CFU-ECs) counting, CACs percentage/apoptosis, cytokine production and migratory/differentiation assay] were performed, together with endothelial dependent dilatation (EDD) test in vivo. The addition of serial concentrations IL-8 and anti-IL8 in vitro cultures was performed to assess IL-8 mediated cell survival/apoptosis. Results- The percentage of alive and apoptotic CACs did not differ among the 3 groups. CFU-ECs absolute number obtained from T1D, but not from ITA, was reduced compared to C (C=7.3±1.9, T1D=0.9±0.4 and ITA=4.7±1.9; p

Diabetes mellitus accelerates atherosclerotic processes, and it is known that inflammation plays a key role in atherosclerosis. The aim of the present study was to evaluate in patients with Type 2 diabetes whether serum levels of CRP (C-reactive protein) are associated with cytokine production in whole blood. A total of 89 outpatients with Type 2 diabetes were enrolled, and blood pressure, body mass index, fasting blood glucose, glycated haemoglobin, cholesterol, triacylglycerols (triglycerides) and hs-CRP (high-sensitivity CRP) were measured. IL-6 (interleukin-6), IL-1beta (interleukin-1beta) and TNF-alpha (tumour necrosis factor-alpha) were measured before and after 24 h of incubation of whole blood with LPS (lipopolysaccharide) or saline. The basal values of IL-1beta, IL-6 and TNF-alpha were low and were not significantly related to hs-CRP levels. A univariate analysis showed that the level of IL-1beta and IL-6, obtained after 24 h of incubation of whole blood with LPS, increased significantly with increasing levels of hs-CRP and, after adjusting for potential confounders, IL-1beta still remained statistically significant. In our sample of patients with Type 2 diabetes, there was no association between serum hs-CRP levels and basal levels of IL-6, IL-1beta and TNF-alpha. Conversely, a significant association was observed between serum hs-CRP levels and IL-1beta and IL-6 production after 24 h of incubation of whole blood with LPS. In conclusion, our data suggest that patients with Type 2 diabetes and high hs-CRP levels may have an enhanced reactivity in response to specific stimuli that produce different interleukins, with possible implications in inflammatory atherosclerotic processes.

During the last few years, the incidence of microvascular complications in diabetes mellitus has rapidly increased as a consequence of both an increase in incidence of type 2 and type 1 diabetes mellitus. The pathogenesis of diabetic microvascular complications is still largely unknown. Among the many hypotheses, a dysfunction in angiogenesis has been suggested as a common origin for retinopathy, nephropathy, and neuropathy. Based on this hypothesis, inhibition of vascular endothelial growth factor (VEGF) has been tested as a potential therapeutic approach to prevent and cure diabetic microvascular complications. Several VEGF inhibitors are currently under evaluation or are approved for the treatment of wet age-related macular degeneration and macular edema. These include inhibitors of intracellular transcription of VEGF (e.g. bevasiranib), inhibitors of extracellular VEGF (e.g. pegaptanib), inhibitors of VEGF receptor expression (e.g. aflibercept [VEGF-TRAP]) and inhibitors of the intracellular signaling cascade activating VEGF (e.g. midostaurin). According to the existing evidence base, although inhibition of VEGF results in a better outcome in the case of diabetic retinopathy and also, despite some discrepant results, in the case of diabetic nephropathy, there is no final confirmation that VEGF inhibition is a valid approach for diabetic neuropathy. The latter complication actually, in line with other chronic neuropathies, seems to improve with stimulation of angiogenesis through increased expression of VEGF.