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Objective: Dyslipidemia is a significant risk factor for progression of diabetic kidney disease (DKD). Determining the changes in individual lipids and lipid networks across a spectrum of DKD severity may identify lipids that are pathogenic to DKD progression. Methods: We performed untargeted lipidomic analysis of kidney cortex tissue from diabetic db/db and db/db eNOS−/− mice along with non-diabetic littermate controls. A subset of mice were treated with the renin-angiotensin system (RAS) inhibitors, lisinopril and losartan, which improves the DKD phenotype in the db/db eNOS−/− mouse model. Results: Of the three independent variables in this study, diabetes had the largest impact on overall lipid levels in the kidney cortex, while eNOS expression and RAS inhibition had smaller impacts on kidney lipid levels. Kidney lipid network architecture, particularly of networks involving glycerolipids such as triacylglycerols, was substantially disrupted by worsening kidney disease in the db/db eNOS−/− mice compared to the db/db mice, a feature that was reversed with RAS inhibition. This was associated with decreased expression of the stearoyl-CoA desaturases, Scd1 and Scd2, with RAS inhibition. Conclusions: In addition to the known salutary effect of RAS inhibition on DKD progression, our results suggest a previously unrecognized role for RAS inhibition on the kidney triacylglycerol lipid metabolic network. [ABSTRACT FROM AUTHOR]

Diabetes is the most frequent cause of chronic kidney disease (CKD), leading to nearly half of all cases of kidney failure requiring replacement therapy. The principal cause of death among patients with diabetes and CKD is cardiovascular disease (CVD). Sodium/glucose cotransporter 2 (SGLT2) inhibitors were developed to lower blood glucose levels by inhibiting glucose reabsorption in the proximal tubule. In clinical trials designed to demonstrate the CVD safety of SGLT2 inhibitors in type 2 diabetes mellitus (T2DM), consistent reductions in risks for secondary kidney disease end points (albuminuria and a composite of serum creatinine doubling or 40% estimated glomerular filtration rate decline, kidney failure, or death), along with reductions in CVD events, were observed. In patients with CKD, the kidney and CVD benefits of canagliflozin were established by the CREDENCE (Canagliflozin and Renal Events in Diabetes With Established Nephropathy Clinical Evaluation) trial in patients with T2DM, urinary albumin-creatinine ratio >300 mg/g, and estimated glomerular filtration rate of 30 to <90 mL/min/1.73 m2 To clarify and support the role of SGLT2 inhibitors for treatment of T2DM and CKD, the National Kidney Foundation convened a scientific workshop with an international panel of more than 80 experts. They discussed the current state of knowledge and unanswered questions in order to propose therapeutic approaches and delineate future research. SGLT2 inhibitors improve glomerular hemodynamic function and are thought to ameliorate other local and systemic mechanisms involved in the pathogenesis of CKD and CVD. SGLT2 inhibitors should be used when possible by people with T2DM to reduce risks for CKD and CVD in alignment with the clinical trial entry criteria. Important risks of SGLT2 inhibitors include euglycemic ketoacidosis, genital mycotic infections, and volume depletion. Careful consideration should be given to the balance of benefits and harms of SGLT2 inhibitors and risk mitigation strategies. Effective implementation strategies are needed to achieve widespread use of these life-saving medications. [ABSTRACT FROM AUTHOR]

Regulation of mitochondrial dynamics and energetics in the diabetic renal proximal tubule by the b2-adrenergic receptor agonist formoterol. Am J Physiol Renal Physiol 319: F773-F779, 2020. First published September 21, 2020; doi:10.1152/ajprenal.00427.2020.--Diabetes is a prevalent metabolic disease that contributes to 50% of all endstage renal disease and has limited treatment options. We previously demonstrated that the b2-adrenergic receptor agonist formoterol induced mitochondrial biogenesis and promoted recovery from acute kidney injury. Here, we assessed the effects of formoterol on mitochondrial dysfunction and dynamics in renal proximal tubule cells (RPTCs) treated with high glucose and in a mouse model of type 2 diabetes. RPTCs exposed to 17 mM glucose exhibited increased electron transport chain (ETC) complex I, II, III, and V protein levels and reduced ATP levels and uncoupled oxygen consumption rate compared with RPTCs cultured in the absence of glucose or osmotic controls after 96 h. ETC proteins, ATP, and oxygen consumption rate were restored in RPTCs treated with formoterol. RPTCs exposed to high glucose had increased phosphodynamin- related protein 1 (Drp1), a mitochondrial fission protein, and decreased mitofusin 1 (Mfn1), a mitochondrial fusion protein. Formoterol treatment restored phospho-Drp1 and Mfn1 to control levels. Db/db and nondiabetic (db/m) mice (10 wk old) were treated with formoterol or vehicle for 3 wk and euthanized. Db/db mice showed increased renal cortical ETC protein levels in complexes I, III, and V and decreased ATP; these changes were prevented by formoterol. Phospho-Drp1 was increased and Mfn1 was decreased in db/db mice, and formoterol restored both to control levels. Together, these findings demonstrate that hyperglycemic conditions in vivo and exposure of RPTCs to high glucose similarly alter mitochondrial bioenergetic and dynamics profiles and that treatment with formoterol can reverse these effects. Formoterol may be a promising strategy for treating early stages of diabetic kidney disease. [ABSTRACT FROM AUTHOR]

To define the components of the metabolic syndrome that contribute to diabetic polyneuropathy (DPN) in type 2 diabetes mellitus (T2DM), we treated the BKS db/db mouse, an established murine model of T2DM and the metabolic syndrome, with the thiazolidinedione class drug pioglitazone. Pioglitazone treatment of BKS db/db mice produced a significant weight gain, restored glycemic control, and normalized measures of serum oxidative stress and triglycerides but had no effect on LDLs or total cholesterol. Moreover, although pioglitazone treatment normalized renal function, it had no effect on measures of large myelinated nerve fibers, specifically sural or sciatic nerve conduction velocities, but significantly improved measures of small unmyelinated nerve fiber architecture and function. Analyses of gene expression arrays of large myelinated sciatic nerves from pioglitazone-treated animals revealed an unanticipated increase in genes related to adipogenesis, adipokine signaling, and lipoprotein signaling, which likely contributed to the blunted therapeutic response. Similar analyses of dorsal root ganglion neurons revealed a salutary effect of pioglitazone on pathways related to defense and cytokine production. These data suggest differential susceptibility of small and large nerve fibers to specific metabolic impairments associated with T2DM and provide the basis for discussion of new treatment paradigms for individuals with T2DM and DPN. [ABSTRACT FROM AUTHOR]

Murine models are valuable instruments in defining the pathogenesis of diabetic nephropathy (DN), but they only partially recapitulate disease manifestations of human DN, limiting their utility. To define the molecular similarities and differences between human and murine DN, we performed a cross-species comparison of glomerular transcriptional networks. Glomerular gene expression was profiled in patients with early type 2 DN and in three mouse models (streptozotocin DBA/2, C57BLKS db/db, and eNOS-deficient C57BLKS db/db mice). Species-specific transcriptional networks were generated and compared with a novel network-matching algorithm. Three shared human--mouse cross-species glomerular transcriptional networks containing 143 (Human-DBA STZ), 97 (Human-BKS db/db), and 162 (Human-BKS eNOS-/- db/db) gene nodes were generated. Shared nodes across all networks reflected established pathogenic mechanisms of diabetes complications, such as elements of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and vascular endothelial growth factor receptor (VEGFR) signaling pathways. In addition, novel pathways not previously associated with DN and cross-species gene nodes and pathways unique to each of the human--mouse networks were discovered. The human--mouse shared glomerular transcriptional networks will assist DN researchers in selecting mouse models most relevant to the human disease process of interest. Moreover, they will allow identification of new pathways shared between mice and humans. [ABSTRACT FROM AUTHOR]

Increased expression of the facilitative glucose transporter, GLUT1, leads to glomerulopathy that resembles diabetic nephropathy, whereas prevention of enhanced GLUT1 expression retards nephropathy. While many of the GLUT1-mediated effects are likely due to mesangial cell effects, we hypothesized that increased GLUT1 expression in podocytes also contributes to the progression of diabetic nephropathy. Therefore, we generated two podocyte-specific GLUT1 transgenic mouse lines (driven by a podocin promoter) on a db/m C57BLKS background. Progeny of the two thunders were used to generate diabetic db/db and control db/m littermate mice. Immunoblots of glomerular lysates showed that transgenic mice had a 3.5-fold (line 1) and 2.1-fold (line 2) increase in GLUT1 content compared with wild-type mice. Both lines showed similar increases in fasting blood glucose and body weights at 24 wk of age compared with wild-type mice. Mesangial index (percent PAS-positive material in the mesangial tuft) increased 88% (line 1) and 75% (line 2) in the wild-type diabetic mice but only 48% (line 1) and 39% (line 2) in the diabetic transgenic mice (P < 0.05, transgenic vs. wild-type mice). This reduction in mesangial expansion was accompanied by a reduction in fibronectin accumulation, and vascular endothelial growth factor (VEGF) levels increased only half as much in the transgenic diabetic mice as in wild-type diabetic mice. Levels of nephrin, neph1, CD2AP, podocin, and GLUT4 were not significantly different in transgenic compared with wild-type mice. Taken together, increased podocyte GLUT1 expression in diabetic mice does not contribute to early diabetic nephropathy; surprisingly, it protects against mesangial expansion and fibronectin accumulation possibly by blunting podocyte VEGF increases. [ABSTRACT FROM AUTHOR]

Enhanced expression of the facilitative glucose transporter, GLUT1, has been shown to inhibit apoptosis in several cell systems including vascular smooth muscle cells (VSMC5). A decrease in apoptosis could lead to increased VSMC numbers in neointimal and medial arterial layers under several pathologic conditions. The hypothesis underlying these studies is that GLUT 1 induces expression of antiapoptotic and prosurvival genes that increase VSMC survival. Transcriptomic analysis of A7r5 VSMCs, in which GLUT1 was acutely overexpressed, showed a 2.14-fold increase in c-FLICE inhibitory protein (cFLIP), which promotes cellular growth and prevents apoptosis through caspase 8 binding. We confirmed that overexpression of GLUT1 induced mRNA and protein expression of both the long and short isoforms of cFLIP (cFLIPL and cFLIPs) in primary and stable immortalized VSMC lines as well as in aortas from GLUT1 transgenic mice. Increased GLUT1 reduced VSMC death by more than twofold after serum withdrawal, as evidenced by decreased caspase 3 activity and Trypan blue exclusion studies. GLUT1 overexpression resulted in a greater than twofold increase in proliferating cell nuclear antigen expression and live cell numbers, consistent with augmented VSMC proliferation. Lentiviral knockdown of cFLIPL showed that cFLIPL was necessary for the proproliferative and antiapoptotic effects of GLUT1 overexpression. Taken together, these data suggest that GLUT1 induction of cFLTPL expression augments proliferation and prevents apoptosis in VSMCs. [ABSTRACT FROM AUTHOR]

Recent studies suggest that thiazolidinediones ameliorate diabetic nephropathy (DN) independently of their effect on hyperglycemia. In the current study, we confirm and extend these findings by showing that rosiglitazone treatment prevented the development of DN and reversed multiple markers of oxidative injury in DBA/2J mice made diabetic by low- dose streptozotocin. These diabetic mice developed a 14.2-fold increase in albuminuria and a 53% expansion of renal glomerular extracellular matrix after 12 wk of diabetes. These changes were largely abrogated by administration of rosiglitazone beginning 2 wk after the completion of streptozotocin injections. Rosiglitazone had no effect on glycemic control. Rosiglitazone had similar effects on insulin-treated diabetic mice after 24 wk of diabetes. Podocyte loss and glomerular fibronectin accumulation, other markers of early DN, were prevented by rosiglitazone in both 12- and 24-wk diabetic models. Surprisingly, glomerular GLUT1 levels did not increase and nephrin levels did not decrease in the diabetic animals; neither changed with rosiglitazone. Plasma and kidney markers of protein oxidation and lipid peroxidation were significantly elevated in the 24-wk diabetic animals despite insulin treatment and were reduced to near-normal levels by rosiglitazone. Finally, urinary metabolites were markedly altered by diabetes. Of 1,988 metabolite features identified by electrospray ionization time of flight mass spectrometry, levels of 56 were altered more than twofold in the urine of diabetic mice. Of these, 21 were returned to normal by rosiglitazone. Thus rosiglitazone has direct effects on the renal glomerulus to reduce reactive oxygen species accumulation to prevent type 1 diabetic mice from development of DN. [ABSTRACT FROM AUTHOR]

Glucose transport is a highly regulated process and is dependent on a variety of signaling events. Glycogen synthase kinase-3 (GSK-3) has been implicated in various aspects of the regulation of glucose transport, but the mechanisms by which GSK-3 activity affects glucose uptake have not been well defined. We report that basal glycogen synthase kinase-3 (GSK-3) activity regulates glucose transport in several cell types. Chronic inhibition of basal GSK-3 activity (8-24 h) in several cell types, including vascular smooth muscle cells, resulted in an approximately twofold increase in glucose uptake due to a similar increase in protein expression of the facilitative glucose transporter 1 (GLUT1). Conversely, expression of a constitutively active form of GSK-3β resulted in at least a twofold decrease in GLUT1 expression and glucose uptake. Since GSK-3 can inhibit mammalian target of rapamycin (mTOR) signaling via phosphorylation of the tuberous sclerosis complex subunit 2 (TSC2) tumor suppressor, we investigated whether chronic GSK-3 effects on glucose uptake and GLUT1 expression depended on TSC2 phosphorylation and TSC inhibition of mTOR. We found that absence of functional TSC2 resulted in a 1.5-to 3-fold increase in glucose uptake and GLUT1 expression in multiple cell types. These increases in glucose uptake and GLUT1 levels were prevented by inhibition of mTOR with rapamycin. GSK-3 inhibition had no effect on glucose uptake or GLUT1 expression in TSC2 mutant cells, indicating that GSK-3 effects on GLUT1 and glucose uptake were mediated by a TSC2/mTOR-dependent pathway. The effect of GSK-3 inhibition on GLUT1 expression and glucose uptake was restored in TSC2 mutant cells by transfection of a wild-type TSC2 vector, but not by a TSC2 construct with mutated GSK-3 phosphorylation sites. Thus, TSC2 and rapamycin-sensitive mTOR function downstream of GSK-3 to modulate effects of GSK-3 on glucose uptake and GLUT1 expression. GSK-3 therefore suppresses glucose uptake via TSC2 and mTOR and may serve to match energy substrate utilization to cellular growth. [ABSTRACT FROM AUTHOR]

The authors review pathogenic processes that appear to be critical in the development of diabetic glomerulosclerosis. The general consensus about major signaling mechanisms involved in stimulating mesangial cell synthesis of extracellular matrix (ECM) emerged during the 1990s. They note reports which found that isolated podocyte damage and loss leads to glomerulosclerosis. Also described are new insights into mechanisms of diabetic glomerulosclerosis.

Background: Hypoxia inducible factor-1 (HIF-1) is a transcription factor that functions to maintain cellular homeostasis in response to hypoxia. There is evidence that HIF-1 can also trigger apoptosis, possibly when cellular responses are inadequate to meet energy demands under hypoxic conditions. Methods: Cardiac derived H9c2 and renal tubular epithelial HK-2 cells expressing either the wild type oxygen regulated subunit of HIF-1 (pcDNA3-Hif-1α) or a dominant negative version that lacked both DNA binding and transactivation domains (pcDNA3-DN-Hif-1α), were maintained in culture and exposed to hypoxia. An RNA interference approach was also employed to selectively knockdown expression of Hif-1α. Apoptosis was analyzed in both H9c2 and HK-2 cells by Hoechst and TUNEL staining, caspase 3 activity assays and activation of pro-apoptotic Bc12 family member Bax. Results: Overexpression of pcDNA3-DN-Hif-1α led to a significant reduction in hypoxia -induced apoptosis (17 ± 2%, P < 0.01) in H9c2 cells compared to both control-transfected and wild type Hif-1α transfected cells. Moreover, selective ablation of HIF-1α protein expression by RNA interference in H9c2 cells led to 55% reduction of caspase 3 activity and 46% reduction in the number of apoptotic cells as determined by Hoechst 33258 staining, after hypoxia. Finally, upregulation of the pro-apoptotic protein, Bax, was found in H9c2 cells overexpressing full-length pcDNA3-HA-HIF-1α exposed to hypoxia. In HK-2 cells overexpression of wild-type Hif-1α led to a two-fold increase in Hif-1α levels during hypoxia. This resulted in a 3.4-fold increase in apoptotic cells and a concomitant increase in caspase 3 activity during hypoxia when compared to vector transfected control cells. HIF-1α also induced upregulation of Bax in HK-2 cells. In addition, introduction of dominant negative Hif-1α constructs in both H9c2 and HK-2 -cells led to decreased active Bax expression. Conclusion: These data demonstrate that HIF-1α is an important component of the apoptotic signaling machinery in the two cell types. [ABSTRACT FROM AUTHOR]

Background: Selenoproteins contain selenocysteine (Sec), commonly considered the 21st genetically encoded amino acid. Many selenoproteins, such as the glutathione peroxidases and thioredoxin reductases, protect cells against oxidative stress by functioning as antioxidants and/or through their roles in the maintenance of intracellular redox balance. Since oxidative stress has been implicated in the pathogenesis of diabetic nephropathy, we hypothesized that selenoproteins protect against this complication of diabetes. Methods: C57BL/6 mice that have a podocyte-specific inability to incorporate Sec into proteins (denoted in this paper as PodoTrsp-/-) and control mice were made diabetic by intraperitoneal injection of streptozotocin, or were injected with vehicle. Blood glucose, body weight, microalbuminuria, glomerular mesangial matrix expansion, and immunohistochemical markers of oxidative stress were assessed. Results: After 3 and 6 months of diabetes, control and PodoTrsp-/- mice had similar levels of blood glucose. There were no differences in urinary albumin/creatinine ratios. Periodic acid-Schiff staining to examine mesangial matrix expansion also demonstrated no difference between control and PodoTrsp-/- mice after 6 months of diabetes, and there were no differences in immunohistochemical stainings for nitrotyrosine or NAD(P)H dehydrogenase, quinone 1. Conclusion: Loss of podocyte selenoproteins in streptozotocin diabetic C57BL/6 mice does not lead to increased oxidative stress as assessed by nitrotyrosine and NAD(P)H dehydrogenase, quinone 1 immunostaining, nor does it lead to worsening nephropathy. [ABSTRACT FROM AUTHOR]

Numerous studies have investigated specific pathways that link diabetes and high extracellular glucose exposure to glomerulosclerosis and mesangial cell extracellular matrix production. However, only in the past ten years has a role for glucose transporters in this process been addressed. Many different glucose transporters are expressed in glomeruli; of these, the GLUT1 facilitative glucose transporter is upregulated in the diabetic renal cortex and in response to glomerular hypertension, as well as in cultured mesangial cells exposed to high glucose. Transgenic mouse and cell models have recently been developed to test the role of GLUT1 in the pathogenesis of glomerulosclerosis with and without diabetes. Clinical studies of GLUT1 alleles performed in humans have identified GLUT1 susceptibility alleles for diabetic nephropathy. Studies are also currently under way to assess the potential role of GLUT1 in nondiabetic renal disorders. [ABSTRACT FROM PUBLISHER]

Background: A reduction in the number of podocytes and podocyte density has been documented in the kidneys of patients with diabetes mellitus. Additional studies have shown that podocyte injury and loss occurs in both diabetic animals and humans. However, most studies in animals have examined relatively long-term changes in podocyte number and density and have not examined effects early after initiation of diabetes. We hypothesized that streptozotocin diabetes in rats and mice would result in an early reduction in podocyte density and that this reduction would be prevented by antioxidants. Methods: The number of podocytes per glomerular section and the podocyte density in glomeruli from rats and mice with streptozotocin (STZ)-diabetes mellitus was determined at several time points based on detection of the glomerular podocyte specific antigens, WT-1 and GLEPP1. The effect of insulin administration or treatment with the antioxidant, a-lipoic acid, on podocyte number was assessed. Results: Experimental diabetes resulted in a rapid decline in apparent podocyte number and podocyte density. A significant reduction in podocytes/glomerular cross-section was found in STZ diabetes in rats at 2 weeks (14%), 6 weeks (18%) and 8 weeks (34%) following STZ injection. Similar declines in apparent podocyte number were found in STZ diabetes in C57BL/6 mice at 2 weeks, but not at 3 days after injection. Treatment with a-lipoic acid substantially prevented podocyte loss in diabetic rats but treatment with insulin had only a modest effect. Conclusion: STZ diabetes results in reduction in apparent podocyte number and in podocyte density within 2 weeks after onset of hyperglycemia. Prevention of these effects with antioxidant therapy suggests that this early reduction in podocyte density is due in part to increased levels of reactive oxygen species as well as hyperglycemia. [ABSTRACT FROM AUTHOR]

The hyperglycemia of maternal diabetes suppresses the glucose transporter-1 (GLUT1) facilitative glucose transporter 49-66% in preimplantation embryos. Glucose uptake is reduced and apoptosis is activated. We hypothesized that the reduction of embryonic GLUT1 may play a key role in the malformations of diabetic embryopathy. Therefore, we produced GLUT1-deficient transgenic mice [i.e., antisense-GLUT1 (GTIA5)] to determine whether GLUT1 deficiency alone could reproduce the growth defects. Early cell division of fertilized mouse eggs injected with GT1AS was markedly impaired; P < 0.001 vs. controls. Two populations of preimplantation embryos obtained from GT1AS x GT1AS heterozygote matings exhibited reduction of the 2-deoxyglucose uptake rate: one by 50% (presumed heterozygotes, P < 0.001 vs. control) and the other by 95% (presumed homozygotes, P < 0.001 vs. heterozygotes). Embryonic GLUT1 deficiency in the range reported with maternal diabetes was associated with growth retardation and developmental malformations similar to those described in diabetes-exposed embryos: intrauterine growth retardation (31.1%), caudal regression (9.8%), anencephaly with absence of the head (6.6%), microphthalmia (4.9%), and micrognathia (1.6%). Reduced body weight !(small embryos, <70% of the nontransgenic body weight) was accompanied by other malformations and a 56% reduction of GLUT1 protein, P < 0.001 vs. nonsmall embryos (body weight ≥70% normal). The heart, brain, and kidneys of embryonic day 18.5 GT1AS embryos exhibited 24-51% reductions of GLUT1 protein. The homozygous GT1A5 genotype was lethal during gestation. Reduced embryonic GLUT1 was associated with the appearance of apoptosis. Therefore, GLUT1 deficiency may play a role in producing embryonic malformations resulting from the hyperglycemia of maternal diabetes. Late gestational macrosomia was absent, apparently requiring a different mechanism. [ABSTRACT FROM AUTHOR]

Recent experimental work indicates that the hyperglycemia-induced increase in mesangial matrix production, which is a hallmark in the development of diabetic nephropathy, is mediated by increased expression of GLUTI. Mesangial cells stably transfected with human GLUT1 mimic the effect of hyperglycemia on the production of the extracellular matrix proteins, particularly fibronectin, when cultured under normoglycemic conditions. Our investigation of the molecular mechanism of this effect has revealed that the enhanced fibronectin production was not mediated by the prosclerotic cytokine transforming growth factor (TGF)-β1. We found markedly increased nuclear content in Jun proteins, leading to enhanced DNA-binding activity of activating protein 1 (AP-1). AP-1 inhibition reduced fibronectin production in a dosage-dependent manner. Moreover, inhibition of classic protein kinase C (PKC) isoforms prevented both the activation of AP-1 and the enhanced fibronectin production. In contrast to mesangial cells exposed to high glucose, no activation of the hexosamine biosynthetic, p38, or extracellular signal-related kinase 1 and 2 mitogen-activated protein kinase pathways nor any increase in TGF-β1 synthesis could be detected, which could be explained by the absence of oxidative stress in cells transfected with the human GLUT1 gene. Our data indicate that increased glucose uptake and metabolism induce PKC-dependent AP-1 activation that is sufficient for enhanced fibronectin production, but not for increased TGF-β1 expression. [ABSTRACT FROM AUTHOR]

Examines the inducement of apoptosis by hypoxia via independent pathways in Jurkat cells. Methods in the conduct of the study; Role of glucose in prevention of cytochrome c releases and activation of caspase 9; Attenuation of hypoxia-induced cell death in Jurkat cells in the presence of glucose.

Background: Serotonin (5-HT, 5-hydroxytryptamine) activates the Extracellular Signal-Regulated Kinase (ERK)/ Mitogen-Activated Protein Kinase (MAPK) pathways, in vascular smooth muscle cells. Parallel MAPK pathways, the c-Jun N-terminal Kinase (JNK) and p38 pathway, are activated by stimulators of the ERK/MAPK pathway. We hypothesized that 5-HT would activate the JNK and p38 pathways in rat vascular smooth muscle cells. Results: Results were determined using standard Western analysis and phosphospecific JNK and p38 antibodies. No significant activation by 5-HT (10-9 - 10-5 M; 30 min) of the JNK or p38 pathways, as measured by protein phosphorylation, was observed in any of these experiments. These experiments were repeated in the presence of the serine/threonine phosphatase inhibitor okadaic acid (1 uM) and the tyrosine phosphatase inhibitor sodium orthovanadate (1 uM) to maximize any observable signal. Even under these optimized conditions, no activation of the JNK or p38 pathways by 5-HT was observed. Time course experiments (5-HT 10-5 M; 5 min, 15 min, 30 min and 60 min) showed no significant activation of JNK after incubation with 5-HT at any time point. However, we detected strong activation of JNK p54 and p46 (5- and 7 fold increases in bands p54 and p46, respectively over control levels) by anisomycin (500 ng/ml, 30 min). Similarly, a JNK activity assay failed to reveal activation of JNK by 5-HT, in contrast to the strong stimulation by anisomycin. Conclusion: Collectively, these data support the conclusion that 5-HT does not activate the JNK or p38 pathways in rat vascular smooth muscle cells. [ABSTRACT FROM AUTHOR]

Describes the characteristics of the AE anion exchanger mRNA and protein expression in vascular smooth muscle cells (VSMC), aorta and renal microvessels. Expression of AE isoforms in cultured VSMC and in rat blood vessels; AE transcript expression in vascular smooth muscle; Ribonuclease protection analysis of AE3 in aorta.

Simple Summary: Type 1 and type 2 diabetes can lead to serious health problems that affect many organs including the nerve, the eye, and the kidney. These health problems can be disabling or even life-threatening, and to date, there are no effective therapies that can slow or prevent their development. We studied the effect of empagliflozin, a medication that reduces blood sugar levels and the risk of cardiovascular disease, on the nerve, eye, and kidney in a side-by-side comparison of type 1 and type 2 mouse models. Empagliflozin had no beneficial effects on disease progression in type 2 diabetic mice, but improved nerve function in the type 1 diabetic model, an effect that was accompanied by reduced markers of oxidative injury. These findings support the concept that different mechanisms contribute to nerve damage in type 1 and type 2 diabetes. Our results further support our view that the concept of one therapy will benefit all complications should be abandoned as we pursue more personalized, tissue-specific, diabetes type-specific treatments. Microvascular complications account for the significant morbidity associated with diabetes. Despite tight glycemic control, disease risk remains especially in type 2 diabetes (T2D) patients and no therapy fully prevents nerve, retinal, or renal damage in type 1 diabetes (T1D) or T2D. Therefore, new antidiabetic drug classes are being evaluated for the treatment of microvascular complications. We investigated the effect of empagliflozin (EMPA), an inhibitor of the sodium/glucose cotransporter 2 (SGLT2), on diabetic neuropathy (DPN), retinopathy (DR), and kidney disease (DKD) in streptozotocin-induced T1D and db/db T2D mouse models. EMPA lowered blood glycemia in T1D and T2D models. However, it did not ameliorate any microvascular complications in the T2D model, which was unexpected, given the protective effect of SGLT2 inhibitors on DKD progression in T2D subjects. Although EMPA did not improve DKD in the T1D model, it had a potential modest effect on DR measures and favorably impacted DPN as well as systemic oxidative stress. These results support the concept that glucose-centric treatments are more effective for DPN in T1D versus T2D. This is the first study that provides an evaluation of EMPA treatment on all microvascular complications in a side-by-side comparison in T1D and T2D models. [ABSTRACT FROM AUTHOR]