The role of TGF-β and epithelial-to mesenchymal transition in diabetic nephropathy

Transforming Growth Factor-beta (TGF-β) is a pro-sclerotic cytokine widely associated with the development of fibrosis in diabetic nephropathy. Central to the underlying pathology of tubulointerstitial fibrosis is epithelial-to-mesenchymal transition (EMT), or the trans-differentiation of tubular epithelial cells into myofibroblasts. This process is accompanied by a number of key morphological and phenotypic changes culminating in detachment of cells from the tubular basement membrane and migration into the interstitium. Ultimately these cells reside as activated myofibroblasts and further exacerbate the state of fibrosis. A large body of evidence supports a role for TGF-β and downstream Smad signalling in the development and progression of renal fibrosis. Here we discuss a role for TGF-β as the principle effector in the development of renal fibrosis in diabetic nephropathy, focusing on the role of the TGF-β1 isoform and its downstream signalling intermediates, the Smad proteins. Specifically we review evidence for TGF-β1 induced EMT in both the proximal and distal regions of the nephron and describe potential therapeutic strategies that may target TGF-β1 activity.     

Beneficial effect of dl-α-lipoic acid on cyclosporine A induced hyperlipidemic nephropathy in rats

Cyclosporine A (CsA)-induced dyslipidemia is one of the most important risk factors for morbidity and mortality after solid organ transplantation. Reducing this side effect of CsA by dietary agents may be safe, cost-effective, and attractive to both patients and health professionals. Hence the present study was designed to evaluate the role of dl-α-Lipoic acid (LA) in deteriorating the lipid abnormalities induced by CsA in rat kidney. Male albino Wistar rats were divided into four groups. CsA administered at a dose of 25 mg/kg body weight, orally for 21 days showed abnormal changes in the levels of lipoprotein fractions (LDL, HDL and VLDL) and lipid profile in both plasma and renal tissue. Significant alterations were also observed in the activities of lipid metabolizing enzymes. Co-treatment with LA (20 mg/kg body weight, oral gavage, for 21 days) reverted the levels of lipid profile (P < 0.001, P < 0.01) and lipoprotein fractions (P < 0.001, P < 0.01) to near control. The activities of lipid metabolizing enzymes also showed considerable restoration on LA supplementation. The outcome of this study provides evidence that LA (a natural metabolic antioxidant) treatment acts as a potent antilipemic agent against CsA-induced lipid abnormalities.     

The effect of ATP-sensitive K+ channels on the electrical burst activity and insulin secretion in pancreatic β-cells

In recent years, the electrical burst activity of the insulin releasing pancreatic β-cells has attracted many experimentalists and theoreticians, largely because of its functional importance, but also because of the nonlinear nature of the burst activity. The ATP-sensitive K⁺ channels are believed to play an important role in electrical activity and insulin release. In this paper, we show by computer simulation how ATP and antidiabetic drugs can lengthen the plateau fraction of bursting and how these chemicals can increase the intracellular Ca²⁺ level in the pancreatic β-cell.     

The effect of anaerobiosis on the induced synthesis of β-galactosidase in non-growingEscherichia coli

Depending on conditions of aeration maltose and glucose were found to exhibit different effects on the inducible synthesis of β-galactosidase in aerobically grown cells ofEscherichia coli starving for an exogenous source of nitrogen; both saccharides repressed the synthesis of the enzyme under aerobic conditions, while the above-mentioned saccharides were essential for the enzyme synthesis under anaerobic conditions. The presence of maltose in the medium resulted in the repression of the enzyme synthesis in anaerobically grown cells starving for an exogenous nitrogen source under anaerobic conditions. The synthesis of β-galactosidase-specific messenger RNA was completely blocked and the synthesis of the enzyme proper considerably inhibited in aerobically grown cells incubated anaerobically in a medium without nitrogen and carbon sources.     

Intracellular proton-mediated activation of TRPV3 channels accounts for the exfoliation effect of α-hydroxyl acids on keratinocytes

α-Hydroxyl acids (AHAs) from natural sources act as proton donors and topical compounds that penetrate skin and are well known in the cosmetic industry for their use in chemical peels and improvement of the skin. However, little is known about how AHAs cause exfoliation to expose fresh skin cells. Here we report that the transient receptor potential vanilloid 3 (TRPV3) channel in keratinocytes is potently activated by intracellular acidification induced by glycolic acid. Patch clamp recordings and cell death assay of both human keratinocyte HaCaT cells and TRPV3-expressing HEK-293 cells confirmed that intracellular acidification led to direct activation of TRPV3 and promoted cell death. Site-directed mutagenesis revealed that an N-terminal histidine residue, His-426, known to be involved in 2-aminoethyl diphenylborinate-mediated TRPV3 activation, is critical for sensing intracellular proton levels. Taken together, our findings suggest that intracellular protons can strongly activate TRPV3, and TRPV3-mediated proton sensing and cell death in keratinocytes may serve as a molecular basis for the cosmetic use of AHAs and their therapeutic potential in acidic pH-related skin disorders.     

¹H-NMR-based metabonomic analysis of metabolic profiling in diabetic nephropathy rats induced by streptozotocin

Elucidation of the metabolic profiling in diabetic nephropathy (DN) rats is of great assistance for understanding the pathogenesis of DN. In this study, 1H-nuclear magnetic resonance (NMR)-based metabonomics combined with HPLC measurements was used to quantitatively analyze the metabolic changes in urine and kidney extracts from diabetic 2-wk and 8-wk rats induced by streptozotocin (STZ). Pattern recognition analysis of either urine or kidney extracts indicated that the two diabetic groups were separated obviously from the control group, suggesting that the metabolic profiles of the diabetic groups were markedly different from the control. The diabetic 8-wk rats showed lower levels of creatine, dimethylamine, and higher levels of ascorbate, succinate, lactate, citrate, allantoin, 2-ketoglutarate, and 3-hydrobutyrate (3-HB) in the urine samples. Moreover, the diabetic 8-wk rats displayed lower levels of succinate, creatine, myo-inositol, alanine, lactate, and ATP, and higher levels of 3-HB and glucose in the kidney extracts. The observed metabolic changes imply the enhanced pathways of either lipid or ketone body synthesis and decreased pathways of either tricarboxylic acid cycle or glycolysis in DN rats compared with the control. Our results suggest that the energy metabolic changes are associated with the pathogenic process of DN.     

Prevention of renal apoB retention is protective against diabetic nephropathy: role of TGF-β inhibition

Animal studies demonstrate that hyperlipidemia and renal lipid accumulation contribute to the pathogenesis of diabetic nephropathy (DN). We previously demonstrated that renal lipoproteins colocalize with biglycan, a renal proteoglycan. The purpose of this study was to determine whether prevention of renal lipid (apoB) accumulation attenuates DN. Biglycan-deficient and biglycan wild-type Ldlr^−/− mice were made diabetic via streptozotocin and fed a high cholesterol diet. As biglycan deficiency is associated with elevated transforming growth factor-β (TGF-β), in some experiments mice were injected with either the TGF-β-neutralizing antibody, 1D11, or with 13C4, an irrelevant control antibody. Biglycan deficiency had no significant effect on renal apoB accumulation, but led to modest attenuation of DN with ∼30% reduction in albuminuria; however, biglycan deficiency caused a striking elevation in TGF-β. Use of 1D11 led to sustained suppression of TGF-β for approximately 8 weeks at a time. The 1D11 treatment caused decreased renal apoB accumulation, decreased albuminuria, decreased renal hypertrophy, and improved survival, compared with the 13C4 treatment. Thus, prevention of renal apoB accumulation is protective against development of DN. Furthermore, this study demonstrates that prevention of renal apoB accumulation is a mechanism by which TGF-β inhibition is nephroprotective.     

The dual role of the cystathionine γ-lyase/hydrogen sulfide pathway in CVB3-induced myocarditis in mice

The present study found that serum H₂S level, H₂S production rate, CSE mRNA and CSE protein levels were increased in CVB3-induced myocarditis. dl-proparglygylcine (PAG), an irreversible CSE inhibitor, decreased the infected myocardium titers on postinfection day 4, while NaHS, a H₂S donor, alleviated myocardial injury and necrosis, inflammatory cell infiltration and interstitial edema on postinfection day 10. These data reveal that the CSE/H₂S pathway is upregulated in the heart in a murine model of CVB3-induced myocarditis and that inhibition of endogenous H₂S is beneficial to treatment early in the disease while administration of exogenous H₂S is protective to infected myocardium during the later stage.     

The effect of nucleosides on the induced synthesis of β-galactosidase in non-growing cells ofEscherichia coli

The induced synthesis of β-galactosidase in non-growing cells ofEscherichia coli starving for exogenous carbon and nitrogen sources was stimulated markedly by the addition of any of four nucleosides tested: adenosine, guanosine, cytidine, and uridine. Adenosine was used as a representative of this group of compounds in most experiments. The decrease of ability of the cells to synthesize β-galactosidase, resulting from a prolonged starvation for exogenous carbon and nitrogen, was removed by adenosine. This compound also considerably reduced the inhibitory effect of metabolic poisons on the induced synthesis of β-galactosidase. The blockade of induced β-galactosidase synthesis evoked in aerobically grown cells by anaerobic starvation for exogenous sources of carbon and nitrogen was also significantly reduced by adenosine. The weak transient catabolic repression of induced synthesis of β-galactosidase evoked by glucose in non-growing cells ofEscherichia coli deprived of exogenous carbon and nitrogen sources was prevented by adenosine. The total repression caused by higher glucose concentrations was not influenced by this compound. The results are discussed from the point of view of the role of the energy state ofEscherichia coli cells in the regulation of β-galactosidase synthesis.     

The protective effect of cannabinoid type 2 receptor activation on renal ischemia–reperfusion injury

Natural products from medicinal plants have always attracted a lot of attention due to their diverse and interesting therapeutic properties. We have employed the principles of green chemistry involving isomerization, coupling and condensation reaction to synthesize a class of compounds derived from eugenol, a naturally occurring bioactive phytophenol. The compounds were characterized structurally by ¹H-, ¹³C-NMR, FT-IR spectroscopy and mass spectrometry analysis. The purity of compounds was detected by HPLC. The synthesized compounds exhibited anti-cancer activity. A 10–12-fold enhancement in efficiency of drug molecules (~?1 µM) was observed when delivered with graphene oxide (GO) as a nanovehicle. Our data suggest cell death via apoptosis in a dose-dependent manner due to increase in calcium levels in specific cancer cell lines. Interestingly, the benzoxazine derivatives of eugenol with GO nanoparticle exhibited enhanced therapeutic potential in cancer cells. In addition to anti-cancer effect, we also observed significant role of these derivatives on parasite suggesting its multi-pharmacological capability.

     

The role of helices 5 and 6 on the human β1-adrenoceptor activation mechanism

The human β₁-adrenoceptor (β₁AR) is a G-protein-coupled receptor (GPCR) involved in sympathetic system regulation through agonist-induced activation. The conserved CWXP-motif in helix 6 (rotamer toggle switch) is one of the most important activation switches in Class A GPCRs. In order to investigate how the agonist binding disturbs this switch, we carried out molecular dynamics simulations of a hβ₁AR model in the apo and R-noradrenaline-bound forms. The results show that the agonist binding changes the β₁-angle distribution of Cys336, Trp337 and Phe341 residues and increases the helix 6 bending. Overall, we provide a functional hβ₁AR model, showing how the rotamer toggle switch mechanism works at atomic level.     

The protective effect of peroxiredoxin II on oxidative stress induced apoptosis in pancreatic β-cells

Cell signaling mediated by morphogens is essential to coordinate growth and patterning, two key processes that govern the formation of a complex multi-cellular organism. During growth and patterning, cells are specified by both quantitative and directional information. While quantitative information regulates cell proliferation and differentiation, directional information is conveyed in the form of cell polarities instructed by local and global cues. Major morphogens like Wnts play critical roles in embryonic development and they are also important in maintaining tissue homeostasis. Abnormal regulation of these signaling events leads to a diverse array of devastating diseases including cancer. Wnts transduce their signals through several distinct pathways and they regulate vertebrate embryonic development by providing both quantitative and directional information. Here, taking the developing skeletal system as an example, we review our work on Wnt signaling pathways in various aspects of development. We focus particularly on our most recent findings that showed that in vertebrates, Wnt5a acts as a global cue to establishing planar cell polarity (PCP). Our work suggests that Wnt morphogens regulate development by integrating quantitative and directional information. Our work also provides important insights in disease like Robinow syndrome, brachydactyly type B1 (BDB1) and spina bifida, which can be caused by human mutations in the Wnt/PCP signaling pathway.     

Essential role of ERK activation in neurite outgrowth induced by α-lipoic acid

Background

Neurite outgrowth is an important aspect of neuronal plasticity and regeneration after neuronal injury. Alpha-lipoic acid (LA) has been used as a therapeutic approach for a variety of neural disorders. We recently reported that LA prevents local anesthetics-induced neurite loss. In this study, we hypothesized that LA administration promotes neurite outgrowth.

Methods

To test our hypothesis, we treated mouse neuroblastoma N2a cells and primary neurons with LA. Neurite outgrowth was evaluated by examination of morphological changes and by immunocytochemistry for β-tubulin-3. ROS production was examined, as were the phosphorylation levels of ERK and Akt. In separate experiments, we determined the effects of the inhibition of ERK or PI3K/Akt as well as ROS production on LA-induced neurite outgrowth.

Results

LA promoted significantly neurite outgrowth in a time- and concentration-dependent manner. LA stimulation significantly increased the phosphorylation levels of both Akt and ERK and transiently induced ROS production. PI3K/Akt inhibition did not affect LA-induced neurite outgrowth. However, the inhibition of ERK activation completely abolished LA-induced neurite outgrowth. Importantly, the prevention of ROS production by antioxidants attenuated LA-stimulated ERK activation and completely abolished LA-promoted neurite outgrowth.

Conclusion

Our data suggest that LA stimulates neurite outgrowth through the activation of ERK signaling, an effect mediated through a ROS-dependent mechanism.     

Anti-diabetic effect of a novel N-Trisaccharide isolated from Cucumis prophetarum on streptozotocin–nicotinamide induced type 2 diabetic rats

Ethnopharmacological relevanceCucumis prophetarum (L.) is used in traditional Indian medicine for the treatment of inflammation related problems.Aim of the studyThe present investigation was designed to study the effect of N-Trisaccharide (a new compound isolated from the fruit of C. prophetarum (L.)) on hyperglycemia in streptozotocin (STZ)–nicotinamide (NA) induced type 2 diabetic rats.Materials and methodsDifferent doses of N-Trisaccharide (25 and 50 mg/kg b.w.) were administered once daily for 28 days to STZ–NA induced diabetic rats. Plasma insulin and glycogen levels were measured. The activities of hexokinase, glucose-6-phosphatase, fructose-1,6-bisphosphatase, glucose-6-phosphate dehydrogenase, glycogen synthase and glycogen phosphorylase were measured. Further, histological studies on pancreas were also carried out.ResultsThe active compound at doses of 25 and 50 mg/kg b.w. given orally for 14 days showed 47.7% and 69.3% antihyperglycemic activity, respectively. Treatment at the same doses for 28 days provided complete protection against STZ–NA challenge (65 and 230 mg/kg b.w., respectively), intraperitoneally. N-Trisaccharide significantly (p ≤ 0.05) increased the plasma insulin and liver glycogen levels in diabetic rats. The altered enzyme activities of carbohydrate metabolism in the liver and kidney of the diabetic rats were significantly (p ≤ 0.05) improved. Additionally, N-Trisaccharide increased glycogen synthase and decreased glycogen phosphorylase activity in diabetic rats. Histological studies confirmed an increase in insulin level is due to stimulation of injured pancreatic β-cells.ConclusionThe results of the study suggested that N-Trisaccharide possesses propitious effect on STZ–NA induced type 2 diabetes, indicating its usefulness in diabetes management.     

The effect of cathepsin K deficiency on airway development and TGF-β1 degradation

Background

Human immunodeficiency virus (HIV) infected patients are at increased risk for the development of pulmonary arterial hypertension (PAH). Recent reports have demonstrated that HIV associated viral proteins induce reactive oxygen species (ROS) with resultant endothelial cell dysfunction and related vascular injury. In this study, we explored the impact of HIV protein induced oxidative stress on production of hypoxia inducible factor (HIF)-1α and platelet-derived growth factor (PDGF), critical mediators implicated in the pathogenesis of HIV-PAH.

Methods

The lungs from 4-5 months old HIV-1 transgenic (Tg) rats were assessed for the presence of pulmonary vascular remodeling and HIF-1α/PDGF-BB expression in comparison with wild type controls. Human primary pulmonary arterial endothelial cells (HPAEC) were treated with HIV-associated proteins in the presence or absence of pretreatment with antioxidants, for 24 hrs followed by estimation of ROS levels and western blot analysis of HIF-1α or PDGF-BB.

Results

HIV-Tg rats, a model with marked viral protein induced vascular oxidative stress in the absence of active HIV-1 replication demonstrated significant medial thickening of pulmonary vessels and increased right ventricular mass compared to wild-type controls, with increased expression of HIF-1α and PDGF-BB in HIV-Tg rats. The up-regulation of both HIF-1α and PDGF-B chain mRNA in each HIV-Tg rat was directly correlated with an increase in right ventricular/left ventricular+septum ratio. Supporting our in-vivo findings, HPAECs treated with HIV-proteins: Tat and gp120, demonstrated increased ROS and parallel increase of PDGF-BB expression with the maximum induction observed on treatment with R5 type gp-120_CM. Pre-treatment of endothelial cells with antioxidants or transfection of cells with HIF-1α small interfering RNA resulted in abrogation of gp-120_CM mediated induction of PDGF-BB, therefore, confirming that ROS generation and activation of HIF-1α plays critical role in gp120 mediated up-regulation of PDGF-BB.

Conclusion

In summary, these findings indicate that viral protein induced oxidative stress results in HIF-1α dependent up-regulation of PDGF-BB and suggests the possible involvement of this pathway in the development of HIV-PAH.     

Investigations on unconventional hydrogen bonds in RNA binding proteins: The role of CH⋯OC interactions

We have investigated the roles played by CH⋯OC interactions in RNA binding proteins. There was an average of 78 CH⋯OC interactions per protein and also there was an average of one significant CH⋯OC interaction for every 6 residues in the 59 RNA binding proteins studied. Main chain–Main chain (MM) CH⋯OC interactions are the predominant type of interactions in RNA binding proteins. The donor atom contribution to CH⋯OC interactions was mainly from aliphatic residues. The acceptor atom contribution for MM CH⋯OC interactions was mainly from Val, Phe, Leu, Ile, Arg and Ala. The secondary structure preference analysis of CH⋯OC interacting residues showed that, Arg, Gln, Glu and Tyr preferred to be in helix, while Ala, Asp, Cys, Gly, Ile, Leu, Lys, Met, Phe, Trp and Val preferred to be in strand conformation. Most of the CH⋯OC interacting polar amino acid residues were solvent exposed while, majority of the CH⋯OC interacting non polar residues were excluded from the solvent. Long and medium-range CH⋯OC interactions are the predominant type of interactions in RNA binding proteins. More than 50% of CH⋯OC interacting residues had a higher conservation score. Significant percentage of CH⋯OC interacting residues had one or more stabilization centers. Sixty-six percent of the theoretically predicted stabilizing residues were also involved in CH⋯OC interactions and hence these residues may also contribute additional stability to RNA binding proteins.     

The effect of context on the folding of β-hairpins

Small β-hairpin peptides have been widely used as models for the folding of β-sheets. But how applicable is the folding of such models to β-structure in larger proteins with conventional hydrophobic cores? Here we present multiple unfolding simulations of three such proteins that contain the WW domain double hairpin β-sheet motif: cold shock protein A (CspA), cold shock protein B (CspB) and glucose permease IIA domain. We compare the behavior of the free motif in solution and in the context of proteins of different size and architecture. Both Csp proteins lost contacts between the double-hairpin motif and the protein core as the first step of unfolding and proceeded to unfold with loss of the third β-strand, similar to the isolated WW domain. The glucose permease IIA domain is a larger protein and the contacts between the motif and the core were not lost as quickly. Instead the unfolding pathway of glucose permease IIA followed a different pathway with β1 pulling away from the sheet first. Interestingly, when the double hairpin motif was excised from the glucose permease IIA domain and simulated in isolation in water it unfolded by the same pathway as the WW domain, indicating that it is tertiary interactions with the protein that alter the motif’s unfolding not a sequence dependent effect on its intrinsic unfolding behavior. With respect to the unfolding of the hairpins, there was no consistent order to the loss of hydrogen bonds between the β-strands in the hairpins in any of the systems. Our results show that while the folding behavior of the isolated WW domain is generally consistent with the double hairpin motif’s behavior in the cold shock proteins, it is not the case for the glucose permease IIA domain. So, one must be cautious in extrapolating findings from model systems to larger more complicated proteins where tertiary interactions can overwhelm intrinsic behavior.     

The effect of tachykinin neuropeptides on amyloid β aggregation

A hallmark of Alzheimer’s disease is production of amyloid β peptides resulting from aberrant cleavage of the amyloid precursor protein. Amyloid β assembles into fibrils under physiological conditions, through formation of neurotoxic intermediate oligomers. Tachykinin peptides are known to affect amyloid β neurotoxicity in cells. To understand the mechanism of this effect, we studied how tachykinins affect Aβ(1–40) aggregation in vitro. Fibrils grown in the presence of tachykinins exhibited reduced thioflavin T (ThT) fluorescence, while their morphology, observed in transmission electron microscopy (TEM), did not alter. Cross linking studies revealed that the distribution of low molecular weight species was not affected by tachykinins. Our results suggest that there may be a specific interaction between tachykinins and Aβ(1–40) that allows them to co-assemble. This effect may explain the reduction of Aβ(1–40) neurotoxicity in cells treated with tachykinins.