Development of a Drosophila melanogaster based model for the assessment of cadmium and mercury mediated renal tubular toxicity.

Xenobiotic mediated renal toxicity is one of the major health concerns to the organisms, including humans. New chemicals with nephrotoxic potential are continuously being added to the list of existing nephrotoxicants. To predict the nephrotoxicity of these new chemicals, reliable and cost-effective alternative animal models are required. It is a prerequisite for the identification and assessment of these compounds as potential nephrotoxicants to prevent renal toxicity in the exposed population. Drosophila melanogaster, a genetically tractable invertebrate animal model, has a renal system functionally analogous to humans. The Malpighian tubules (MTs) of D. melanogaster are similar to the tubular part of nephron of the human kidney. Besides, it recapitulates the renal toxicity hallmark with mammals when exposed to known nephrotoxicants. In this study, first instar larvae of D. melanogaster (Oregon R) were exposed to different concentrations of two well-known nephrotoxicants, cadmium (Cd) and mercury (Hg). Akin to higher organisms, Cd and Hg exposure to D. melanogaster produce similar phenotypes. MTs of exposed D. melanogaster larvae exhibited increased oxidative stress, activated cellular antioxidant defense mechanism, GSH depletion, increased cleaved caspase-3 expression, increased DEVDase activity and increased cell death. The functional status of MTs was assessed by fluid secretion rate (FSR), efflux activity of transporter protein, mitochondrial membrane potential (MMP), ATP level and expression of junctional protein (Dlg). All the phenotypes observed in MTs of D. melanogaster larvae recapitulate the phenotypes observed in higher organisms. Increased uric acid level, the hallmark of renal dysfunction, was also observed in exposed larvae. Taken together, the study suggests that MTs of D. melanogaster may be used as a functional model to evaluate xenobiotic mediated nephrotoxicity.

Ecdysone regulates morphogenesis and function of Malpighian tubules in Drosophila melanogaster through EcR-B2 isoform.

Malpighian tubules are the osmoregulatory and detoxifying organs of Drosophila and its proper development is critical for the survival of the organism. They are made up of two major cell types, the ectodermal principal cells and mesodermal stellate cells. The principal and stellate cells are structurally and physiologically distinct from each other, but coordinate together for production of isotonic fluid. Proper integration of these cells during the course of development is an important pre-requisite for the proper functioning of the tubules. We have conclusively determined an essential role of ecdysone hormone in the development and function of Malpighian tubules. Disruption of ecdysone signaling interferes with the organization of principal and stellate cells resulting in malformed tubules and early larval lethality. Abnormalities include reduction in the number of cells and the clustering of cells rather than their arrangement in characteristic wild type pattern. Organization of F-actin and ß-tubulin also show aberrant distribution pattern. Malformed tubules show reduced uric acid deposition and altered expression of Na(+)/K(+)-ATPase pump. B2 isoform of ecdysone receptor is critical for the development of Malpighian tubules and is expressed from early stages of its development.

Cancer stem cell in prostate cancer progression, metastasis and therapy resistance.

Prostate cancer (PCa) is the most diagnosed malignancy in the men worldwide. Cancer stem cells (CSCs) are the sub-population of cells present in the tumor which possess unique properties of self-renewal and multilineage differentiation thus thought to be major cause of therapy resistance, disease relapse, and mortality in several malignancies including PCa. CSCs have also been shown positive for the common stem cells markers such as ALDH EZH2, OCT4, SOX2, c-MYC, Nanog etc. Therefore, isolation and characterization of CSCs specific markers which may discriminate CSCs and normal stem cells are critical to selectively eliminate CSCs. Rapid advances in the field offers a theoretical explanation for many of the enduring uncertainties encompassing the etiology and an optimism for the identification of new stem-cell targets, development of reliable and efficient therapies in the future. The emerging reports have also provided unprecedented insights into CSCs plasticity, quiescence, renewal, and therapeutic response. In this review, we discuss the identification of PCa stem cells, their unique properties, stemness-driving pathways, new diagnostics, and therapeutic interventions.

Hsp27 over expression protect against cadmium induced nephrotoxicity in Drosophila melanogaster.

Cadmium (Cd) exposure to the animals including humans is reported as nephrotoxic compounds i.e., disturbing redox status (increase oxidative stress), mitochondrial dysfunction, renal cell death and altered transporters in the renal system. Hsp27 (a small heat shock protein) has been shown as one of the modulators in the renal dysfunction and increased against the Cd induced toxicity. However, no studies are reported on the genetic modulation of stress protein against the Cd-induced nephrotoxicity. The current study aimed to examine the protective role of hsp27 overexpression against the Cd-induced nephrotoxicity using Drosophila melanogaster as an animal model. D. melanogaster renal system includes nephrocytes and Malpighian tubules (MTs) that show the functional similarity with mammalian kidney nephron. Overexpression of the hsp27 was found to reduce the Cd induced oxidative stress, rescue cell death in MTs of Cd exposed D. melanogaster larvae. The rescued GSH level, NADPH level and glucose 6 phosphate dehydrogenase (G6PD) activity were also observed in the MTs of the Cd exposed organism. Function (efflux activity and fluid secretion rate) of the MTs was restored in Cd exposed hsp27 overexpressed larvae. Further, results were confirmed by restored brush border microvilli density and reduced uric acid level. Tissue specific knockdown of hsp27 developed Cd like phenotypes in MTs and the phenotypes enhanced in Cd exposed condition. The present study clearly shows the role of hsp27 overexpression in restoration of the MTs function and protection against the Cd induced renal toxicity.

Genotypic Analysis of COL4A1 Gene in Diabetic Nephropathy and Type 2 Diabetes Mellitus Patients: A Comparative Genetic Study.

Diabetic nephropathy (DN) is specified by microalbuminuria, glomerular lesions, and renal fibrosis leading to end-stage renal disease. The pathophysiology of DN is multifactorial as a result of gene-environment interaction. Clinical studies suggested that gene mutations affect various pathways involved in DN, including extracellular matrix (ECM). During chronic hyperglycemia, collagen type-4-mediated ECM overproduction occurs, leading to renal fibrosis and DN development. In this study, COL4A1 gene variant rs605143 (G/A) was analyzed in diabetes and DN patients from the study population. We genotyped 386 study subjects, comprising 120 type 2 diabetes mellitus (T2DM) patients, 120 DN, and 146 healthy controls. All study subjects were analyzed for biochemical assays by commercially available kits and genotypic analysis by polymerase chain reaction-restriction fragment length polymorphism and confirmed by Sanger sequencing. Statistical analyses were done using SPSS and GraphPad. Anthroclinicopathological parameters showed a significant association between T2DM and DN. Genotype AA of COL4A1 gene variant rs605143 (G/A) showed a significant association with T2DM and DN compared with controls with 5.87- and 8.01-folds risk, respectively. Mutant allele A also significantly associated with T2DM and DN independently compared with healthy controls with 2.29- and 2.81-time risk in the study population. This study's findings suggested that COL4A1 gene variant rs605143 (G/A) can be used as predictive biomarkers for T2DM and DN independently. However, this gene variant needs to be analyzed in a large sample to explore the shared genetic association between T2DM and DN.

Single and combined effect of bisphenol A with high sucrose diet on the diabetic and renal tubular dysfunction phenotypes in Drosophila melanogaster.

In the present study, effect of exposure of bisphenol A (BPA) and combined exposure of BPA + HSD has been investigated on the glucose homeostasis and associated renal complications in Drosophila. Exposure of 1.0 mM BPA alone induced type 2 diabetes like condition (T2D) in adult male D. melanogaster via oxidative stress. Elevated TGF-ß signaling was evident by increased expression of baboon (babo) in BPA exposed organism that stimulated the modulation of extracellular matrix (ECM) component collagen IV resulting in the fibrosis of the Malpighian tubules (MTs). Combined exposure of BPA + HSD (high sucrose diet) resulted in the increased magnitude of T2D and MTs dysfunction parameters. Taken together, the study illustrates that BPA has diabetogenic potential in exposed Drosophila that caused adverse effects on their MTs and combined exposure with BPA and HSD could aggravate the renal tubular dysfunction. The study further suggests the use of Drosophila model to study the environmental chemicals induced diabetes mediated renal dysfunction.

Copper-mediated ß-amyloid toxicity and its chelation therapy in Alzheimer's disease.

The link between bio-metals, Alzheimer's disease (AD), and its associated protein, amyloid-ß (Aß), is very complex and one of the most studied aspects currently. Alzheimer's disease, a progressive neurodegenerative disease, is proposed to occurs due to the misfolding and aggregation of Aß. Dyshomeostasis of metal ions and their interaction with Aß has largely been implicated in AD. Copper plays a crucial role in amyloid-ß toxicity, and AD development potentially occurs through direct interaction with the copper-binding motif of APP and different amino acid residues of Aß. Previous reports suggest that high levels of copper accumulation in the AD brain result in modulation of toxic Aß peptide levels, implicating the role of copper in the pathophysiology of AD. In this review, we explore the possible mode of copper ion interaction with Aß, which accelerates the kinetics of fibril formation and promote amyloid-ß mediated cell toxicity in Alzheimer's disease and the potential use of various copper chelators in the prevention of copper-mediated Aß toxicity. KEYWORDS: Short Twitter Statement: Authors explore copper ion interaction w/ Aß and kinetics of fibril formation in promoting amyloid-ß mediated cell toxicity in Alzheimer's disease and the potential use of copper chelators in the prevention of copper-mediated Aß toxicity. SHORT TWITTER STATEMENT: Authors explore copper ion interaction w/Aß and kinetics of fibril formation in promoting amyloid-ß mediated cell toxicity in Alzheimer's disease and the potential use of copper chelators in the prevention of copper-mediated Aß toxicity.

High sucrose diet induces morphological, structural and functional impairments in the renal tubules of Drosophila melanogaster: A model for studying type-2 diabetes mediated renal tubular dysfunction.

Continuous feeding of high dietary sugar is strongly associated with type 2 diabetes (T2D) and its secondary complications. Diabetic nephropathy (DN) is a major secondary complication that leads to glomerular and renal tubular dysfunction. The present study is aimed to investigate the effects of chronic exposure of high sugar diet (HSD) on renal tubules. Malpighian tubules (MTs), a renal organ of Drosophila, were used as a model in the study. Feeding of HSD develops T2D condition in Drosophila. The MTs showed structural abnormalities in 20 days of HSD fed flies. Impaired insulin signaling, oxidative stress, enhanced levels of AGE-RAGE and induction of apoptosis were observed in the MTs of these flies. Further, altered expression of transporters, enhanced uric acid level and reduced fluid secretion rate confirmed the impaired function of MTs in these flies. RNA-seq and RT-PCR analyses in the MTs of HSD fed-and control-flies revealed the altered expression of candidate genes that regulate several important pathways including extracellular matrix (ECM), advanced glycation end products-receptor for advanced glycation end products (AGE-RAGE), transforming growth factor ß (TGF-ß), galactose, starch and sucrose metabolism that are well known mediators of renal tubular dysfunction in DN patients. Disruption of insulin signaling in the MTs also causes renal tubular dysfunction similar to HSD fed flies. Overall, the study suggests that phenotypes observed in the MTs of HSD fed flies recapitulate several hallmarks of renal tubular dysfunction in DN patients. Therefore, we conclude that MTs of HSD fed flies may be used for deciphering the underlying mechanisms of T2D mediated renal tubular dysfunction.

Candle soot derived carbon nanoparticles: An assessment of cellular and progressive toxicity using Drosophila melanogaster model.

The biomedical demand of the nanomaterials is continuously increasing due to their wide range of applications in the field. However, before the implementation of these nanomaterials, toxicity assessment is essential for its safe usage. In the present study, the toxicity of carbon nanoparticles (CNPs) was investigated which was derived from candle soot and compared with commercially available multi-walled carbon nanotubes (CNTs) by using Drosophila melanogaster as a model system. First instar Drosophila larvae were exposed to CNPs as well as CNTs, and the toxic effects of these nanomaterials were compared. The result shows that both nanomaterials enhance the level of reactive oxygen species and oxidative stress in the Drosophila, which leads to the upregulation of heat shock proteins that may cause cytotoxicity in exposed Drosophila larvae. In contrast, exposure to CNPs and CNTs did not affect the developmental period of the larvae. Morphology of the internal organs, brain, gut and Malpighian tubules was also not altered in the exposed larvae. Similarly, no change observed in the cytoskeleton (F-actin) of these organs. Reproductive performance was slightly reduced in the case of CNPs compare to control. However, CNTs exposure did not show any significant effect on the reproductive performance of the flies that emerged from exposed larvae in comparison to control. Hence the study concludes that exposure to CNPs and CNTs cause a moderate level of cytotoxicity in Drosophila. The study also indicates that the inexpensive CNPs may use as an alternative to expensive CNTs for biomedical and biological applications.

Drosophila Renal Organ as a Model for Identification of Targets and Screening of Potential Therapeutic Agents for Diabetic Nephropathy.

Diabetic nephropathy is one of the major causes of kidney failure, accounting for ~44% of all cases. In spite of the significant mortality rate of diabetic nephropathy, specific and effective treatment is still eluding. Identification of genetic determinants and understanding their role in the progression of disease are essential for developing diagnostic tools and effective therapy. Drosophila melanogaster is a genetically tractable model organism and is being used for understanding the genetic basis of several human diseases. Drosophila has a well developed renal system and shares conserved developmental and functional processes with humans. Apart from similarities in renal system, type 1 and type 2 diabetes can be induced in Drosophila following mechanisms similar to those in human. This review discusses the current therapies available for diabetic nephropathy and examines the potential of Drosophila renal system as a model for identifying drug targets for diabetic nephropathy and screening of the potential drugs for their efficacy.

Presence of Zearalenone in Cereal Grains and Its Exposure Risk Assessment in Indian Population.

Zearalenone (ZEA) is a toxic metabolite of Fusarium genera that frequently contaminates cereal grains. India being a tropical country provides suitable conditions for fungal invasion to the cereals. In the absence of any regulatory limits for ZEA in India, the present study was carried out to analyze the contamination levels of ZEA in different cereal samples consumed by Indian population and its exposure assessment through intake. Out of 117 cereal samples comprising of wheat, rice, corn, and oats, 70 (84%) were found to be positive for ZEA contamination, among which 24 (33%) samples exceeded the permissible limits proposed by European Union when analyzed by high-performance liquid chromatography. The positive samples were further validated by Liquid Chromatography-Mass Spectroscopy (LC-MS) analysis. Based on the quantitative estimation of ZEA contamination in cereals and their daily consumption values, the probable daily intake of ZEA was found to be 16.9- and 7.9-fold higher in rice and wheat samples, respectively, than the tolerable daily intake prescribed by European Food Safety Authority. The presence of ZEA at high levels indicates a higher exposure risk for Indian population as wheat and rice are staple foods in India. Thus, there is an immediate need to set the permissible levels of ZEA in India to safeguard the health of 1.34 billion people. PRACTICAL APPLICATION: High levels of ZEA contaminated wheat and rice samples suggest that the consumers are at a greater exposure risk. The study will help the Indian regulatory bodies to set the permissible level of ZEA in different cereal grains so as to safeguard the health of common masses. This can happen by simply adopting to European Food Safety Authority standards or depending on the consumption pattern of food and its occurrence, the new safe limit can be prescribed in India like in other Asian countries.

Drosophila Malpighian Tubules: A Model for Understanding Kidney Development, Function, and Disease.

The Malpighian tubules of insects are structurally simple but functionally important organs, and their integrity is important for the normal excretory process. They are functional analogs of human kidneys which are important physiological organs as they maintain water and electrolyte balance in the blood and simultaneously help the body to get rid of waste and toxic products after various metabolic activities. In addition, it receives early indications of insults to the body such as immune challenge and other toxic components and is essential for sustaining life. According to National Vital Statistics Reports 2016, renal dysfunction has been ranked as the ninth most abundant cause of death in the USA. This chapter provides detailed descriptions of Drosophila Malpighian tubule development, physiology, immune function and also presents evidences that Malpighian tubules can be used as a model organ system to address the fundamental questions in developmental and functional disorders of the kidney.

Ecdysone signaling is required for proper organization and fluid secretion of stellate cells in the Malpighian tubules of Drosophila melanogaster.

Drosophila development is a tightly regulated process involving metamorphosis of a relatively less mobile larva to a highly motile adult, triggered by secretion of 20-hydroxyecdysone. Under the influence of ecdysone, most of the larval tissues degenerate, while the imaginal cells differentiate and form adult specific structures. Although the larval Malpighian tubules do not seem to be affected by ecdysone during metamorphosis, we show that ecdysone signaling plays an important role in the early development and functioning of Malpighian tubules. Disruption of ecdysone receptor function, using targeted expression of dominant negative ecdysone receptor in stellate cells, results in disruption of organization of Malpighian tubules. The number of stellate cells is reduced in such Malpighian tubules. Further, they get clustered rather than distributed in their characteristic wild type pattern. We also demonstrate that expression of Drosophila integrin protein (DRIP), an aquaporin responsible for trans-cellular water transport, is also reduced in stellate cells when ecdysone signaling is disrupted. Our results show that of the three ecdysone receptor isoforms, only EcR-B2 rescues these phenotypes. A similar pattern of stellate cell clustering and reduced expression of DRIP is observed in ecd(1), a temperature sensitive mutant, under non-permissive conditions. These results suggest that ecdysone signaling is required for proper patterning and functioning of stellate cells and that EcR-B2 may be the primary isoform required for ecdysone signaling in stellate cells.