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Drosophila metamorphosis is associated with substantial metabolic activity involving cell death and cell proliferation leading to differentiation of adult tissues and structures. Unlike other larval tissues, Malpighian tubules (MTs) exhibit apoptotic immunity and do not undergo cell death but are carried over to the adult with some cell reorganisation. They persist despite the fact that they express apoptotic proteins and caspases. In the present study, we analysed the global transcription changes in MTs and compared with salivary glands, to decipher the biology of MTs. Gene set enrichment analysis indicated reduced expression of many ecdysone induced genes, including the critical regulator of cell death, E93 in MTs. We hypothesize that reduction of E93 could be because of over expression of ecdysone oxidase, which is high in MTs and is responsible for regulation of hormone titer by degradation of ecdysone. Ectopic expression of E93 in MTs results in cell death through autophagy. Fork head, which is crucial for survival, is enriched in the MT transcriptome, and its down regulation in MTs could be consequent to over expression of E93. Together our data suggests that the cascade of events initiated by ecdysone mediates survival of MTs through concerted action of multiple factors.

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.

Background: Polyglutamine (polyQ) disorders are caused by expanded CAG (Glutamine) repeats in neurons in the brain. The expanded repeats are also expressed in the non-neuronal cells, however, their contribution to disease pathogenesis is not very well studied. In the present study, we have expressed a stretch of 127 Glutamine repeats in Malpighian tubules (MTs) of Drosophila melanogaster as these tissues do not undergo ecdysone induced histolysis during larval to pupal transition at metamorphosis., Results: Progressive degeneration, which is the hallmark of neurodegeneration is also observed in MTs. The mutant protein forms inclusion bodies in the nucleus resulting in expansion of the nucleus and affect chromatin organization which appear loose and open, eventually resulting in DNA fragmentation and blebbing. A virtual absence of tubule lumen was observed followed by functional abnormalities. As development progressed, severe abnormalities affecting pupal epithelial morphogenesis processes were observed resulting in complete lethality. Distribution of heterogeneous RNA binding protein (hnRNP), HRB87F, Wnt/wingless and JNK signaling and expression of Relish was also found to be affected. Expression of resistance genes following polyQ expression was up regulated., Conclusion: The present study gives an insight into the effects of polyQ aggregates in non-neuronal tissues., (Copyright © 2015. Published by Elsevier Inc.)

In insects, humoral response to injury is accomplished by the production of antimicrobial peptides (AMPs) which are secreted in the hemolymph to eliminate the pathogen. Drosophila Malpighian tubules (MTs), however, are unique immune organs that show constitutive expression of AMPs even in unchallenged conditions and the onset of immune response is developmental stage dependent. Earlier reports have shown ecdysone positively regulates immune response after pathogenic challenge however, a robust response requires prior potentiation by the hormone. Here we provide evidence to show that MTs do not require prior potentiation with ecdysone hormone for expression of AMPs and they respond to ecdysone very fast even without immune challenge, although the different AMPs Diptericin, Cecropin, Attacin, Drosocin show differential expression in response to ecdysone. We show that early gene Broad complex (BR-C) could be regulating the IMD pathway by activating Relish and physically interacting with it to activate AMPs expression. BR-C depletion from Malpighian tubules renders the flies susceptible to infection. We also show that in MTs ecdysone signaling is transduced by EcR-B1 and B2. In the absence of ecdysone signaling the IMD pathway associated genes are down regulated and activation and translocation of transcription factor Relish is also affected., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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 β-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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Systemic immune response via the Immune deficiency pathway requires Drosophila inhibitor of apoptosis protein 2 to activate the NF-κB transcription factor Relish. Malpighian tubules (MTs), simple epithelial tissue, are the primary excretory organs, performing additional role in providing protection to Drosophila against pathogenic infections. MTs hold a strategic position in Drosophila as one of the larval tissues that are carried over to adults, unlike other larval tissues that are histolysed during pupation. In this paper we show that Diap2 is an important regulator of local epithelial immune response in MTs and depletion of Diap2 from MTs, increases susceptibility of flies to infection. In the absence of Diap2, activation and translocation of Relish to the nucleus is abolished and as a consequence the production of IMD pathway dependent AMPs are reduced. Ion channels, (Na(+)/K(+))-ATPase and V-ATPase, are important for the immune response of MTs and expression of AMPs and the IMD pathway genes are impaired on inhibition of transporters, and they restrict the translocation of Relish into the nucleus. We show that Diap2 could be regulating ion channels, as loss of Diap2 consequently reduces the expression of ion channels and affects the balance of ion concentrations which results in reduced uric acid deposition. Thus Diap2 seems to be a key regulator of epithelial immune response in MTs, perhaps by modulating ion channels., (© 2013 Wiley Periodicals, Inc.)

Malpighian tubules (MT) of Drosophila melanogaster are osmoregulatory organs that maintain the ionic balance and remove toxic substances from the body. Additionally they act as autonomous immune sensing organs, which secrete antimicrobial peptides in response to invading microbial pathogens. We show that the antimicrobial peptides (AMP) diptericin, cecropinA, drosocin and attacinA are constitutively expressed and are regulated in developmental stage specific manner. Their developmental expression begins from 3(rd) instar larval stage and an immune challenge increases the expression several folds. Spatial variations in the level of expression along the MT tissue are observed. The mortality of 3(rd) instar larvae fed on bacterial food is much less than that of the earlier larval stages, coinciding with the onset of innate immunity response in MT. Ectopic expression of AMP imparts better resistance to infection while, loss of function of one of the AMP through directed RNAi reduces host survival after immune challenge. The AMP secreted from the MT exhibit bactericidal activity. Expression of the NF-κB transcription factor, Relish, also coincides with activation of immune responsive genes in MT, demonstrating that immune regulation in MT is under developmental control and is governed by the Imd pathway.

Drosophila metamorphosis is characterized by the histolysis of larval structures by programmed cell death, which paves the way for the establishment of adult-specific structures under the influence of the steroid hormone ecdysone. Malpighian tubules function as an excretory system and are one of the larval structures that are not destroyed during metamorphosis and are carried over to adulthood. The pupal Malpighian tubules evade destruction in spite of expressing apoptotic proteins, Reaper, Hid, Grim, Dronc and Drice. Here we show that in the Malpighian tubules expression of apoptotic proteins commences right from embryonic development and continues throughout the larval stages. Overexpression of these proteins in the Malpighian tubules causes larval lethality resulting in malformed tubules. The number and regular organization of principal and stellate cells of Malpighian tubules is disturbed, in turn disrupting the physiological functioning of the tubules as well. Strikingly, the localization of beta-tubulin, F-actin and Disclarge (Dlg) is also disrupted. These results suggest that the apoptotic proteins could be having non-apoptotic function in the development of Malpighian tubules.

Drosophila development proceeds through three larval stages, before it pupates to reach adulthood. During pupation, larval tissues are destructed by programmed cell death and replaced by adult structures. Programmed cell death is a tightly regulated process accomplished by the induction of three closely linked pro-apoptotic genes reaper, hid and grim, ultimately leading to the activation of caspases, DRONC and DRICE and results in cell death. Unlike other larval tissues, Malpighian tubules are unique in not undergoing characteristic ecdysone-induced apoptosis and are carried to the adults. In this paper we show that apoptotic proteins, HID, GRIM, DRONC and DRICE are expressed in the Malpighian tubules, however they are sequestered in the nucleus. Significantly DRONC and DRICE are not enzymatically processed to active forms in the Malpighian tubules, however, ectopic expression of pro-apoptotic proteins leads to malformed Malpighian tubules and lethality. We also show that the Drosophila inhibitor of apoptotic protein 1, DIAP1, is localized and processed differently in Malpighian tubules. These results provide first evidence in favor of differential activity of apoptotic proteins in Malpighian tubules., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

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.