Mechanisms of podocyte injury and implications for diabetic nephropathy.

Albuminuria is the hallmark of both primary and secondary proteinuric glomerulopathies, including focal segmental glomerulosclerosis (FSGS), obesity-related nephropathy, and diabetic nephropathy (DN). Moreover, albuminuria is an important feature of all chronic kidney diseases (CKDs). Podocytes play a key role in maintaining the permselectivity of the glomerular filtration barrier (GFB) and injury of the podocyte, leading to foot process (FP) effacement and podocyte loss, the unifying underlying mechanism of proteinuric glomerulopathies. The metabolic insult of hyperglycemia is of paramount importance in the pathogenesis of DN, while insults leading to podocyte damage are poorly defined in other proteinuric glomerulopathies. However, shared mechanisms of podocyte damage have been identified. Herein, we will review the role of haemodynamic and oxidative stress, inflammation, lipotoxicity, endocannabinoid (EC) hypertone, and both mitochondrial and autophagic dysfunction in the pathogenesis of the podocyte damage, focussing particularly on their role in the pathogenesis of DN. Gaining a better insight into the mechanisms of podocyte injury may provide novel targets for treatment. Moreover, novel strategies for boosting podocyte repair may open the way to podocyte regenerative medicine.

Protective Role of the M-Sec-Tunneling Nanotube System in Podocytes.

BACKGROUND: Podocyte dysfunction and loss are major determinants in the development of proteinuria. FSGS is one of the most common causes of proteinuria, but the mechanisms leading to podocyte injury or conferring protection against FSGS remain poorly understood. The cytosolic protein M-Sec has been involved in the formation of tunneling nanotubes (TNTs), membrane channels that transiently connect cells and allow intercellular organelle transfer. Whether podocytes express M-Sec is unknown and the potential relevance of the M-Sec-TNT system in FSGS has not been explored. METHODS: We studied the role of the M-Sec-TNT system in cultured podocytes exposed to Adriamycin and in BALB/c M-Sec knockout mice. We also assessed M-Sec expression in both kidney biopsies from patients with FSGS and in experimental FSGS (Adriamycin-induced nephropathy). RESULTS: Podocytes can form TNTs in a M-Sec-dependent manner. Consistent with the notion that the M-Sec-TNT system is cytoprotective, podocytes overexpressed M-Sec in both human and experimental FSGS. Moreover, M-Sec deletion resulted in podocyte injury, with mitochondrial abnormalities and development of progressive FSGS. In vitro, M-Sec deletion abolished TNT-mediated mitochondria transfer between podocytes and altered mitochondrial bioenergetics. Re-expression of M-Sec reestablishes TNT formation and mitochondria exchange, rescued mitochondrial function, and partially reverted podocyte injury. CONCLUSIONS: These findings indicate that the M-Sec-TNT system plays an important protective role in the glomeruli by rescuing podocytes via mitochondrial horizontal transfer. M-Sec may represent a promising therapeutic target in FSGS, and evidence that podocytes can be rescued via TNT-mediated horizontal transfer may open new avenues of research.

MicroRNA 146a is associated with diabetic complications in type 1 diabetic patients from the EURODIAB PCS.

BACKGROUND: MicroRNA-146a-5p (miR-146a-5p) is a key regulator of inflammatory processes. Expression of miR-146a-5p is altered in target organs of diabetic complications and deficiency of miR-146a-5p has been implicated in their pathogenesis. We investigated if serum miR-146a-5p levels were independently associated with micro/macrovascular complications of type 1 diabetes (DM1). METHODS: A nested case-control study from the EURODIAB PCS of 447 DM1 patients was performed. Cases (n = 294) had one or more complications of diabetes, whereas controls (n = 153) did not have any complication. Total RNA was isolated from all subjects and miR-146a-5p levels measured by qPCR. Both the endogenous controls U6 snRNA and the spike (Cel-miR-39) were used to normalize the results. Logistic regression analysis was carried out to investigate the association of miR-146a-5p with diabetes complications. RESULTS: MiR-146a-5p levels were significantly lower in cases [1.15 (0.32-3.34)] compared to controls [1.74 (0.44-6.74) P = 0.039]. Logistic regression analysis showed that levels of miR-146a-5p in the upper quartile were inversely associated with reduced odds ratio (OR) of all complications (OR 0.34 [95% CI 0.14-0.76]) and particularly with cardiovascular diseases (CVD) (OR 0.31 [95% CI 0.11-0.84]) and diabetic retinopathy (OR 0.40 [95% CI 0.16-0.99]), independently of age, sex, diabetes duration, A1c, hypertension, AER, eGFR, NT-proBNP, and TNF-α. CONCLUSIONS: In this large cohort of DM1 patients, we reported an inverse and independent association of miR-146a-5p with diabetes chronic complications and in particular with CVD and retinopathy, suggesting that miR-146a-5p may be a novel candidate biomarker of DM1 complications.

Neuronal Glutamate Transporters Control Dopaminergic Signaling and Compulsive Behaviors.

There is an ongoing debate on the contribution of the neuronal glutamate transporter EAAC1 to the onset of compulsive behaviors. Here, we used behavioral, electrophysiological, molecular, and viral approaches in male and female mice to identify the molecular and cellular mechanisms by which EAAC1 controls the execution of repeated motor behaviors. Our findings show that, in the striatum, a brain region implicated with movement execution, EAAC1 limits group I metabotropic glutamate receptor (mGluRI) activation, facilitates D1 dopamine receptor (D1R) expression, and ensures long-term synaptic plasticity. Blocking mGluRI in slices from mice lacking EAAC1 restores D1R expression and synaptic plasticity. Conversely, activation of intracellular signaling pathways coupled to mGluRI in D1R-containing striatal neurons of mice expressing EAAC1 leads to reduced D1R protein level and increased stereotyped movement execution. These findings identify new molecular mechanisms by which EAAC1 can shape glutamatergic and dopaminergic signals and control repeated movement execution.SIGNIFICANCE STATEMENT Genetic studies implicate Slc1a1, a gene encoding the neuronal glutamate transporter EAAC1, with obsessive-compulsive disorder (OCD). EAAC1 is abundantly expressed in the striatum, a brain region that is hyperactive in OCD. What remains unknown is how EAAC1 shapes synaptic function in the striatum. Our findings show that EAAC1 limits activation of metabotropic glutamate receptors (mGluRIs) in the striatum and, by doing so, promotes D1 dopamine receptor (D1R) expression. Targeted activation of signaling cascades coupled to mGluRIs in mice expressing EAAC1 reduces D1R expression and triggers repeated motor behaviors. These findings provide new information on the molecular basis of OCD and suggest new avenues for its treatment.

The role of cannabinoid signaling in acute and chronic kidney diseases.

The endogenous cannabinoids anandamide and 2-arachidonoylglycerol bind to the cannabinoid receptors of type 1 and 2. These receptors are also the binding sites for exogenous, both natural and synthetic, cannabinoids that are used for recreation purposes. Until recently, cannabinoids and cannabinoid receptors have attracted little interest among nephrologists; however, a full endocannabinoid system (ECS) is present in the kidney and it has recently emerged as an important player in the pathogenesis of diabetic nephropathy, drug nephrotoxicity, and progressive chronic kidney disease. This newly established role of the ECS in the kidney might have therapeutic relevance, as pharmacological modulation of the ECS has renoprotective effects in experimental animals, raising hope for future potential applications in humans. In addition, over the last years, there has been a number of reported cases of acute kidney injury (AKI) associated with the use of synthetic cannabinoids that appear to have higher potency and rate of toxicity than natural Cannabis. This poorly recognized cause of renal injury should be considered in the differential diagnosis of AKI, particularly in young people. In this review we provide an overview of preclinical evidence indicating a role of the ECS in renal disease and discuss potential future therapeutic applications. Moreover, we give a critical update of synthetic cannabinoid-induced AKI.

Heat Shock Proteins in Vascular Diabetic Complications: Review and Future Perspective.

Heat shock proteins (HSPs) are a large family of proteins highly conserved throughout evolution because of their unique cytoprotective properties. Besides assisting protein refolding and regulating proteostasis under stressful conditions, HSPs also play an important role in protecting cells from oxidative stress, inflammation, and apoptosis. Therefore, HSPs are crucial in counteracting the deleterious effects of hyperglycemia in target organs of diabetes vascular complications. Changes in HSP expression have been demonstrated in diabetic complications and functionally related to hyperglycemia-induced cell injury. Moreover, associations between diabetic complications and altered circulating levels of both HSPs and anti-HSPs have been shown in clinical studies. HSPs thus represent an exciting therapeutic opportunity and might also be valuable as clinical biomarkers. However, this field of research is still in its infancy and further studies in both experimental diabetes and humans are required to gain a full understanding of HSP relevance. In this review, we summarize current knowledge and discuss future perspective.

An inherited duplication at the gene p21 Protein-Activated Kinase 7 (PAK7) is a risk factor for psychosis.

Identifying rare, highly penetrant risk mutations may be an important step in dissecting the molecular etiology of schizophrenia. We conducted a gene-based analysis of large (>100 kb), rare copy-number variants (CNVs) in the Wellcome Trust Case Control Consortium 2 (WTCCC2) schizophrenia sample of 1564 cases and 1748 controls all from Ireland, and further extended the analysis to include an additional 5196 UK controls. We found association with duplications at chr20p12.2 (P = 0.007) and evidence of replication in large independent European schizophrenia (P = 0.052) and UK bipolar disorder case-control cohorts (P = 0.047). A combined analysis of Irish/UK subjects including additional psychosis cases (schizophrenia and bipolar disorder) identified 22 carriers in 11 707 cases and 10 carriers in 21 204 controls [meta-analysis Cochran-Mantel-Haenszel P-value = 2 × 10(-4); odds ratio (OR) = 11.3, 95% CI = 3.7, ∞]. Nineteen of the 22 cases and 8 of the 10 controls carried duplications starting at 9.68 Mb with similar breakpoints across samples. By haplotype analysis and sequencing, we identified a tandem ~149 kb duplication overlapping the gene p21 Protein-Activated Kinase 7 (PAK7, also called PAK5) which was in linkage disequilibrium with local haplotypes (P = 2.5 × 10(-21)), indicative of a single ancestral duplication event. We confirmed the breakpoints in 8/8 carriers tested and found co-segregation of the duplication with illness in two additional family members of one of the affected probands. We demonstrate that PAK7 is developmentally co-expressed with another known psychosis risk gene (DISC1) suggesting a potential molecular mechanism involving aberrant synapse development and plasticity.

Deficiency of cannabinoid receptor of type 2 worsens renal functional and structural abnormalities in streptozotocin-induced diabetic mice.

A functionally active endocannabinoid system is present within the kidney. The cannabinoid receptor type 2 (CB2) is expressed by both inflammatory cells and podocytes, and its activation has beneficial effects in experimental diabetic nephropathy. To further explore the role of CB2 in diabetic nephropathy, we studied renal functional and structural abnormalities in streptozotocin-induced diabetic CB2 knockout mice. In diabetic mice, deletion of the CB2 receptor albuminuria, the downregulation of podocin and nephrin, mesangial expansion, overexpression of extracellular matrix components, monocyte infiltration, and reduced renal function were all exacerbated. To investigate the relative contributions of podocytes and monocytes to the phenotype of diabetic knockout mice, bone marrow transplantation experiments were performed. The lack of CB2 on bone marrow-derived cells was shown to be important in driving the enhanced glomerular monocyte accrual found in diabetic knockout mice. Absence of CB2 on resident glomerular cells had a major role in worsening diabetic nephropathy, both functional and structural abnormalities, likely by enhanced MCP-1 and CB1 signaling. Studies in cultured podocytes demonstrated that CB2 expression is not altered by a high glucose milieu but is downregulated by mechanical stretch, mimicking glomerular capillary hypertension. Thus, CB2 deletion worsens diabetic nephropathy, independent of bone marrow-derived cells.

Serum MicroRNA-191-5p Levels in Vascular Complications of Type 1 Diabetes: The EURODIAB Prospective Complications Study.

CONTEXT: MicroRNA-191-5p regulates key cellular processes involved in the pathogenesis of diabetic complications such as angiogenesis, extracellular matrix deposition, and inflammation. However, no data on circulating microRNA-191-5p in the chronic complications of diabetes are available. OBJECTIVE: To assess whether serum levels of microRNA-191-5p were associated with micro- and macrovascular disease in a large cohort of subjects with type 1 diabetes mellitus (DM1) from the EURODIAB Prospective Complication Study. DESIGN AND SETTING: Levels of microRNA-191-5p were measured by quantitative PCR in 420 patients with DM1 recruited as part of the cross-sectional analysis of the EURODIAB Prospective Complication Study. Cases (n = 277) were subjects with nephropathy and/or retinopathy and/or cardiovascular disease (CVD). Controls (n = 143) were patients without complications. Logistic regression analysis was performed to evaluate the potential independent association of microRNA-191-5p levels with chronic complications of diabetes. RESULTS: Levels of microRNA-191-5p were significantly reduced (P < .001) in cases compared with controls even after adjustment for age, sex, and diabetes duration. Logistic regression analysis revealed that microRNA-191-5p was negatively associated with a 58% reduced odds ratio (OR) of chronic diabetes complications, specifically CVD, micro-macroalbuminuria, and retinopathy (OR, 0.42; 95% CI, 0.23-0.77), independent of age, sex, physical activity, educational levels, diabetes duration, glycated hemoglobin, total insulin dose, hypertension, smoking, total cholesterol, albumin excretion rate, estimated glomerular filtration rate, serum vascular cell adhesion molecule-1, and tumor necrosis factor-α. Analyses performed separately for each complication demonstrated a significant independent association with albuminuria (OR, 0.36; 95% CI, (0.18-0.75) and CVD (OR, 0.34; 95% CI, 0.16-0.70). CONCLUSIONS: In DM1 subjects, microRNA-191-5p is inversely associated with vascular chronic complications of diabetes.

Novel Insight into the Mechanisms of the Bidirectional Relationship between Diabetes and Periodontitis.

Periodontitis and diabetes are two major global health problems despite their prevalence being significantly underreported and underestimated. Both epidemiological and intervention studies show a bidirectional relationship between periodontitis and diabetes. The hypothesis of a potential causal link between the two diseases is corroborated by recent studies in experimental animals that identified mechanisms whereby periodontitis and diabetes can adversely affect each other. Herein, we will review clinical data on the existence of a two-way relationship between periodontitis and diabetes and discuss possible mechanistic interactions in both directions, focusing in particular on new data highlighting the importance of the host response. Moreover, we will address the hypothesis that trained immunity may represent the unifying mechanism explaining the intertwined association between diabetes and periodontitis. Achieving a better mechanistic insight on clustering of infectious, inflammatory, and metabolic diseases may provide new therapeutic options to reduce the risk of diabetes and diabetes-associated comorbidities.

Association of serum MicroRNA-145-5p levels with microvascular complications of type 1 Diabetes: The EURODIAB prospective complications study.

AIMS: To investigate whether serum miR-145-5p levels were associated with micro-macrovascular chronic complications in patients with type 1 diabetes (DM1). METHODS: A nested case-control study from the EURODIAB Prospective Complications Study was performed. Cases (n = 289) had one or more complications of diabetes, whereas controls (n = 153) did not have any complication. We measured miR-145-5p levels by qPCR and investigated the association with diabetes complications. RESULTS: Mean miR-145-5p levels were significantly lower in cases with microangiopathy [2.12 (0.86-4.94)] compared to controls [3.15 (1.21-7.36), P < 0.05] even after adjustment for age, gender, and diabetes duration. In logistic regression analysis, miR-145-5p levels in the lowest tertile were associated with an over three-fold increased odds ratio (OR) of albuminuria [3.22 (1.17-8.81)], independently of both demographic and diabetes-related factors. In addition, mir145-5p levels in the lowest tertile were independently and inversely associated with arterial hypertension [1.96 (1.08-3.56)] and hypertension was the mediator of the relationship between miR-145-5p and albuminuria. CONCLUSIONS: In this large cohort of DM1 patients, we found an inverse association between miR-145-5p and albuminuria that was mediated by systemic hypertension.

Novel biomarkers of diabetic kidney disease: current status and potential clinical application.

Diabetic kidney disease (DKD) is a leading cause of end-stage renal disease (ESRD). Although both albuminuria and glomerular filtration rate (GFR) are well-established diagnostic/prognostic biomarkers of DKD, they have important limitations. There is, thus, increasing quest to find novel biomarkers to identify the disease in an early stage and to improve risk stratification. In this review, we will outline the major pitfalls of currently available markers, describe promising novel biomarkers, and discuss their potential clinical relevance. In particular, we will focus on the importance of recent advancements in multi-omic technologies in the discovery of new DKD biomarkers. In addition, we will provide an update on new emerging approaches to explore renal function and structure, using functional tests and imaging.

The future of diabetic kidney disease management: what to expect from the experimental studies?

Diabetic kidney disease (DKD) is a major cause of end-stage renal disease. Intensive blood glucose and blood pressure control, particularly using inhibitors of the renin-angiotensin system, have long been mainstays of therapy in patients with DKD. Moreover, new anti-hyperglycemic drugs have recently shown renoprotective effects and this represents a major progress in the management of DKD. However, the risk of progression is still substantial and additional drugs are required. Recent preclinical studies have identified novel therapeutic targets that may optimize renoprotection in the near future. Besides strategies aimed to reduce oxidative stress and inflammation in the kidney, novel extra-renal approaches targeting stem cells, extracellular vesicles, and the microbiota are on the horizon with promising preclinical data. Herein, we will review these lines of research and discuss potential clinical applications. Given the poor yield of experimental studies in DKD in the past years, we will also discuss strategies to improve translation of preclinical research to humans.

Expression of nuclear Methyl-CpG binding protein 2 (Mecp2) is dependent on neuronal stimulation and application of Insulin-like growth factor 1.

Methyl-CpG binding protein 2 (MECP2) is a chromosome-binding protein that regulates the development and maintenance of brain circuits. Altered function of the protein product of MECP2 plays an important role in the etiology of many neurodevelopmental disorders. Mutations involving a loss of function are implicated in the etiology of Rett syndrome, intellectual disability, psychosis and severe encephalopathy. Conversely, MECP2 duplications have been identified in autism and intellectual disability. MECP2 action is dependent on neuronal function, as the DNA binding is modulated by activity, and it is phosphorylated in response to stimulation. Although MECP2 is considered a major risk factor for neurodevelopmental disorders, and it is a mediator of activity-dependent mechanisms, the expression levels in response to neuronal activity have never been measured. We studied the expression of Mecp2 protein and RNA in mice neuronal cultures in response to different stimulation conditions and in the presence of insulin-like growth factor1 (IGF1): a growth factor involved in brain development and plasticity. The stimulation protocols were selected according to their ability to induce different forms of synaptic plasticity: rapid depolarization, feed-forward plasticity (LTP, LTD) and feedback forms of plasticity (TTX, KCl). We find a significant reduction of Mecp2 protein nuclear expression in neurons in response to stimuli that induce a potentiation of neuronal response, suggesting that Mecp2 protein expression is modulated by neuronal activation. Application of IGF1 to the cultures induces an increase in the expression of Mecp2 transcript and nuclear Mecp2 protein in neurons. These results show that Mecp2 is responsive to neuronal stimulation and IGF1, and different stimuli have different effects on Mecp2 expression; this differential response may have downstream effects on functional mechanisms regulating brain development and plasticity.

Substituted E-3-(3-indolylmethylene)-1,3-dihydroindol-2-ones with antitumor activity. In depth study of the effect on growth of breast cancer cells.

The synthesis of new substituted E-3-(3-indolylmethylene)-1,3-dihydroindol-2-ones is reported. The antitumor activity was evaluated according to protocols available at the National Cancer Institute (NCI), Bethesda, MD. Structure-activity relationships are discussed. The action of selected compounds was investigated in MCF-7 breast cancer cells. The ability of these derivatives to inhibit cellular proliferation was accompanied by increased level of p53 and its transcriptional targets p21 and Bax, interference in the cell cycle progression with cell accumulation in the G2/M phase, and activation of apoptosis.

Bis-guanylhydrazone diimidazo[1,2-a:1,2-c]pyrimidine as a novel and specific G-quadruplex binding motif.

A bis-guanylhydrazone derivative of diimidazo[1,2-a:1,2-c]pyrimidine has unexpectedly been found to be a potent stabiliser of several quadruplex DNAs, whereas there is no significant interaction with duplex DNA. Molecular modeling suggests that the guanylhydrazone groups play an active role in quadruplex binding.