Alteration of autophagy-related protein 5 (ATG5) levels and Atg5 gene expression in diabetes mellitus with and without complications.

BACKGROUND: Autophagy is a catabolic mechanism that involves lysosomal-dependent degradation of unnecessary intracellular components and responsible for normal cellular homeostasis. Autophagy pathway and its key participant ATG5/LC3 are associated with several pathologies such as diabetes mellitus and its complications. METHODS: Levels and expression of autophagy key components ATG5 and LC3B were analyzed in both human model and murine tissues. One hundred and twenty human subjects were divided into four groups: Healthy (control), diabetes mellitus without complications, diabetic nephropathy, and diabetic retinopathy. Additionally, we used kidneys from WT healthy and diabetic nephropathy mice. Lysate derived from human peripheral blood mononuclear cells and murine renal cortex lysates were subjected to western blot and immunohistochemical analysis. RESULTS: Western blot and immunohistochemical analysis demonstrate that ATG5 protein levels were significantly decreased in diabetes mellitus, diabetic nephropathy (DN), and diabetic retinopathy patients versus healthy controls and in DN mice compared to healthy mice (0.65 ± 0.04; 1.15 ± 0.13 A.U. units, respectively). Quantification of staining area (%) of ATG5 mice tissue expression also decreased in DN versus healthy mice (4.42 ± 1.08%; 10.87 ± 1.01%, respectively). LC3B LEVELS AND EXPRESSION: Significant reduction in peripheral blood mononuclear cells in diabetic patients (with or without complications) vs. healthy controls. Renal LC3B levels were lower in DN versus healthy mice (0.36 ± 0.03; 0.68 ± 0.07 A.U. units). Renal LC3B staining quantification revealed significant reduction in DN versus healthy mice (1.7 ± 0.23%; 8.56 ± 1.7%). CONCLUSION: We conclude that ATG5, as well as LC3B, are down regulated in diabetic patients with or without complications. This diminution contributes to deficiencies in the autophagy process.

The Therapeutic Effect of Active Vitamin D Supplementation in Preventing the Progression of Diabetic Nephropathy in a Diabetic Mouse Model.

BACKGROUND: Diabetic nephropathy (DN) is one of the most common microvascular complications of diabetes and is the leading cause of end-stage renal disease (ESRD) and replacement therapy worldwide. Vitamin D levels in DN patients are very low due to the decrease in the synthesis and activity of 1-α hydroxylase in the proximal tubule cells and decrease in the vitamin D receptor abundance. To date, few studies have shown the antioxidant effects of 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] on hyperglycemia-induced renal injury. The selective activator of the vitamin D receptor, paricalcitol, reduces proteinuria and slows the progression of kidney injury. The precise mechanism through which vitamin D affects diabetic status and provides kidney protection remains to be determined. METHODS: Diabetes mellitus (DM) was induced in 94 8-week-old DBA/2J mice by intraperitoneal injection of streptozotocin (STZ). DM mice were randomly divided into receiving vehicle or treatment with paricalcitol, the active vitamin D analog, 1 week after DM induction or paricalcitol treatment 3 weeks after DM induction. An additional control group of healthy wild-type mice was not treated. Urine albumin, blood urea nitrogen, and creatinine levels were measured before and at the end of the paricalcitol treatment. Periodic acid-Schiff, immunohistochemistry staining, and western blot of the renal tissues of vitamin D receptor, villin, nephrin, and podocin expressions, were analyzed. RESULTS: Paricalcitol treatment restored villin, nephrin, and podocin protein levels that were downregulated upon DM induction, and reduced fibronectin protein level. Vitamin D receptor activation by paricalcitol may reduce proteinuria of DN in mice and alleviate high-glucose-induced injury of kidney podocytes by regulating the key molecules such nephrin-podocin. CONCLUSIONS: Paricalcitol treatment was associated with improved structural changes in type 1 diabetic mice including upregulation of vitamin D receptor expression, and decreased fibrosis markers such as fibronectin. These effects may contribute to the consistent benefit of vitamin D analog to slow the deterioration in glomerular function and reduce the risk of ESRD in patients with type 1 and 2 diabetes mellitus. Our results suggest that additional use of paricalcitol may be beneficial in treating patients with diabetes under standard therapeutic strategies.

The potential use of natural products to negate hepatic, renal and neuronal toxicity induced by cancer therapeutics.

Different types of chemotherapeutics are used for cancer treatment. These drugs act on several signal pathways, lead to programmed cell death, and damage cancer cells. Although many specific mechanisms of action have been suggested for chemotherapeutics, there are still gaps in understanding their effects. They may affect different components of the cell, particularly proteins with specific functions, such as enzymes. Recently, targeted and immuno therapies were introduced for treatment of different cancers. However, many cancer patients still depend on traditional and well-known drugs. Doxorubicin and platinum-based drugs are among the most frequently used chemotherapeutics. They are highly cytotoxic for cancer cells, but they also act on healthy cells. Hence, it is crucial to understand the mechanisms involved in order to decrease their side effects. Natural products, many of which are also available over-the-counter, may be considered to decrease various cancer drug-induced side effects. This review focuses on the use of these compounds to overcome side effects of chemotherapeutics, primarily doxorubicin and cisplatin, in the liver, kidney, and neuronal systems.

The Iron-Klotho-VDR Axis Is a Major Determinant of Proximal Convoluted Tubule Injury in Haptoglobin 2-2 Genotype Diabetic Nephropathy Patients and Mice.

The haptoglobin (Hp) genotype (1-1 and 2-2) is a major determinant of nephropathy progression in diabetes mellitus patients. Hp 2-2 diabetic mice have impaired Hb clearance and increased iron deposits and oxidative stress in the proximal tubules (PCT), leading to increased renal injury. However, the precise mechanism of the PCT injury in diabetic nephropathy (DN) remains elusive. In the kidney, 1,25(OH)2D3 suppresses the inflammatory response to renal tubular injury and requires normal renal expression of the α-klotho protein. In this study, we set out to test the hypothesis that the increased renal iron deposits in the PCT of Hp 2-2 DN affect the α-klotho-vitamin D receptor (VDR) axis and thereby exacerbates the PCT injury generated by the iron deposits. Immunohistochemical analysis of human and mouse kidney biopsies along with western blot analysis showed that the increased iron deposits in the PCT of the Hp 2-2 genotype were accompanied with significantly decreased α-klotho and VDR renal expression but significantly increased 1-α-hydroxylase renal expression. In conclusion, the iron-klotho-VDR axis is a major player in the mechanism contributing to iron-mediated PCT injury in diabetic Hp 2-2 mice and patients. Targeting this axis may open the way for new ideas regarding the pathogenesis and treatment of DN.

aPKCζ affects directed cell migration through the regulation of myosin light chain phosphorylation.

Cell motility is an essential cellular process for a variety of biological events. It requires cross-talk between the signaling and the cytoskeletal systems. Despite the recognized importance of aPKCζ for cell motility, there is little understanding of the mechanism by which aPKCζ mediates extracellular signals to the cytoskeleton. In the present study, we report that aPKCζ is required for the cellular organization of acto-non-muscle myosin II (NMII) cytoskeleton, for proper cell adhesion and directed cell migration. We show that aPKCζ mediates EGF-dependent RhoA activation and recruitment to the cell membrane. We also show that aPKCζ mediates EGF-dependent myosin light chain (MRLC) phosphorylation that is carried out by Rho-associated protein kinase (ROCK), and that aPKCζ is required for EGF-dependent phosphorylation and inhibition of the myosin phosphatase targeting subunit (MYPT). Finally, we show that aPKCζ mediates the spatial organization of the acto-NMII cytoskeleton in response to EGF stimulation. Our data suggest that aPKCζ is an essential component regulator of acto-NMII cytoskeleton organization leading to directed cell migration, and is a mediator of the EGF signal to the cytoskeleton.

[HAPTOGLOBIN POLYMORPHISM AS AN INDEPENDENT PREDICTOR OF DIABETIC NEPHROPATHY AND RETINOPATHY].

INTRODUCTION: The antioxidant protein haptoglobin (Hp) plays a major role in the development of diabetic complications such as diabetic nephropathy and retinopathy. In humans, two alleles of Hp were identified: 1 and 2 with three possible genotypes: 1-1, 2-1, and 2-2. The Hp protein products differ in their biochemical and biophysical properties, such as their antioxidant capacity. The Hp1 protein is superior to the Hp2 protein in binding to free hemoglobin and neutralizing its oxidative potential and the accompanying renal and retinal injury. Hence, diabetic patients with different Hp phenotypes have variable susceptibility to developing diabetic nephropathy and retinopathy. In diabetes, the kidney and the retinal injury progress gradually over time. Thus, understanding the factors that mediate the aggravation and progression of these complications is of critical importance. One of the latest hypotheses regarding the involvement of haptoglobin in the development of diabetic complications is its contribution to impaired vitamin D activation in the kidney. Over the last few years, great efforts were made in the field to explore this notion and decrypt the mechanism behind it. The goal in this area is that the research findings will be translated into clinical practice and lead to the development of a pharmacogenomics clinical approach that will deal with diabetic complications by selective administration of vitamin D according to the Hp genotype.

Interaction between the Haptoglobin 2 Phenotype and Diabetes Mellitus on Systolic Pulmonary Arterial Pressure and Nitric Oxide Bioavailability in Hemodialysis Patients.

Elevated systolic pulmonary artery pressure (s-PAP, ≥35 mmHg) serves as an independent predictor of mortality in hemodialysis (HD) and diabetic (DM) patients. A polymorphism in the antioxidant Haptoglobin (Hp) gene has been shown to regulate the bioavailability of nitric oxide (NO), a major mediator of pulmonary vascular tone. We therefore set out to test the hypothesis that the Hp polymorphism may be a determinant of developing elevated s-PAP specifically in the DM state due to a decreased bioavailability of NO. To test our hypothesis we Hp typed and performed transthoracic echocardiography on a series of HD patients and stratified them into elevated and normal s-PAP groups and then evaluated whether there was a significant association between the Hp type, elevated s-PAP, and decreased NO bioavailability as defined by low plasma nitrite. We found a statistically significant interaction between the Hp type and DM on the prevalence of elevated s-PAP and lower mean nitrite levels with the combination of elevated s-PAP and low nitrite levels being significantly more prevalent in Hp 2-2 DM individuals. We conclude that the Hp 2 type is associated with elevated s-PAP levels and low plasma nitrite levels in HD patients specifically in the DM state.

The tumor suppressor Lgl1 forms discrete complexes with NMII-A and Par6α-aPKCζ that are affected by Lgl1 phosphorylation.

Non-muscle myosin IIA (NMII-A) and the tumor suppressor lethal giant larvae 1 (Lgl1) play a central role in the polarization of migrating cells. Mammalian Lgl1 interacts directly with NMII-A, inhibiting its ability to assemble into filaments in vitro. Lgl1 also regulates the cellular localization of NMII-A, the maturation of focal adhesions and cell migration. In Drosophila, phosphorylation of Lgl affects its association with the cytoskeleton. Here we show that phosphorylation of mammalian Lgl1 by aPKCζ prevents its interaction with NMII-A both in vitro and in vivo, and affects its inhibition of NMII-A filament assembly. Phosphorylation of Lgl1 affects its cellular localization and is important for the cellular organization of the acto-NMII cytoskeleton. We further show that Lgl1 forms two distinct complexes in vivo, Lgl1-NMIIA and Lgl1-Par6α-aPKCζ, and that the formation of these complexes is affected by the phosphorylation state of Lgl1. The complex Lgl1-Par6α-aPKCζ resides in the leading edge of the cell. Finally, we show that aPKCζ and NMII-A compete to bind directly to Lgl1 at the same domain. These results provide new insights into the mechanism regulating the interaction between Lgl1, NMII-A, Par6α and aPKCζ in polarized migrating cells.

The tumor suppressor Lgl1 regulates NMII-A cellular distribution and focal adhesion morphology to optimize cell migration.

The Drosophila tumor suppressor Lethal (2) giant larvae (Lgl) regulates the apical-basal polarity in epithelia and asymmetric cell division. However, little is known about the role of Lgl in cell polarity in migrating cells. In this study we show direct physiological interactions between the mammalian homologue of Lgl (Lgl1) and the nonmuscle myosin II isoform A (NMII-A). We demonstrate that Lgl1 and NMII-A form a complex in vivo and provide data that Lgl1 inhibits NMII-A filament assembly in vitro. Furthermore, depletion of Lgl1 results in the unexpected presence of NMII-A in the cell leading edge, a region that is not usually occupied by this protein, suggesting that Lgl1 regulates the cellular localization of NMII-A. Finally, we show that depletion of Lgl1 affects the size and number of focal adhesions, as well as cell polarity, membrane dynamics, and the rate of migrating cells. Collectively these findings indicate that Lgl1 regulates the polarity of migrating cells by controlling the assembly state of NMII-A, its cellular localization, and focal adhesion assembly.