Carlo Pesce

Deletion of p66Shc Longevity Gene Protects Against Experimental Diabetic Glomerulopathy by Preventing Diabetes-Induced Oxidative Stress

p66Shc regulates both steady-state and environmental stress-dependent reactive oxygen species (ROS) generation. Its deletion was shown to confer resistance to oxidative stress and protect mice from aging-associated vascular disease. This study was aimed at verifying the hypothesis that p66Shc deletion also protects from diabetic glomerulopathy by reducing oxidative stress. Streptozotocin-induced diabetic p66Shc knockout (KO) mice showed less marked changes in renal function and structure, as indicated by the significantly lower levels of proteinuria, albuminuria, glomerular sclerosis index, and glomerular and mesangial areas. Glomerular content of fibronectin and collagen IV was also lower in diabetic KO versus wild-type mice, whereas apoptosis was detected only in diabetic wild-type mice. Serum and renal tissue advanced glycation end products and plasma isoprostane 8-epi-prostaglandin F2α levels and activation of nuclear factor κB (NF-κB) were also lower in diabetic KO than in wild-type mice. Mesangial cells from KO mice grown under high-glucose conditions showed lower cell death rate, matrix production, ROS levels, and activation of NF-κB than those from wild-type mice. These data support a role for oxidative stress in the pathogenesis of diabetic glomerulopathy and indicate that p66Shc is involved in the molecular mechanism(s) underlying diabetes-induced oxidative stress and oxidant-dependent renal injury.

Galectin-3/AGE-receptor 3 knockout mice show accelerated AGE-induced glomerular injury: evidence for a protective role of galectin-3 as an AGE receptor

We previously showed that mice lacking galectin‐3/AGE‐receptor 3 develop accelerated diabetic glomerulopathy. To further investigate the role of galectin‐3/AGE‐receptor function in the pathogenesis of diabetic renal disease, galectin‐3 knockout (KO) and coeval wild‐type (WT) mice were injected for 3 months with 30 μg/day of Nε‐carboxymethyllysine (CML)‐modified or unmodified mouse serum albumin (MSA). Despite receiving equal doses of CML, KO had higher circulating and renal AGE levels and showed more marked renal functional and structural changes than WT mice, with significantly higher proteinuria, albuminuria, glomerular, and mesangial area and glomerular sclerosis index. Renal 4‐hydroxy‐2‐nonenal content and NFκB activation were also more pronounced in KO‐CML vs. WT‐CML. Kidney mRNA levels of fibronectin, laminin, collagen IV, and TGF‐β were up‐regulated, whereas those of matrix metalloproteinase‐2 and ‐14 were down‐regulated, again more markedly in KO‐CML than WT‐CML mice. Basal and CML‐induced RAGE and 80K‐H mRNA levels were higher in KO vs. WT mice. MSA injection did not produce any significant effect in both genotypes. The association of galectin‐3 ablation with enhanced susceptibility to AGE‐induced renal disease, increased AGE levels and signaling, and altered AGE‐receptor pattern indicates that galectin‐3 is operating in vivo as an AGE receptor to afford protection toward AGE‐dependent tissue injury.

Advanced lipoxidation end-products mediate lipid-induced glomerular injury: role of receptor-mediated mechanisms

Atherosclerosis and renal disease are related conditions, sharing several risk factors. This includes hyperlipidaemia, which may result in enhanced lipoprotein accumulation and chemical modification, particularly oxidation, with formation of advanced lipoxidation endproducts (ALEs). We investigated whether increased lipid peroxidation plays a major role in the pathogenesis of lipid‐induced renal disease, via receptor‐mediated mechanisms involving the scavenger and advanced glycation endproduct (AGE) receptors. Mice knocked out for galectin‐3 (Gal3−/−), an AGE receptor previously shown to protect from AGE‐induced renal injury, and the corresponding wild‐type (Gal3+/+) animals, were fed an atherogenic high‐fat diet (HFD; 15% fat, 1.25% cholesterol and 0.5% sodium cholate); mice fed a normal‐fat diet (NFD; 4% fat) served as controls. Gal3+/+ mice fed a HFD developed glomerular disease, as indicated by proteinuria, mesangial expansion and glomerular hypertrophy and sclerosis. Glomerular injury was associated with increased glomerular matrix protein expression, ALE and oxidized LDL content, oxidative stress, AGE and scavenger receptor expression and macrophage infiltration, with only modest renal/glomerular fat accumulation and changes in lipid metabolism. Fibrotic and inflammatory changes, together with accumulation of ALEs, such as 4‐hydroxy‐2‐nonenal adducts and Nε‐carboxymethyllysine, oxidative stress and expression of the receptor of AGEs (RAGE), were significantly more marked in Gal3−/− animals, whereas fat deposition and abnormalities in lipid metabolism remained modest. Thus, lipid‐induced renal damage is mainly dependent on lipid peroxidation with formation of carbonyl reactive species and ALEs, which accumulate within the kidney tissue, thus triggering receptor‐mediated pro‐inflammatory signalling pathways, as in atherogenesis. Moreover, galectin‐3 exerts a significant role in the uptake and effective removal of modified lipoproteins, with diversion of these products from RAGE‐dependent pro‐inflammatory pathways associated with downregulation of RAGE expression. Copyright

Role of angiotensin‐converting enzyme 2 and angiotensin(1–7) in 17β‐oestradiol regulation of renal pathology in renal wrap hypertension in rats

17β‐Oestradiol (E2)‐mediated inhibition of angiotensin‐converting enzyme (ACE) protects the E2‐replete kidney from the progression of hypertensive renal disease. Angiotensin‐converting enzyme 2 (ACE2), a homologue of ACE, counters the actions of ACE by catalysing the conversion of angiotensin II (Ang II) to angiotensin(1–7) [Ang(1–7)]. We investigated E2 regulation of ACE2 in the renal wrap (RW) model of hypertension in rats. After 6 weeks on a high‐sodium diet (4% NaCl), the activity of ACE2 was reduced in the renal cortex by 31%, which was mirrored by similar decreases in ACE2 protein (30%) and mRNA expression (36%) in the ovariectomized RW rat (RW‐OVX); E2 replacement prevented these effects. The RW‐OVX rats exhibited greater renal injury, including 1.7‐fold more tubulointerstitial fibrosis and 1.6‐fold more glomerulosclerosis than E2‐replete females (RW‐Intact and RW‐OVX+E2). Angiotensin(1–7) infusion prevented these exacerbating effects of ovariectomy on renal pathology; no differences in indicators of renal injury were observed between RW‐OVX‐Ang(1–7) and RW‐Intact rats. These renal protective effects of Ang(1–7) infusion were not attributable to increased ACE2 activity or to changes in heart rate or body weight, since these parameters were unchanged by Ang(1–7) infusion. Furthermore, Ang(1–7) infusion did not attenuate renal injury by reducing mean arterial pressure (MAP), since infusion of the peptide did not lower MAP but rather caused a slight increase during a 6 week chronic treatment for Ang(1–7). These results suggest that E2‐mediated upregulation of renal ACE2 and the consequent increased Ang(1–7) production contribute to E2‐mediated protection from hypertensive renal disease. These findings have implications for E2‐deficient women with hypertensive renal disease and suggest that therapeutics targeted towards increasing ACE2 activity and Ang(1–7) levels will be renal protective.

Female Protection in Progressive Renal Disease Is Associated with Estradiol Attenuation of Superoxide Production

BACKGROUND Several types of renal disease progress at a faster rate in men compared with women, but the reasons for this sex difference are not well understood. Chronic renal disease is associated with elevated levels of toxic reactive oxygen species (ROS). Superoxide, the major ROS in the kidney, is generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. OBJECTIVE To determine if female protection from renal disease progression is consistent with 17beta-estradiol (E2) attenuation of superoxide production, this study was conducted to assess superoxide production in the renal cortex of male and female control and renal wrap (RW) rats, as well as in ovariectomized rats treated with vehicle or E2. METHODS Sprague-Dawley rats were divided into 2 sham operation male (Sham-M) and female (Sham-F) control groups, and 4 RW hypertensive groups: RW-M; RW-F; RW ovariectomized females treated with vehicle (RW-OVX); and RW ovariectomized females treated with E2, supplied as a 0.24 mg/60-day release pellet (RW-OVX+E2). All groups were maintained on a high-sodium (4% NaCl) diet for 6 weeks. RESULTS Mean (SEM) markers of renal injury and oxidative stress, including urinary protein (mg/24 h: RW-M, 298 [31] vs RW-F, 169 [22]; P < 0.001), microalbuminuria (RW/Sham arbitrary units [AU]/24 h: M, 8.78 [0.58] vs F, 4.31 [1.0]; P < 0.005), and malondialdehyde (nmol/24 h: RW-M, 167 [23] vs RW-F, 117 [8.5]; P < 0.05) levels, as well as mean glomerular volume (microm3 x 10(6): RW-M, 2.25 [0.16] vs RW-F, 1.25 [0.04]; P < 0.001) and the glomerulosclerotic index (AU: RW-M, 2.64 [0.19] vs RW-F, 1.10 [0.09]; P < 0.001) were greater in both control and RW males compared with females in the same treatment groups. Though RW surgery increased mean arterial pressure in both male and female rats, no sex difference was observed. Under these conditions, mean (SEM) renal cortical NADPH oxidase activity was 1.3-fold higher in RW males compared with RW females (relative light units [RLU]/180 sec: RW-M, 4080 [240] vs RW-F, 3200 [260]; P < 0.05). Ovariectomy increased NADPH oxidase activity by 1.4-fold (RLU/180 sec: RW-OVX, 4520 [184]; P < 0.01) under conditions in which the mean glomerular volume and glomerulosclerotic index were both increased by 1.5-fold, whereas E2 replacement (RLU/180 sec: RW-OVX+E2, 2745 [440]) prevented these effects. Furthermore, the effects on NADPH oxidase activity were mirrored by changes in the protein abundance of NADPH oxidase subunit p22P(phox). CONCLUSION These results suggest that E2 protects the female kidney in part by attenuating injury-induced increases in renal superoxide production.

The galectin-3/RAGE dyad modulates vascular osteogenesis in atherosclerosis

AIMS Vascular calcification correlates with inflammation and plaque instability in a dual manner, depending on the spotty/granular (micro) or sheet-like/lamellated (macro) pattern of calcification. Modified lipoproteins trigger both inflammation and calcification via receptors for advanced lipoxidation/glycation endproducts (ALEs/AGEs). This study compared the roles of galectin-3 and receptor for AGEs (RAGE), two ALEs/AGEs-receptors with diverging effects on inflammation and bone metabolism, in the process of vascular calcification. METHODS AND RESULTS We evaluated galectin-3 and RAGE expression/localization in 62 human carotid plaques and its relation to calcification pattern, plaque phenotype, and markers of inflammation and vascular osteogenesis; and the effect of galectin-3 ablation and/or exposure to an ALE/AGE on vascular smooth muscle cell (VSMC) osteogenic differentiation. While RAGE co-localized with inflammatory cells in unstable regions with microcalcification, galectin-3 was expressed also by VSMCs, especially in macrocalcified areas, where it co-localized with alkaline phosphatase. Expression of galectin-3 and osteogenic markers was higher in macrocalcified plaques, whereas the opposite occurred for RAGE and inflammatory markers. Galectin-3-deficient VSMCs exhibited defective osteogenic differentiation, as shown by altered expression of osteogenic transcription factors and proteins, blunted activation of pro-osteoblastogenic Wnt/β-catenin signalling and proliferation, enhanced apoptosis, and disorganized mineralization. These abnormalities were associated with RAGE up-regulation, but were only in part prevented by RAGE silencing, and were partially mimicked or exacerbated by treatment with an AGE/ALE. CONCLUSION These data indicate a novel molecular mechanism by which galectin-3 and RAGE modulate in divergent ways, not only inflammation, but also vascular osteogenesis, by modulating Wnt/β-catenin signalling, and independently of ALEs/AGEs.

Glomerular Cell Replication and Cell Loss through Apoptosis in Experimental Diabetes mellitus

Aim: To evaluate changes in the glomerular cell balance between replication and apoptosis in experimental diabetes mellitus (DM) in relation to morphometric data. Methods: Adult Sprague-Dowley rats with streptozotocin-induced DM and controls of the same age and strain were sacrificed 4 and 8 weeks and 6 months after disease onset. Cell replication was demonstrated with MIB-5, and apoptosis with the terminal uridine nick end labeling technique. Glomerular size and glomerular cell population were estimated morphologically. Results: Diabetic and control rats showed irrelevant MIB-5 positivity at all time points. Glomerular apoptosis was minimal in rats with 4 and 8 weeks of DM and in controls. Rats with 6 months of DM showed significantly higher glomerular apoptosis values than controls (2.49 ± 0.25 vs. 0.65 ± 0.16%; p < 0.001). The mean cell count per glomerular profile was significantly lower in these diabetic rats (64.02 ± 1.93 vs. 78.27 ± 0.99; p < 0.001), a change that correlated with that in apoptosis. The glomerular cell density was further decreased in diabetic rats because of the diabetic increase in mean glomerular volume (1.598 vs. 0.927 106 µm). Conclusions: Apoptosis is associated with loss of glomerular cells in rats with long-term, streptozotocin-induced DM and – to a considerably lower degree – in controls of the same age and strain. These changes could be relevant to glomerulosclerosis associated with long-term, streptozotocin-induced DM.

Induction of alopecia in mice exposed to cigarette smoke

Besides being responsible for a high proportion of those chronic degenerative diseases that are the leading causes of death in the population, tobacco smoking has been associated with skin diseases. Smoke genotoxicants are metabolized in hair follicle cells, where they form DNA adducts and cause DNA damage. The suspicion was raised that, in humans, a link may exist between smoking and both premature grey hair and hair loss. In order to check this hypothesis, we carried out a study in C57BL/6 mice exposed whole-body to a mixture of sidestream and mainstream cigarette smoke. After 3 months exposure, most mice developed areas of alopecia and grey hair, while no such lesions occurred either in sham-exposed mice or in smoke-exposed mice receiving the chemopreventive agent N-acetylcysteine with drinking water. Cell apoptosis occurred massively in the hair bulbs at the edge of alopecia areas. Smoke-exposed mice had extensive atrophy of the epidermis, reduced thickness of the subcutaneous tissue, and scarcity of hair follicles. On the whole, exposure to smoke genotoxic components appears to alter the hair cycle with a dystrophic anagen pattern. Although this mechanism is different from that of genotoxic cytostatic drugs, N-acetylcysteine appears to exert protective effects in both conditions.

Early, but Not Advanced, Glomerulopathy Is Reversed by Pancreatic Islet Transplants in Experimental Diabetic Rats: Correlation With Glomerular Extracellular Matrix mRNA Levels

In this study, we investigated 1) whether long-term restoration of euglycemia by means of pancreatic islet transplants is capable of preventing and/or reversing renal functional and structural alterations in an experimental model of insulin-deficient diabetes, and 2) whether changes in extracellular matrix (ECM) and cell turnover at the glomerular level and biochemical abnormalities associated with hyperglycemia correlate with the renal outcome after transplantation. Male Lewis rats, rendered diabetic by intravenous injection of streptozotocin, underwent homologous islet transplantation via the portal vein at 2 weeks (study A), at 4 months (study B), and at 8 months (study C) after the induction of diabetes and killed 12 months after transplantation in study A and 4 months after transplantation in studies B and C. Age-matched nondiabetic and untreated diabetic rats were used as control animals and were studied at 4, 8, and 12 months. In the untreated diabetic animals, metabolic derangement was associated with increased erythrocyte polyol and fructose levels, tail-tendon content of advanced glycation end products (AGEs), total proteinuria, albuminuria, kidney weight, and mean glomerular volume as well as with marked glomerular and extraglomerular lesions. Glomerular gene expression for the ECM components fibronectin and collagen IV and for TGF-β was also increased, whereas glomerular cell proliferation was unaffected by diabetes. In study A, changes in renal function and structure observed in diabetic rats at 12 months were completely prevented by successful islet transplants. In study B, all functional and structural abnormalities detected in diabetic rats at 4 months of disease duration were virtually reversed by 4 months of euglycemia in transplanted animals, whereas they progressed further in untreated diabetic rats. In study C, the course of functional and structural changes observed in untreated diabetic rats was not reversed by islet transplantation. Likewise, tissue AGE accumulation and particularly upregulation of glomerular ECM and transforming growth factor (TGF)-β gene expression, which are believed to play a role in the pathogenesis of altered renal function and structure in diabetes, were normalized in transplanted rats from study A and study B, but not in those from study C. These experiments show that restoration of euglycemia by islet transplants is capable of preventing experimental diabetic glomerulopathy and reversing early changes in renal function and structure induced by diabetes. In a later phase of the disease, when glomerular matrix gene expression becomes independent of hyperglycemia, possibly because of the persistent increase in tissue AGE accumulation, metabolic control is not capable of reversing renal abnormalities.

Galectin-3: an emerging all-out player in metabolic disorders and their complications

Galectin-3 has been increasingly recognized as an important modulator of several biological functions, by interacting with several molecules inside and outside the cell, and an emerging player in numerous disease conditions. Galectin-3 exerts various and sometimes contrasting effects according to its location, type of injury or site of damage. Strong evidence indicates that galectin-3 participates in the pathogenesis of diabetic complications via its receptor function for advanced glycation end-products (AGEs) and advanced lipoxidation end-products (ALEs). AGEs/ALEs are produced to an increased extent in target organs of complications, such as kidney and vessels; here, lack of galectin-3 impairs their removal, leading to accelerated damage. In contrast, in the liver, AGE/ALE tissue content and injury are decreased, because lack of galectin-3 results in reduced uptake and tissue accumulation of these by-products. Some of these effects can be explained by changes in the expression of receptor for AGEs (RAGE), associated with galectin-3 deletion and consequent changes in AGE/ALE tissue levels. Furthermore, galectin-3 might exert AGE/ALE- and RAGE-independent effects, favoring resolution of inflammation and modulating fibrogenesis and ectopic osteogenesis. These effects are mediated by intracellular and extracellular galectin-3, the latter via interaction with N-glycans at the cell surface to form lattice structures. Recently, galectin-3 has been implicated in the development of metabolic disorders because it favors glucose homeostasis and prevents the deleterious activation of adaptive and innate immune response to obesogenic/diabetogenic stimuli. In conclusion, galectin-3 is an emerging all-out player in metabolic disorders and their complications that deserves further investigation as the potential target of therapeutic intervention.