Protective effect of the tunneling nanotube-TNFAIP2/M-sec system on podocyte autophagy in diabetic nephropathy.

Podocyte injury leading to albuminuria is a characteristic feature of diabetic nephropathy (DN). Hyperglycemia and advanced glycation end products (AGEs) are major determinants of DN. However, the underlying mechanisms of podocyte injury remain poorly understood. The cytosolic protein TNFAIP2/M-Sec is required for tunneling nanotubes (TNTs) formation, which are membrane channels that transiently connect cells, allowing organelle transfer. Podocytes express TNFAIP2 and form TNTs, but the potential relevance of the TNFAIP2-TNT system in DN is unknown. We studied TNFAIP2 expression in both human and experimental DN and the renal effect of tnfaip2 deletion in streptozotocin-induced DN. Moreover, we explored the role of the TNFAIP2-TNT system in podocytes exposed to diabetes-related insults. TNFAIP2 was overexpressed by podocytes in both human and experimental DN and exposre of podocytes to high glucose and AGEs induced the TNFAIP2-TNT system. In diabetic mice, tnfaip2 deletion exacerbated albuminuria, renal function loss, podocyte injury, and mesangial expansion. Moreover, blockade of the autophagic flux due to lysosomal dysfunction was observed in diabetes-injured podocytes both in vitro and in vivo and exacerbated by tnfaip2 deletion. TNTs allowed autophagosome and lysosome exchange between podocytes, thereby ameliorating AGE-induced lysosomal dysfunction and apoptosis. This protective effect was abolished by tnfaip2 deletion, TNT inhibition, and donor cell lysosome damage. By contrast, Tnfaip2 overexpression enhanced TNT-mediated transfer and prevented AGE-induced autophagy and lysosome dysfunction and apoptosis. In conclusion, TNFAIP2 plays an important protective role in podocytes in the context of DN by allowing TNT-mediated autophagosome and lysosome exchange and may represent a novel druggable target.Abbreviations: AGEs: advanced glycation end products; AKT1: AKT serine/threonine kinase 1; AO: acridine orange; ALs: autolysosomes; APs: autophagosomes; BM: bone marrow; BSA: bovine serum albumin; CTSD: cathepsin D; DIC: differential interference contrast; DN: diabetic nephropathy; FSGS: focal segmental glomerulosclerosis; HG: high glucose; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; LMP: lysosomal membrane permeabilization; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PI3K: phosphoinositide 3-kinase; STZ: streptozotocin; TNF: tumor necrosis factor; TNFAIP2: tumor necrosis factor, alpha-induced protein 2; TNTs: tunneling nanotubes; WT: wild type.

Regression of diabetic complications by islet transplantation in the rat.

AIMS/HYPOTHESIS: Type 1 diabetes is a chronic disease leading to complications such as peripheral neuropathies, nephropathy and cardiovascular disease. Pancreatic islet transplantation is being extensively investigated for blood glucose control in animals and in human type 1 diabetic patients, but the question of whether it can reverse long-term diabetic complications has not been fully explored. We investigated the effects of islet transplantation on diabetic complications in a rat model of streptozotocin-induced diabetes. METHODS: Three groups of rats were used: healthy controls, diabetic and diabetic rats transplanted with microencapsulated islets at 2 months after diabetes induction, when neuropathy was detectable by a decrease in tail nerve conduction velocity (NCV) and impaired nociceptive thresholds. Blood glucose levels and body weight were measured weekly. The variables considered were: thermal (hot plate test) and mechanical sensitivity (Randal-Selitto paw withdrawal test), NCV and Na+, K+-ATPase activity in the sciatic nerve. At the end of the experiments hearts were removed for morphometric determination and myocyte number, and kidneys removed for histological examination. RESULTS: Islet transplantation in diabetic rats induced normoglycaemia in a few days, accompanied by a rapid rise in body weight and amelioration of impaired nociceptive thresholds, as well as normalisation of NCV and Na(+), K(+)-ATPase, which were both about 25% below normal in diabetic rats. Myocyte loss was reduced (-34%) by islet transplantation and the observed mild kidney damage of diabetic rats was prevented. CONCLUSIONS/INTERPRETATION: Besides controlling glycaemia, transplantation of microencapsulated pancreatic islets induced almost complete regression of neuropathy and prevented cardiovascular alterations.

Hyperglycemia activates p53 and p53-regulated genes leading to myocyte cell death.

To determine whether enzymatic p53 glycosylation leads to angiotensin II formation followed by p53 phosphorylation, prolonged activation of the renin-angiotensin system, and apoptosis, ventricular myocytes were exposed to levels of glucose mimicking diabetic hyperglycemia. At a high glucose concentration, O-glycosylation of p53 occurred between 10 and 20 min, reached its peak at 1 h, and then decreased with time. Angiotensin II synthesis increased at 45 min and 1 h, resulting in p38 mitogen-activated protein (MAP) kinase-driven p53 phosphorylation at Ser 390. p53 phosphorylation was absent at the early time points, becoming evident at 1 h, and increasing progressively from 3 h to 4 days. Phosphorylated p53 at Ser 18 and activated c-Jun NH(2)-terminal kinases were identified with hyperglycemia, whereas extracellular signal-regulated kinase was not phosphorylated. Upregulation of p53 was associated with an accumulation of angiotensinogen and AT(1) and enhanced production of angiotensin II. Bax quantity also increased. These multiple adaptations paralleled the concentrations of glucose in the medium and the duration of the culture. Myocyte death by apoptosis directly correlated with glucose and angiotensin II levels. Inhibition of O-glycosylation prevented the initial synthesis of angiotensin II, p53, and p38-MAP kinase (MAPK) phosphorylation and apoptosis. AT(1) blockade had no influence on O-glycosylation of p53, but it interfered with p53 phosphorylation; losartan also prevented phosphorylation of p38-MAPK by angiotensin II. Inhibition of p38-MAPK mimicked at a more distal level the consequences of losartan. In conclusion, these in vitro results support the notion that hyperglycemia with diabetes promotes myocyte apoptosis mediated by activation of p53 and effector responses involving the local renin-angiotensin system.

IGF-1 overexpression inhibits the development of diabetic cardiomyopathy and angiotensin II-mediated oxidative stress.

Stimulation of the local renin-angiotensin system and apoptosis characterize the diabetic heart. Because IGF-1 reduces angiotensin (Ang) II and apoptosis, we tested whether streptozotocin-induced diabetic cardiomyopathy was attenuated in IGF-1 transgenic mice (TGM). Diabetes progressively depressed ventricular performance in wild-type mice (WTM) but had no hemodynamic effect on TGM. Myocyte apoptosis measured at 7 and 30 days after the onset of diabetes was twofold higher in WTM than in TGM. Myocyte necrosis was apparent only at 30 days and was more severe in WTM. Diabetic nontransgenic mice lost 24% of their ventricular myocytes and showed a 28% myocyte hypertrophy; both phenomena were prevented by IGF-1. In diabetic WTM, p53 was increased in myocytes, and this activation of p53 was characterized by upregulation of Bax, angiotensinogen, Ang type 1 (AT(1)) receptors, and Ang II. IGF-1 overexpression decreased these biochemical responses. In vivo accumulation of the reactive O(2) product nitrotyrosine and the in vitro formation of H(2)O(2)-(.)OH in myocytes were higher in diabetic WTM than TGM. Apoptosis in vitro was detected in myocytes exhibiting high H(2)O(2)-(.)OH fluorescence, and apoptosis in vivo was linked to the presence of nitrotyrosine. H(2)O(2)-(.)OH generation and myocyte apoptosis in vitro were inhibited by the AT(1) blocker losartan and the O(2) scavenger TIRON: In conclusion, IGF-1 interferes with the development of diabetic myopathy by attenuating p53 function and Ang II production and thus AT(1) activation. This latter event might be responsible for the decrease in oxidative stress and myocyte death by IGF-1.

Canine ventricular myocytes possess a renin-angiotensin system that is upregulated with heart failure.

Ventricular pacing leads to a dilated myopathy in which cell death and myocyte hypertrophy predominate. Because angiotensin II (Ang II) stimulates myocyte growth and triggers apoptosis, we tested whether canine myocytes express the components of the renin-angiotensin system (RAS) and whether the local RAS is upregulated with heart failure. p53 modulates transcription of angiotensinogen (Aogen) and AT(1) receptors in myocytes, raising the possibility that enhanced p53 function in the decompensated heart potentiates Ang II synthesis and Ang II-mediated responses. Therefore, the presence of mRNA transcripts for Aogen, renin, angiotensin-converting enzyme, chymase, and AT(1) and AT(2) receptors was evaluated by reverse transcriptase-polymerase chain reaction in myocytes. Changes in the protein expression of these genes were then determined by Western blot in myocytes from control dogs and dogs affected by congestive heart failure. p53 binding to the promoter of Aogen and AT(1) receptor was also determined. Ang II in myocytes was measured by ELISA and by immunocytochemistry and confocal microscopy. Myocytes expressed mRNAs for all the constituents of RAS, and heart failure was characterized by increased p53 DNA binding to Aogen and AT(1). Additionally, protein levels of Aogen, renin, cathepsin D, angiotensin-converting enzyme, and AT(1) were markedly increased in paced myocytes. Conversely, chymase and AT(2) proteins were not altered. Ang II quantity and labeling of myocytes increased significantly with cardiac decompensation. In conclusion, dog myocytes synthesize Ang II, and activation of p53 function with ventricular pacing upregulates the myocyte RAS and the generation and secretion of Ang II. Ang II may promote myocyte growth and death, contributing to the development of heart failure.

Inhibition of p53 function prevents renin-angiotensin system activation and stretch-mediated myocyte apoptosis.

To determine whether stretch-induced activation of p53 is necessary for the up-regulation of the local renin-angiotensin system and angiotensin II (Ang II)-induced apoptosis, ventricular myocytes were infected with an adenoviral vector carrying mutated p53, Adp53m, before 12 hours of stretch. Noninfected myocytes and myocytes infected with AdLacZ served as controls. Stretching of Adp53m-infected myocytes prevented stimulation of p53 function that conditioned the expression of p53-dependent genes; quantity of angiotensinogen (Aogen), AT(1), and Bax decreased, whereas Bcl-2 increased. Ang II generation was not enhanced by stretch. Conversely, stretch produced opposite changes in noninfected and AdLacZ-infected myocytes: Aogen increased twofold, AT(1) increased 2. 1-fold, Bax increased 2.5-fold, and Ang II increased 2.4-fold. These responses were coupled with 4.5-fold up-regulation of wild-type p53. Stretch elicited comparable adaptations in p53-independent genes, in the presence or absence of mutated p53; renin increased threefold, angiotensin-converting enzyme increased ninefold, and AT(2) increased 1.7-fold. Infection with Adp53m inhibited myocyte apoptosis after stretch. Conversely, stretch increased apoptosis by 6.2-fold in myocytes with elevated endogenous wild-type p53. Thus, a competitor of p53 function interfered with both stretch-induced Ang II formation and apoptosis, indicating that p53 is a major modulator of myocyte renin-angiotensin system and cell survival after mechanical deformation.

Up-regulation of AT(1) and AT(2) receptors in postinfarcted hypertrophied myocytes and stretch-mediated apoptotic cell death.

To determine whether up-regulation of AT(1) and AT(2) receptors occurred in hypertrophied myocytes after infarction and whether AT(2) played a role in stretch-mediated apoptosis, left ventricular myocytes were dissociated from the surviving portion of the wall 8 days after coronary occlusion and cardiac failure in rats. Control cells were obtained from sham-operated animals. Myocytes were stretched in an equibiaxial stretch apparatus and angiotensin II (Ang II) formation and cell death were measured 3 and 12 hours later. AT(1) and AT(2) proteins were evaluated in freshly isolated myocytes and after stretch. The effects of AT(1) and AT(2) antagonists on stretch-induced Ang II synthesis and apoptosis were also established. Myocardial infarction increased AT(1) and AT(2) in myocytes and stretch further up-regulated these receptors. Ang II levels were higher in postinfarcted myocytes and this peptide increased with the duration of stretch in both groups of cells. Similarly, apoptosis increased with time in control and postinfarcted myocytes. Absolute values of Ang II and apoptosis were greater in myocytes from infarcted hearts at 3 and 12 hours after stretch. Addition of AT(1) blocker to cultures inhibited stretch-activated apoptosis in both myocyte populations as well as the generation of Ang II in postinfarcted myocytes. In contrast, AT(2) antagonists had no impact on these cellular events. In conclusion, Ang II stimulated cell death through AT(1) receptor activation, whereas ligand binding to AT(2) receptor did not alter Ang II concentration and apoptosis in normal and postinfarcted hypertrophied myocytes.

Myocyte death in streptozotocin-induced diabetes in rats in angiotensin II- dependent.

To determine whether myocyte death and angiotensin II (AT II) formation are implicated in the development of diabetic cardiomyopathy, rats were injected with streptozotocin, and apoptosis and necrosis were measured at 3, 10, and 28 days. Expression of the components of the renin-angiotensin system (RAS) and AT II levels were assessed at 3 days. The percentage of AT II-labeled myocytes and the number and distribution of AT II sites in myocytes were measured at 3 and 10 days. The effects of AT1 blockade on local RAS and cell death were examined at 3 days. Diabetes was characterized by myocyte apoptosis that peaked at 3 days and decreased at 10 and 28 days, in spite of high concentrations of blood glucose. Cell necrosis was absent throughout. Angiotensinogen, renin, and AT1 receptor increased in myocytes from diabetic rat hearts, while angiotensin-converting enzyme and AT2 remained constant. AT II quantity increased severalfold, as did the fraction of AT II positive cells and the number of AT II sites per myocyte. However, AT II labeling decreased at 10 days, which paralleled the reduction in myocyte death. AT1 antagonist inhibited upregulation of this receptor and angiotensinogen, which prevented AT II synthesis and myocyte death at their peaks with diabetes. An aggregate 30% myocyte loss and a 14% increase in the volume of viable cells were found in diabetic rats at 28 days. Thus diabetic cardiomyopathy may be viewed as an AT II-dependent process in which that peptide plays a critical role in myocyte death and hypertrophy.

Left ventricular response to beta-adrenergic stimulation in aging rats.

The incidence of heart failure in the population increases steeply among older people. Overactivation of the sympathetic nervous system is associated to and responsible for worsening of heart failure. This study describes the influence of aging on short-term left ventricular (LV) adaptation to b-adrenergic stimulation in Wistar rats. In controls at 18 mo, interstitial fibrosis was increased with respect to 3-mo-old rats, whereas myocytes dimension and the messenger RNA (mRNA) abundance of atrial natriuretic peptide (ANP), a2(I) procollagen, transforming growth factor (TGF-b1, TGF-b3), and secreted protein, acidic and rich in cysteine (SPARC) were not different. To determine how aging affects LV adaptation to adrenergic stimulation, two groups of animals received isoproterenol (ISO, 1 mg/kg/d) for 3 days. There was no significant difference between young and older rats with respect to increase in LV weight, myocytes dimension, and mRNA abundance of all the genes considered, except a1(III) procollagen. These findings indicate that despite limited compensatory hypertrophy and higher fibrosis, LV from aged nonsenescent rats preserves the capacity to adapt to b-adrenergic stimulation through the upregulation of several genes encoding extracellular matrix-related proteins.

Effects of a DA2/alpha2 agonist and a beta1-blocker in combination with an ACE inhibitor on adrenergic activity and left ventricular remodeling in an experimental model of left ventricular dysfunction after coronary artery occlusion.

Renin-angiotensin-aldosterone and sympathetic nervous systems overactivity play a major role in worsening the extent of heart failure. Attenuation of neurohumoral activation with angiotensin-converting enzyme (ACE) inhibitors and beta-blockers has proven beneficial in congestive heart failure. Because ACE inhibition is a recommended treatment for heart failure, this study was designed to test the effects on neurohumoral activation, hemodynamics, and left ventricular (LV) volume of the combination of an ACE inhibitor (delapril) with a DA2-dopaminergic receptor/alpha2-adrenoceptor agonist (CHF-1024) or a beta1-adrenoceptor antagonist (metoprolol) after a moderate to large myocardial infarction (MI) in rats. MI was induced by left coronary artery ligation in 134 rats, and six were not operated on. After 2 months, the animals with ECG evidence of MI were treated for 1 more month with CHF- 1024, 0.33 mg/kg/day or with metoprolol (10 mg/kg/day), delivered through implanted osmotic minipumps, in addition to delapril (6 mg/kg/day) in the drinking water. Daily urinary excretion of norepinephrine (NE) and circulating concentration were measured. Hemodynamic variables were measured, and three-dimensional morphometric analysis was done on the diastole-arrested hearts to quantify infarct size and LV geometry. In conscious animals, delapril alone or with CHF-1024 or metropolol did not modify heart rate or systolic blood pressure. Both combination treatments, however, significantly reduced heart rate in anesthetized animals compared with the group receiving vehicle. Infarct size was not different between treatments, averaging 20-22% of LV volume. The threefold increase of LV chamber volume in infarcted rats was significantly attenuated by delapril alone or with CHF-1024 or metoprolol (-37 to -44%, p<0.05). Treatment with a combination of the ACEi and CHF-1024 tended to normalize the shape of the LV cavity. Urinary NE excretion was unaffected by delapril alone but was reduced by the addition of CHF-1024 or metoprolol. In conclusion, 1 month of treatment with doses of delapril having no hemodynamic effect, reduced LV volume in a model of chronic heart failure. When CHF-1024 or metoprolol was given with delapril, sympathetic activation decreased with no unwanted effects, such as excessive hypotension.

Remodelling of cardiac extracellular matrix during beta-adrenergic stimulation: upregulation of SPARC in the myocardium of adult rats.

Our objectives were (i) to evaluate the expression of several genes involved in the remodelling of cardiac extracellular matrix (ECM), with a special interest on SPARC (secreted protein acidic and rich in cysteine) a glycoprotein with anti-adhesive properties, and (ii) to characterise structural changes in the left (LV) and right (RV) ventricles of rats subjected to continuous beta-adrenergic stimulation. The rats were infused for 3 or 7 days with isoproterenol (ISO, 4 mg/kg/day) or vehicle. Hybridisation analysis was done for SPARC, atrial natriuretic peptide (ANP),alpha2 (I) [COL-I] and alpha1 (III) [COL-III] procollagens, TGF-beta1 and TGF-beta3 mRNA content. Interstitial and perivascular collagen deposition in both ventricles was measured after specific staining. The mean cross-sectional area of LV cardiomyocytes was evaluated by quantitative histomorphometry. ISO provoked an increase of LV mass, and a progressive enlargement of cardiomyocytes: their cross-sectional area raised from 205+/-8 micrometer2 in vehicle-treated animals to 247+/-4 and 296+/-9 micrometer2 after 3 or 7 days of ISO infusion, respectively (P<0.001). SPARC messenger abundance increased by more than 50% in LV and RV, a first evidence of its expression in the myocardium of adult rats. Transcripts of ANP, COL-III, TGF-beta1 and TGF-beta3 increased in both ventricles. COL-I transcript increased in LV (75 and 116% on days 3 and 7), but not in RV. In LV, collagen accumulated in the interstitium (2.69+/-0.20v 9. 23+/-0.50% of tissue area for vehicle and ISO 7 days groups, P<0.05) and around coronary arteries (1.04+/-0.11v 4.47+/-0.48% of lumen area for vehicle and ISO 7 days,P<0.05). Cardiac fibrosis was less marked in RV. In conclusion, early expression of SPARC, an anti-adhesive protein, and preferential expression of COL-III, a distensible form of collagen, should increase ECM plasticity and facilitate ventricular remodelling.

Age-dependent expression of fibrosis-related genes and collagen deposition in the rat myocardium.

OBJECTIVES: We sought to characterize the evolution, during maturational growth and early ageing, of the messenger abundance of four genes involved in cardiac fibrosis regulation (procollagens alpha2(I) and alpha1(III), transforming growth factors beta1, and beta3) and corroborate it with the alterations in collagen deposition in cardiac interstitium and around coronary arteries. METHODS: Messenger RNA was quantified in LV and RV of 2-, 6-, 12- and 19-month-old male Sprague-Dawley rats (n = 5 per group) with Northern blot analysis. Collagen deposition was quantified with a semi-automated image analyser on Sirius red-stained sections of LV tissue. RESULTS: There was an age-related monotonous decrease of procollagen type I (COL-I) transcript abundance in LV (p < 0.001) but not in RV. Procollagen type III (COL-III) expression decreased rapidly during maturational growth, both in LV and RV. On the other hand, collagen deposition in myocardial interstitium and around coronary arteries was slightly augmented during the maturational period of life (2-12 months), but with a higher rate during early ageing (up to 19 months). This was not accompanied by a significant thickening of the wall of coronary arteries. Transforming growth factor beta1, (TGF-beta1) and transforming growth factor beta3 (TGF-beta3) transcript abundance showed no major variations during ageing. CONCLUSIONS: These results reflect a striking ventricular difference regarding the age-dependent expression of COL-I. The expression of TGF-beta(s), pleiotropic factors known to influence collagen pathway at different levels, does not seem to be profoundly altered during ageing. The discrepancy between protein and COL-I and COL-III mRNA levels indicates differences in age-related mRNA stability and/or regulation of collagen translation.

Comparative efficacy of a DA2/alpha2 agonist and a beta-blocker in reducing adrenergic drive and cardiac fibrosis in an experimental model of left ventricular dysfunction after coronary artery occlusion.

Attenuation of neuroendocrine activation may be beneficial in congestive heart failure. Sympathetic nervous system overactivity can be reduced by receptors blockade or by reducing norepinephrine (NE) spillover. This study evaluated and compared the effects of a DA2-dopaminergic receptor/alpha2-adrenoceptor agonist (CHF-1024) and a beta1-adrenoreceptor antagonist in terms of hemodynamics, ventricular remodeling, beta-adrenergic drive, and cardiac fibrosis after myocardial infarction (MI) in rats. MI was induced by left coronary artery ligation in 213 rats, whereas 12 were left unoperated on. After 2 months, the operated-on animals were treated for 1 more month with CHF-1024 at either 0.33 mg/kg/day (low dose) or 1 mg/kg/day (high dose) or with metoprolol (10 mg/kg/day), delivered through implanted osmotic minipumps. Plasma concentration and urinary excretion of NE were measured before the rats were killed. Hemodynamic variables were measured and morphometric analysis was done on the diastole-arrested hearts to quantify left ventricular remodeling and interstitial collagen density. Metoprolol treatment tended to normalize LV end-diastolic pressure (LVEDP). CHF-1024 at either dose, and metoprolol, significantly reduced collagen deposition in LV of infarcted animals (from 8.8 +/- 0.5% LV area in vehicle-treated rats to 6.6 +/- 0.2% or 6.4 +/- 0.2% after the low or high dose of CHF-1024, respectively; p < 0.05). Similarly, CHF-1024 at either dose reduced the plasma concentration of NE (from 224 +/- 53 pg/ml to 60 +/- 7 pg/ml or 87 +/- 13 pg/ml; p < 0.05) and urinary excretion of NE in rats with MI, whereas beta-blockade did not affect these variables. In conclusion, CHF-1024 infused for 1 month to rats with LV dysfunction reduced heart rate, NE spillover, and collagen deposition, without unwanted effects, only appearing at the higher dose. Effective beta-blockade with metoprotol reduced LVEDP with no effects on heart function. Neither DA2/alpha2 stimulation nor beta-blockade altered LV remodeling after coronary artery ligation.