High Ambient Glucose Concentration Research Articles

Experimental Diabetic Nephropathy is Associated with Increased Expression of Inflammatory Proteins in the Kidney

AimsDiabetic Nephropathy (DN) is the leading cause of end stage renal disease (ESRD) but pathogenesis of DN is far from clear and hence the treatment remains sub‐optimal. Many studies alluded to the role of chronic inflammation in DN. A recent study in human DN identified 17 different inflammatory proteins collectively called Kidney Risk Inflammatory Signature (KRIS) molecules. (Niewczas et al Nat Med 2019) . These proteins, mostly belonging to tumor necrosis factor (TNF) family predicted risk of progression to ESRD. We tested the hypothesis that the expression of some of such KRIS proteins in experimental models including renal cells cultured in high ambient glucose concentration and kidneys from diabetic animal models is increased. 2.MethodsTo test our hypothesis, we used rat mesangial cells (RMC) in culture and ZSF rats —an established animal model for DN. RMC were grown in DMEM media with either low glucose (5.5 mM) or high glucose (25 mM) for 48 hours and expression of these molecules was determined by Western Blot technique and ELISA. Specifically, we examined TNFα R1 and IL15α proteins. In addition RMC grown in low glucose media for a week were incubated in high glucose media for 48 hours and examined for KRIS proteins. ZSF rats (both male and female) were grown on Purina 50008 diet to render them hyperglycemic and euthanized at 40 weeks of age. Male CD rats (non‐diabetic) were used as controls. Blood and urine samples were obtained at the start and end of the study. Kidneys were harvested, tissue was homogenized and homogenates examined for protein expression by WB. 3.ResultsZSF rats developed obesity, diabetes, hyperlipidemia and proteinuria. Male ZSF rats were heavier (650 gm vs 510 gm) and more hyperglycemic (295 mg/dl vs.210 mg/dl) than females. CD rats were normoglycemic with normal kidney function. Kidney homogenates from ZSF rats demonstrated increased expression of both TNFαR1 and IL15α proteins. These changes were more significant in male than female ZSF rats. RMC exposed to high glucose for 48 hours either directly or after chronic exposure to low glucose demonstrated similar increase in expression of TNFαR1 and IL15α proteins compared to low glucose. 4.ConclusionsIn renal cells exposed to high glucose and in ZSF (diabetic) rats with nephropathy, the expression of TNFαR1 and IL15α proteins, which are inflammatory mediators are increased. Male ZSF rats expressed more significant disease and increase in KRIS proteins indicating the need for further investigations into the role of gender in disease progression in murine models of DN. These results suggest that inflammatory proteins from TNF family are overexpressed in diabetic nephropathy models and that renal injury from chronic hyperglycemia may be mediated by at least in part by chronic inflammation. 5.Support or Funding InformationThis research was funded by Larry and Jane Woirhaye Memorial Research FoundationInflammatory Protein Expression in RMCFigure 1

Fluorofenidone Attenuates Diabetic Nephropathy and Kidney Fibrosis in db/db Mice

Background/Aims: Fluorofenidone [1-(3-fluorophenyl)-5-methyl-2-(1H)-pyridone, AKF-PD], a novel pyridone agent, showed potent antifibrotic properties. The aim of the present study was to investigate the effects of AKF-PD on diabetic nephropathy and kidney fibrosis, and to obtain an insight into its mechanisms of action. Methods: We administered AKF-PD to diabetic db/db mice for 12 weeks. Moreover, we performed in vitro cultures using murine mesangial cells exposed to high ambient glucose concentrations. Results: AKF-PD reduced renal hypertrophy, mesangial matrix expansion and albuminuria in the db/db mice. The upregulated expression of α₁(I)- and α₁(IV)-collagen and fibronectin mRNAs, transforming growth factor-β1 (TGF-β₁), α-smooth muscle actin (α-SMA), and tissue inhibitors of metalloproteinase 1 (TIMP-1) mRNAs and proteins was inhibited by AKF-PD treatment in the renal cortex of db/db mice. The maximal effective dose of AKF-PD was about 500 mg/kg body weight. AKF-PD inhibited the upregulated expression of α₁(I)- and α₁(IV)-collagens, TGF-β₁, TIMP-1 and α-SMA induced by high glucose concentrations in cultured mesangial cells. Conclusions: Our data indicate that AKF-PD diminishes the abnormal accumulation of mesangial matrix through the inhibition of upregulated expression of TGF-β target genes in kidneys of db/db mice, resulting in attenuation of renal fibrosis and amelioration of renal dysfunction despite persistent hyperglycemia. Therefore, AKF-PD, a potent antifibrotic agent, holds great promise in the treatment of diabetic nephropathy.

High glucose initiates calpain-induced necrosis before apoptosis in LLC-PK1 cells

Cells exposed to high ambient glucose concentrations are subject to increases in intracellular calcium ([Ca(2+)](i)). We therefore considered it likely that the calcium-dependent cysteine protease calpain would play a role in the development of high glucose-induced cell injury. After 3 and 24 h, high glucose concentrations (25 mM D-glucose) produced almost identical increases in the degree of necrotic cell death in kidney proximal tubular epithelial cells (LLC-PK(1)) compared to cells treated with control glucose (5 mM D-glucose). Necrotic cell death could be restricted by inhibiting the activity of calpain. High glucose-treated LLC-PK(1) cells were found to have significantly elevated [Ca(2+)](i) concentrations within 1 h, and elevated calpain activity within 2 h compared to control treated cells. The DNA nick sensor poly(ADP-ribose) polymerase (PARP) has previously been shown to be an important driver of high glucose-induced cell death, but here we found that although PARP activity was increased after 24 h, it was unaltered after 3 h. Furthermore, PARP inhibition with PJ-34 did not restrict early high glucose-induced necrosis. Using a gene knockdown strategy with small interference RNA, we found that silencing calpain was effective in reducing the degree of early high glucose-induced necrosis. We conclude that high glucose concentrations evoke an early, calpain-mediated necrosis in cultured proximal tubular cells that is PARP-independent, and precedes the previously recognized activation of apoptosis.

Activated IGF-1R inhibits hyperglycemia-induced DNA damage and promotes DNA repair by homologous recombination

The IGF-1R is a genetic determinant of oxidative stress and longevity. Hyperglycemia induces an exponential increase in the production of a key danger signal, reactive oxygen intermediates, which target genomic DNA. Here, we report for the first time that ligand activation of the IGF-1R prevents hyperglycemia-induced genotoxic stress and enhances DNA repair, maintaining genomic integrity and cell viability. We performed single gel electrophoresis (comet assay) to evaluate DNA damage in serum-starved SV40 murine mesangial cells (MMC) and normal human mesangial cells (NHMC), maintained at high ambient glucose concentration. Hyperglycemia inflicted an impressive array of DNA damage in the form of single-strand breaks (SSBs) and double-strand breaks (DSBs). The inclusion of IGF-1 to culture media of MMC and NHMC prevented hyperglycemia-induced DNA damage. To determine whether DNA damage was mediated by reactive oxygen species (ROS), ROS generation was evaluated, in the presence of IGF-1, or the free radical scavenger n-acetyl-cysteine (NAC). IGF-1 and NAC inhibited hyperglycemic-induced ROS production and hyperglycemia-induced DNA damage. We next asked whether IGF-1 promotes the repair of DSB under hyperglycemic conditions, by homologous recombination (HRR) or nonhomologous end joining (NHEJ). Repair of DSB by NHEJ and HRR was operative in MMC maintained under hyperglycemic conditions. IGF-1 increased HRR by nearly twofold, whereas IGF-1 did not affect DNA repair by NHEJ. IGF-1R enhancement of HRR correlated with the translocation of Rad51 to foci of DNA damage. Inhibition of Rad51 expression by short interfering RNA experiments markedly decreased percentage of MMC positive for Rad51 nuclear foci and increased hyperglycemic DNA damage. We conclude that the activated IGF-1R rescues mesangial cells from hyperglycemia-induced danger signals that target genomic DNA by suppressing ROS and enhancing DNA repair by HRR.

IGF-1 inhibits the mitochondrial apoptosis program in mesangial cells exposed to high glucose.

The activated insulin-like growth factor-1 receptor (IGF-1R) protects cells from a wide range of apoptotic stimuli. Hyperglycemia promotes the intracellular generation of superoxide anion and hydrogen peroxide, both of which have been linked to the activation of the mitochondrial apoptosis program. Here, we report for the first time that ligand activation of the IGF-1R protects normal human mesangial cells and SV40 murine mesangial cells from the glycol-oxidant-induced apoptosis program. The IGF-1R antiapoptosis program was dependent on the recruitment of both Akt/PKB and the ERK subfamily of mitogen-activated protein kinases. IGF-1 treatment also protected the redox potential of mesangial cells maintained at high ambient glucose concentration, by inhibiting the generation of reactive oxygen intermediates and preserving mitochondrial transmembrane potential. IGF-1R survival signals targeted the Bcl-2 family of proteins to protect against glucose-induced apoptosis and oxidative stress. IGF-1-treated cells exhibited a decrease in the Bax/Bcl-2 ratio; increased phosphorylation/inactivation of Bad at Ser112 and Ser136; inhibition of cytochrome c release; perturbations directionally opposed to the initiation of the apoptosis program. In addition, we demonstrate IGF-1R-activated ERK signaling modules phosphorylate Ser112 of the mitochondrial Bad protein, establishing a direct link between surface IGF-1R and the survival program in mitochondria. Our findings indicate that in mesangial cells maintained at high ambient glucose concentration, IGF-1 activates a survival program that maintains the integrity of mitochondria and prevents the expression of the genetic program for apoptosis.

Inhibition of the interaction of AGE-RAGE prevents hyperglycemia-induced fibrosis of the peritoneal membrane.

The peritoneal membrane of long-term peritoneal dialysis patients is characterized by a loss of ultrafiltration capacity, associated morphologically with submesothelial fibrosis and neoangiogenesis. Exposure to high glucose concentrations in peritoneal dialysate and the resultant advanced glycation end-products (AGE) accumulation have been implicated in the development of these changes, but their exact pathophysiological role is unknown. We examined the effect of the interaction of AGE with one of their receptors (i.e., RAGE) on the function and structure of the peritoneum exposed to high ambient glucose concentrations. Streptozotocin-induced diabetic rats and control rats were treated during 6 wk with either neutralizing monoclonal anti-RAGE antibodies or control antibodies. The expression of RAGE was strongly enhanced in the peritoneal membrane of the diabetic animals. The diabetic peritonea were characterized by an elevated transport of small solutes, lower ultrafiltration rates, a higher vascular density, and an upregulation of endothelial nitric oxide synthase expression. These parameters were unaffected by treatment with anti-RAGE antibodies. In contrast, anti-RAGE but not control antibodies prevented upregulation of TGF-beta, development of submesothelial fibrosis, and fibronectin accumulation in the peritoneum of diabetic animals. In conclusion, binding of AGE to RAGE increases the expression of TGF-beta and contributes to the development of submesothelial fibrosis. Neoangiogenesis and the resultant loss of ultrafiltration capacity are mediated by different pathogenetic pathways.

High glucose promotes mesangial cell apoptosis by oxidant-dependent mechanism

Reactive oxygen species are recognized as important mediators of biological responses. Hyperglycemia promotes the intracellular generation of superoxide anion and hydrogen peroxide. In several cell lines, oxidant stress has been linked to the activation of death programs. Here, we report for the first time that high ambient glucose concentration induces apoptosis in murine and human mesangial cells by an oxidant-dependent mechanism. The signaling cascade activated by glucose-induced oxidant stress included the heterodimeric redox-sensitive transcription factor NF-kappaB, which exhibited an upregulation in p65/c-Rel binding activity and suppressed binding activity of the p50 dimer. Recruitment of NF-kappaB and mesangial cell apoptosis were both inhibited by antioxidants, implicating oxidant-induced activation of NF-kappaB in the transmission of the death signal. The genetic program for glucose-induced mesangial cell apoptosis was characterized by an upregulation of the Bax/Bcl-2 ratio. In addition, phosphorylation of the proapoptotic protein Bad was attenuated in mesangial cells maintained at high-glucose concentration, favoring progression of the apoptotic process. These perturbations in the expression and phosphorylation of the Bcl-2 family were coupled with the release of cytochrome c from mitochondria and caspase activation. Our findings indicate that in mesangial cells exposed to high ambient glucose concentration, oxidant stress is a proximate event in the activation of the death program, which culminates in mitochondrial dysfunction and caspase-3 activation, as the terminal event.

Gremlin: an example of the re-emergence of developmental programmes in diabetic nephropathy.

The past two decades have yielded major advances in our understanding of the pathogenetic mechanisms that cause diabetic nephropathy. Of particular interest is the emerging paradigm of the recapitulation of developmental programmes within the diabetic kidney. Recently we have used the complementary techniques of suppression subtractive hybridization and Affymetrix GeneChips to assess changes in gene expression in human mesangial cells subjected to high ambient glucose concentrations and cyclic mechanical strain in vitro, the latter being models of hyperglycaemia and glomerular hypertension, respectively. In this review, we will focus on the potential role of one such differentially expressed gene, namely gremlin, in the pathogenesis of diabetic nephropathy. In the context of developmental nephrology, gremlin warrants special mention. Gremlin is a 184 amino acid protein and a member of the cysteine knot superfamily. The protein is highly conserved during evolution and is present in soluble and cell-associated forms. It belongs to a novel family of bone morphogenetic protein (BMP) antagonists that includes the head-inducing factor Cerberus and the tumour suppressor DAN. These proteins play important roles in limb development and neural crest cell differentiation. Evidence will be presented that mesangial cell gremlin expression is up-regulated by high ambient glucose, cyclic mechanical strain and transforming growth factor-beta (TGF-beta) and that gremlin may be an important modulator of mesangial cell proliferation and epithelial-mesenchymal transdifferentiation in a diabetic milieu.

Angiotensin II promotes glucose-induced activation of cardiac protein kinase C isozymes and phosphorylation of troponin I.

Activation of the protein kinase C (PKC) family is a potential signaling mechanism by which high ambient glucose concentration modulates the phenotype and physiological function of cells. Recently, the cardiac renin angiotensin system (RAS) has been reported to promote PKC translocation in the diabetic heart via the angiotensin (ANG) II type 1 receptor (AT-1R). To evaluate the molecular events coupled with high glucose-induced PKC translocation and to examine the role of endogenously released ANG II in myocyte PKC signaling, primary cultures of adult rat ventricular myocytes were exposed to normal (5 mmol/l) or high (25 mmol/l) glucose for 12-24 h. Western blot analysis indicated that adult rat ventricular myocytes coexpress six PKC isozymes (alpha, beta(1,) beta(2,) delta, epsilon, and zeta). Translocation of five PKC isozymes (beta(1), beta(2), delta, epsilon, and zeta) was detected in response to 25 mmol/l glucose. Inhibition of phospholipase C with tricyclodecan-9-yl-xanthogenate blocked glucose-induced translocation of PKC-beta(2), -delta, and -zeta. Inhibition of tyrosine kinase with genistein blocked glucose-induced translocation of PKC-beta(1) and -delta, whereas chelation of intracellular Ca(2+) with 1,2-bis(2-aminophenoxy)ethane N,N,N,'N'-tetraacetic acid blocked translocation of PKC-beta(1) and -beta(2). Enzyme-linked immunosorbent assay performed on culture media from myocytes maintained in 25 mmol/l glucose detected a twofold increase in ANG II. Addition of an AT-1R antagonist (losartan; 100 nmol/l) to myocyte cultures blocked translocation of PKC-beta(1), -beta(2), -delta, and -epsilon. Phosphorylation of troponin (Tn) I was increased in myocytes exposed to 25 mmol/l glucose. Losartan selectively inhibited Tn I serine phosphorylation but did not affect phosphorylation at threonine residues. We concluded that 1) 25 mmol/l glucose triggers the release of ANG II by myocytes, resulting in activation of the ANG II autocrine pathway; 2) differential translocation of myocyte PKC isozymes occurs in response to 25 mmol/l glucose and ANG II; and 3) AT-1R-dependent PKC isozymes (beta(1), beta(2), delta, and epsilon) target Tn I serine residues.

Antibodies against vascular endothelial growth factor improve early renal dysfunction in experimental diabetes.

Vascular endothelial growth factor (VEGF) is a cytokine that potently stimulates angiogenesis, microvascular hyperpermeability, and endothelium-dependent vasodilation, effects that are largely mediated by endothelial nitric oxide synthase (eNOS). The expression of VEGF is pronounced in glomerular visceral epithelial cells, but its function in renal physiology and pathophysiology is unknown. VEGF expression is upregulated by high ambient glucose concentrations in several cell types in vitro and in glomeruli of diabetic rats. To assess the role of VEGF in the pathophysiology of early renal dysfunction in diabetes, monoclonal anti-VEGF antibodies (Ab) were administered to control and streptozotocin-induced diabetic rats for 6 wk after induction of diabetes. Based on in vitro binding studies, an adequate serum VEGF inhibitory activity was achieved during the entire course of anti-VEGF Ab administration. Anti-VEGF Ab treatment but not administration of isotype-matched control Ab decreased hyperfiltration, albuminuria, and glomerular hypertrophy in diabetic rats. VEGF blockade also prevented the upregulation of eNOS expression in glomerular capillary endothelial cells of diabetic rats. Antagonism of VEGF had no effect on GFR and glomerular volume in control rats. These results identify VEGF as a pathogenetic link between hyperglycemia and early renal dysfunction in diabetes. Targeting VEGF may prove useful as a therapeutic strategy for the treatment of early diabetic nephropathy.

High glucose-induced intercellular adhesion molecule-1 (ICAM-1) expression through an osmotic effect in rat mesangial cells is PKC-NF-kappa B-dependent.

Infiltration of mononuclear cells and glomerular enlargement accompanied by glomerular cell proliferation are very early characteristics of the pathophysiology of diabetes. To clarify the mechanism of early diabetic nephropathy, we measured [3H]-thymidine incorporation and cell numbers to show the influence of a high ambient glucose concentration and the osmotic effect on rat mesangial cell proliferation. We also measured the effect of high glucose on the expression of intercellular adhesion molecule-1 and vascular adhesion molecule-1 by flow cytometry and semiquantitative RT-PCR in mesangial cells and the adhesion of leukocytes to mesangial cells. Cells exposed to high D-glucose (30 mmol/l) caused an increase in [3H]-thymidine incorporation and cell numbers at 24 and 48 h and normalized at 72 h (p < 0.05), whereas these changes were not found in high mannitol (30 mmol/l), IL-1 beta, or TNF alpha-stimulated mesangial cells. Cells exposed to high-glucose (15, 30, or 60 mmol/l) or osmotic agents (L-glucose, raffinose and mannitol) showed that intercellular adhesion molecule-1 expression began to increase after 24 h, reached its maximum at 24 and 48 h and gradually decreased afterwards. The stimulatory effects of high glucose and high mannitol on mRNA expression were observed as early as 6 h and reached its maximum at 12 h. Up-regulation of ICAM-1 protein and mRNA was also found in IL-1-beta and TNF-alpha-stimulated mesangial cells. Neither vascular adhesion molecule-1 protein nor mRNA expression was, however, affected by high glucose and high mannitol. Notably, the protein kinase C inhibitors calphostin C and staurosporine reduced high glucose- or high mannitol-induced intercellular adhesion molecule-1 mRNA expression and high glucose-induced proliferation. Furthermore, the NF-kappa B inhibitor N-tosyl-L-phenylalanine chloromethyl ketone reduced high glucose- or high mannitol-induced intercellular adhesion molecule-1 mRNA expression and high glucose-induced proliferation. Results showed that high glucose (15, 30 mmol/l) or high concentrations of osmotic agents remarkably increased the number of adherent leukocytes to mesangial cells (p < 0.01) compared with control cells (5 mmol/l D-glucose). Functional blocking of intercellular adhesion molecule-1 on mesangial cells with rat intercellular adhesion molecule-1 monoclonal antibody, calphostin C, staurosporine, or N-tosyl-L-phenylalanine chloromethyl ketone significantly inhibited high glucose- or high mannitol-induced increase in leukocyte adhesion (p << 0.05). These results suggest that high glucose can upregulate intercellular adhesion molecule-1 protein and mRNA expression but not vascular adhesion molecule-1 expression in mesangial cells and promote leukocyte adhesion through up-regulation of intercellular adhesion molecule-1 through osmotic effect, possibly depending on the protein kinase C nuclear factor-kappa B (PKC-NF-kappa B) pathway. High glucose itself can also promote mesangial cell proliferation through the PKC-NF-kappa B pathways. We conclude that hyperglycaemia in itself seems to be an important factor in the development of early diabetic nephropathy.

Role of vascular endothelial growth factor in diabetic nephropathy

Vascular endothelial growth factor (VEGF) is a potent cytokine that is considered to be an important mediator in the pathogenesis of endothelial dysfunction in diabetes. This study investigates the effect of high glucose on the signaling and production of VEGF in rat mesangial cells in culture and measures the urinary VEGF level in patients with different stages of diabetic nephropathy. To elucidate the role of VEGF in vivo further, expression of VEGF in control and diabetic kidneys was examined using immunohistochemistry. A high ambient glucose concentration in the culture medium increased VEGF mRNA expression and protein production within 3 h in a concentration-dependent manner. A protein kinase C (PKC) inhibitor and PKC down-regulation inhibited glucose-induced increases in VEGF production. Urinary excretion of VEGF significantly increased according to the degree of proteinuria in patients with diabetes. A weak but significant correlation was found between urinary VEGF excretion and the levels of serum creatinine, creatinine clearance, microalbuminuria, and proteinuria. Immunohistochemistry revealed marked differences in the extent of mesangial VEGF staining between diabetic and control kidneys. Pronounced up-regulation of VEGF was observed in the glomerular epithelial cell in the early phase of diabetic kidney disease, whereas widespread expression of VEGF was found in the tubular segments, especially the proximal segment, in advanced diabetic nephropathy. These results suggest that VEGF may play a role in the pathogenesis of diabetic nephropathy.

Constitutive nitric oxide synthase expression in retinal vascular endothelial cells is suppressed by high glucose and advanced glycation end products.

It has been suggested that increased production of nitric oxide (NO), a potent endothelium-derived vasodilator, may be responsible for increased blood flow in the retinal and renal vascular beds in early diabetes. However, NO-mediated vasodilation has been reported as impaired in diabetes, and there is evidence that the synthesis and release of NO by the vascular endothelium may be flawed in this condition. We examined the effect of high ambient glucose and exposure to exogenous glycated proteins on NO synthesis in cultured retinal microvascular endothelial cells (RMECs), using a polarographic sensor to measure released NO gas. Nitrite (the stable end product of the reaction between NO and molecular oxygen) was measured in tissue culture supernatants. The expression of vascular endothelial constitutive nitric oxide synthase (eNOS), which is responsible for NO synthesis in endothelial cells, was studied by Western blot analysis and Northern hybridization experiments. A dose-dependent reduction of NO synthesis by RMECs occurred 5 days after exposure to 15 and 25 mmol/l glucose, and concomitantly we found that accumulation of nitrite in culture supernatants of high-glucose exposed cells was also reduced. Coincubation of endothelial cells with inhibitors of protein kinase C (PKC) increased the accumulation of nitrite but did not restore it to the levels obtained when cells were cultured in 5 mmol/l glucose. The expression of eNOS by RMECs was markedly reduced by 5 days of exposure to 25 mmol/l glucose and glycated albumin. This study implicates the PKC pathway, which is known to be upregulated on exposure to high ambient glucose concentrations, as a possible factor in the inhibition of eNOS expression in RMECs. This study also suggests that glycated proteins may be involved in the pathogenesis of vascular endothelial dysfunction by modulating the nitric oxide synthase (NOS)/NO pathway in retinal vascular endothelial cells.

The Fibronectin Production Is Increased by Thrombospondin via Activation of TGF-β in Cultured Human Mesangial Cells

Thrombospondin (TSP) is a multifunctional glycoprotein that is synthesized by a variety of cells including mesangial cells (MCs). To clarify the effect of TSP on the pathogenesis of diabetic nephropathy, we studied the effect of glucose concentrations on TSP synthesis in cultured human MCs. Thereafter, the effects of TSP on the activation of transforming growth factor beta (TGF-β) and fibronectin production were investigated in MCs. Incubating MCs with elevated glucose levels for 6 days resulted in an increase in TSP synthesis, measured by an enzyme-linked immunosorbent assay, both in culture media and cell layers. Treatment of MCs with TSP (final concentrations 1 and 5 µg/ml) for 24 h resulted in an increase (1.3- and 2.1-fold, respectively) in active TGF-β, which was determined with an enzyme-linked immunosorbent assay using TGF-β-soluble receptor type II, in the culture media without having any effect on the production of total TGF-β. Exposure of MCs to TSP caused enhancement of fibronectin production in both media and cell layers in a TSP dose-dependent manner with the maximum at a TSP concentration of 1 µg/ml. The TSP-induced increase in fibronectin production from MCs was completely prevented by concomitant treatment with 10 µg/ml anti-TGF-β neutralizing antibody. These results indicate that the TSP production is promoted by a high ambient glucose concentration in human MCs and that TSP, in turn, causes an increase in fibronectin production via activation of TGF-β.

Physiological role of large, Ca2+-activated K+ channels in human glomerular mesangial cells.

1. Contraction assays and patch clamp methods were used to determine the role of K+ channels in the regulation of contractile tone of human mesangial cells (MC) in culture. 2. MC contraction was induced by vasoconstrictor agents, such as angiotensin II (AngII; 100 nmol/L) and glybenclamide (Glyb), but not by iberiotoxin (IbTX), a blocker of large Ca2+-activated K+ channels (BK(Ca)). These results suggest that Glyb-sensitive K+ channels, but not BK(Ca) channels, were active at rest. 3. In the presence of 100 nmol/L IbTX, contraction by AngII was slightly, but not significantly, enhanced, indicating that BK(Ca) has a minimal role as a negative feedback regulator of contraction. Nitroprusside (NP; 100 mu mol/L) a nitric oxide (NO) donor, atrial natriuretic peptide (ANP; 1.0 mu mol/L) and db-cGMP (10 mu mol/L) attenuated AngII-induced contraction in the absence, but not in the presence, of IbTX, suggesting that BK(Ca) channels were activated by cGMP. 4. In patch clamp experiments, three distinct K+-selective channels of 9, 65 and 150 pS (outward currents) were found in excised, inside-out patches. The 150 pS channel was completely inhibited by 100 nmol/L IbTX and displayed voltage- and calcium-dependent gating qualitatively similar to BK(Ca) in other cell types. 5. In cell attached (CA) patches, the response of BK(Ca) to bath AngII (100 nmol/L) was relatively minor in control solutions, but was considerably greater in the presence of db-cGMP. 6. In excised patches, Mg-ATP (1 mmol/L) plus db-cGMP (1 mu mol/L) activated BK(Ca) in the absence, but not the presence, of the non-specific kinase inhibitor, staurosporine. 7. Separate experiments showed that BK(Ca) were also activated by arachidonic acid and high ambient glucose concentrations. 8. These results indicate that: (i) resting MC tone is sensitive to glybenclamide and apamin; and (ii) the role of BK(Ca) as a negative feedback regulator of contraction is minimal under normal conditions but is markedly enhanced by cGMP-stimulating relaxants and arachidonic acid.

High-Glucose–Triggered Apoptosis in Cultured Endothelial Cells

High ambient glucose concentration, linked to vascular complications in diabetes in vivo, modulates mRNA expression of fibronectin, collagen, tissue-type plasminogen activator, and plasminogen activator inhibitor and induces delayed replication and excess cell death in cultured vascular endothelial cells. To determine the role of high ambient glucose (30 mmol/l) in apoptosis, paired cultures of individual isolates of human umbilical vein endothelial cells (HUVECs) were exposed to both high (30 mmol/l) and low (5 mmol/l) concentrations of glucose for short-term (24, 48, and 72 h) and long-term (13 +/- 1 days) experiments. Incubation of HUVECs with high glucose for > 48 h increased DNA fragmentation (13.7 +/- 6.5% of total DNA, mean +/- SD) versus cultures kept in 5 mmol/l glucose (10.9 +/- 5.6%, P < 0.005), as measured by [3H]thymidine assays. Data were confirmed by apoptosis-specific fluorescence-activated cell sorter analysis of confluent HUVEC cultures, which displayed after long-term exposure to 30 mmol/l glucose a 1.5-fold higher prevalence of apoptosis than control cultures exposed to 5 mmol/l glucose (P < 0.005). In contrast, no increase in DNA fragmentation in response to 30 mmol/l glucose was seen for standardized cell lines (K 562, P 815, YT) and fibroblasts. Expression of clusterin mRNA, originally reported to be a molecular marker of apoptosis, was only slightly affected by short-term (24-h) high-glucose exposure but was significantly reduced after long-term incubation in 30 mmol/l glucose (82.2 +/- 13.8% of control) versus 5 mmol/l glucose, which questions the role of clusterin gene expression as a marker of apoptosis.(ABSTRACT TRUNCATED AT 250 WORDS)

High glucose induces cell hypertrophy and stimulates collagen gene transcription in proximal tubule

Tubulointerstitial changes in the diabetic kidney correlate closely with the decline in glomerular filtration. In this study, we used a cell culture system of mouse proximal tubule epithelial cells to test the effects of glucose on cell growth, size, and matrix biosynthesis. [3H]thymidine incorporation was significantly inhibited in cells grown in 450 mg/dl glucose, compared with cells grown in 100 mg/dl glucose. The cells grown in the higher glucose concentration were slightly larger, their protein content and the total protein synthetic rate were significantly increased, and they secreted approximately twice as much procollagens type IV and type I. Concordantly, steady-state procollagen mRNA levels were also increased: 2.6-fold for the alpha 1(IV) and 2.2-fold for the alpha 2(I) procollagens. Additionally, nuclear run-off studies demonstrated that procollagen gene transcription rate was stimulated approximately 50%; beta-actin transcription rate was not altered. We used chloramphenicol acetyltransferase (CAT) reporter gene constructs to determine whether the increased transcription rate of alpha 2(I) gene was associated with activation of its enhancer sequence. Cells transfected with the enhancer demonstrated more than fivefold increase in CAT activity when cultured in the high-glucose medium. These studies demonstrate a multitude of effects of high ambient glucose concentrations on proximal tubule cell growth and collagen biosynthesis; cell proliferation is decreased although cell hypertrophy occurs. Procollagen gene transcription rate is stimulated and this response contributes to the observed increase in procollagen mRNA content. Activation of an enhancer sequence may be one possible mode through which high glucose levels increase the transcription of procollagen type I, presumably involving trans-acting factor(s).

The effect of elevated glucose levels on myo-inositol metabolism in cultured bovine aortic endothelial cells

Bovine aorta endothelial cells were used to determine the effect of high ambient glucose concentrations on myo-inositol metabolism. Culturing the cells for a minimum of 1 week in elevated glucose concentrations caused an increase in the intracellular sorbitol content and a decrease in myo-inositol levels. The accumulation of myo-inositol from the medium and incorporation into phospholipids was reduced 25% to 50% in cells grown in the presence of 30 to 50 mmol/L glucose. This effect was not observed following a short-term exposure of the cells to elevated glucose levels. Kinetic analysis of high-affinity myo-inositol uptake showed that the K'm was significantly increased in cells grown in 30 mmol/L glucose compared to those cultured in 5.6 mmol/L glucose. This would suggest that exposing endothelial cells to high ambient glucose levels for a minimum of 1 week leads to a competitive type of inhibition of high-affinity myo-inositol uptake. The changes in myo-inositol metabolism and content and sorbitol levels mediated by glucose exposure were blocked by addition of the aldose reductase inhibitor, sorbinil, to the media, suggesting that these changes are caused by the accumulation of sorbitol by the cells. Exposure of bovine aorta endothelial cells to high ambient levels of glucose leads to accumulation of sorbitol in the cells, which is responsible for alterations in myo-inositol metabolism. These changes could result in alteration of endothelial cell membrane function and contribute to the pathology of diabetes mellitus.

Renal hypertrophy in experimental diabetes. Effect of diabetes on the pathways of glucose metabolism: differential response in adult and immature rats.

The effect of short-term diabetes, 5 days after the administration of streptozotocin, on renal growth and the activity of alternative pathways of glucose metabolism was studied in immature (21-day-old) rats and in adult rats. The kidney weight increased by 28% in the adult diabetic rats, but by only 10% in the immature diabetic rats, relative to their age-matched control groups. The flux of glucose via the pentose phosphate pathway was increased 2-3-fold in the adult diabetic rats, but was unchanged in the immature diabetic group. Enzymes of this pathway (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) increased by 29% and 77% respectively in adult diabetic rats; in the immature group they showed changes of +5% and +28% respectively. The rate of glucose phosphorylation increased significantly in both groups of diabetic rats; only minor changes were observed in oxidation via the tricarboxylic acid cycle. Increases of 40-50% were found in the activity of enzymes involved in UDP-glucose metabolism (phosphoglucomutase, UDPglucose pyrophosphorylase and UDPglucose dehydrogenase) and in lactate dehydrogenase in both young and adult animals. The results suggest a differential renal response to streptozotocin-diabetes according to the stage of renal growth and development, and it is proposed that the difference is related to the developmental emergence of aldose reductase. Enzymes involved in formation of ribose 5-phosphate and NADPH are strikingly increased in the adult diabetic, whereas metabolic functions dependent on a high ambient glucose concentration, e.g. synthesis of glycogen and glucuronate, are similarly affected in adult and immature diabetic groups, both showing certain aspects of 'glucose overutilization'.

Nature and distribution of two distinct forms of hexokinase within the mammalian lens

Hexokinase activity in calf, rabbit and rat lens represents the summated activities of two distinct forms of hexokinase. These are not unique “lens hexokinases”, but possess the same electrophoretic characteristics as Types I and II of other tissues. Lens is devoid of Types III and IV and IIb. The Michaelis constants of these lens heoxkinases are; Kml: 7·4 × 10−5M, and KmII: 1·0 × 10−3 m . In the calf lens eapsule-epithelium, 24% of the hexokinase is Type I; 33% of the nuclear hexokinase is Type I. The total hexokinase activity of the rat lens increases with age or lens size. The total activity of each member of a pair of lenses is essentially identical and lenses of similar size from rats of different sex have similar activities. The specific activity of lens heoxkinase decreases with increasing lens size and is probably a reflection of proportionally greater synthesis of non-enzymic structural protein. The capsule and epithelium contain 10% of the total lens hexokinase. The equatorial cortex contains approximately 3 times the hexokinase activity of the anterior or posterior cortex, and the nucleus is practically devoid of measureable hexokinase activity. Hexokinase and phosphofructokinase have been shown to be the rate limiting steps in lens glycolysis. Our data suggest that rather than a single form of hexokinase serving as pacemaker, there may actually be two—Type II hexokinase pacing glycolysis in areas of relatively high ambient glucose concentration (epithelium and cortex), and Type I in regions of low ambient glucose concentration (nucleus).