Coordinating cell fate and morphogenesis in Drosophila renal tubules

Using the renal tubules of Drosophila as an example, we explore how cell specification leads to the morphogenetic movements that underlie the generation of tissue architecture. Taking two stages of development, we show first that the tubule cells are allocated by signalling between the endodermal and ectodermal compartments of the posterior gut. Activation of the Wnt pathway patterns the ectodermal anlage, resulting in the expression of tubule genes in a subset of cells and their eversion from the hindgut to form the tubule primordia. We argue that early gene expression directs these morphogenetic movements but not the complete programme of tubule differentiation. In the second example we show that the allocation of the mitogenic tip cell lineage in each tubule is required not only for the normal pattern of cell division but also for the stereotyped three-dimensional arrangement of the mature tubules. Analysis of mutants in which the tip cell lineage is misspecified reveals that both daughters of the tip cell progenitor are required for the tubules to navigate through the body cavity, so that the distal tips locate in their characteristic positions. We show that the regulator of Rac, Myoblast city is essential for this second morphogenetic process.     

Ultrastructure of the Malpighian Tubule Cells in the Mosquito Larvae, Anopheles sinesis

Ultrastructure of epithelial cells constituting the Malpighian tubule of Anopheles sinesis last instar larvae was observed with electron microscope. Malpighian tubule consists of four long and narrow tubule structures with principal cells in typical absorptive cells and regenerative cells forming the simple epithelium. Apical plasma membrane of the principal cell is differentiated into microvilli with one mitochondrion in each microvilli. Basal plasma membrane had extreme infolding to form a canaliculi and a well developed mitochondria was attached in the infoldings. And, rER, ribosomes, and vacuoles were well developed inside the cells. However, there were two main cell types depending on the differentiation of cell organelles. Type 1 cell was cubic, forming the distal portion of Malpighian tubule. The length of microvilli was approximately 4 μm and the basal infoldings were introjected to the depth of 2 μm inside the cell. On the other hand, Type II cell that formed the main proxinal portion was a low squamous type cells with shorter 2 μm of microvilli and the basal infoldings were introjected to the depths of 4 μm inside the cell. As for vacuoles scattered inside the cells, they were regularly observed in both Type I and II and the Type II cells had better developed cellular organelles. Although regenerative cells were extremely small, their cellular organelles were developed and their overall electron density was high that they appeared darker than the principal cells.     

THE EVOLUTION OF FEMALE-BIASED SEXUAL SIZE DIMORPHISM: A POPULATION-LEVEL COMPARATIVE STUDY IN HORNED LIZARDS (PHRYNOSOMA)

Female-biased sexual size dimorphism is uncommon among vertebrates and traditionally has been attributed to asymmetric selective pressures favoring large fecund females (the fecundity-advantage hypothesis) and/or small mobile males (the small-male advantage hypothesis). I use a phylogenetically based comparative method to address these hypotheses for the evolution and maintenance of sexual size dimorphism among populations of three closely related lizard species (Phrynosoma douglasi, P. ditmarsi, and P. hernandezi). With independent contrasts I estimate evolutionary correlations among female body size, male body size, and sexual size dimorphism (SSD) to determine whether males have become small, females have become large, or both sexes have diverged concurrently in body size during the evolutionary Xhistory of this group. Population differences in degree of SSD are inversely correlated with average male body size, but are not correlated with average female body size. Thus, variation in SSD among populations has occurred predominantly through changes in male size, suggesting that selective pressures on small males may affect degree of SSD in this group. I explore three possible evolutionary mechanisms by which the mean male body size in a population could evolve: changes in size at maturity, changes in the variance of male body sizes, and changes in skewness of male body size distributions. Comparative analyses indicate that population differentiation in male body size is achieved by changes in male size at maturity, without changes in the variance or skewness of male and female size distributions. This study demonstrates the potential of comparative methods at lower taxonomic levels (among populations and closely related species) for studying microevolutionary processes that underlie population differentiation.     

The pelvic kidney of male Ambystoma maculatum (Amphibia,urodela, ambystomatidae) with special reference to the sexual collecting ducts

This study details the gross and microscopic anatomy of the pelvic kidney in male Ambystoma maculatum. The nephron of male Ambystoma maculatum is divided into six distinct regions leading sequentially away from a renal corpuscle: (1) neck segment, which communicates with the coelomic cavity via a ventrally positioned pleuroperitoneal funnel, (2) proximal tubule, (3) intermediate segment, (4) distal tubule, (5) collecting tubule, and (6) collecting duct. The proximal tubule is divided into a vacuolated proximal region and a distal lysosomic region. The basal plasma membrane is modified into intertwining microvillus lamellae. The epithelium of the distal tubule varies little along its length and is demarcated by columns of mitochondria with their long axes oriented perpendicular to the basal lamina. The distal tubule possesses highly interdigitating microvillus lamellae from the lateral membranes and pronounced foot processes of the basal membrane that are not intertwined, but perpendicular to the basal lamina. The collecting tubule is lined by an epithelium with dark and light cells. Light cells are similar to those observed in the distal tuble except with less mitochondria and microvillus lamellae of the lateral and basal plasma membrane. Dark cells possess dark euchromatic nuclei and are filled with numerous small mitochondria. The epithelium of the neck segment, pleuroperitoneal funnel, and intermediate segment is composed entirely of ciliated cells with cilia protruding from only the central portion of the apical plasma membrane. The collecting duct is lined by a highly secretory epithelium that produces numerous membrane bound granules that stain positively for neutral carbohydrates and proteins. Apically positioned ciliated cells are intercalated between secretory cells. The collecting ducts anastomose caudally and unite with the Wolffian duct via a common collecting duct. The Wolffian duct is secretory, but not to the extent of the collecting duct, synthesizes neutral carbohydrates and proteins, and is also lined by apical ciliated cells intercalated between secretory cells. Although functional aspects associated with the morphological variation along the length of the proximal portions of the nephron have been investigated, the role of a highly secretory collecting duct has not. Historical data that implicated secretory activity concordant with mating activity, and similarity of structure and chemistry to sexual segments of the kidneys in other vertebrates, lead us to believe that the collecting duct functions as a secondary sexual organ in Ambystoma maculatum. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Autophagy plays a critical role in kidney tubule maintenance,aging and ischemia-reperfusion injury

Autophagy is responsible for the degradation of protein aggregates and damaged organelles. Several studies have reported increased autophagic activity in tubular cells after kidney injury. Here, we examine the role of tubular cell autophagy in vivo under both physiological conditions and stress using two different tubular-specific Atg5-knockout mouse models. While Atg5 deletion in distal tubule cells does not cause a significant alteration in kidney function, deleting Atg5 in both distal and proximal tubule cells results in impaired kidney function. Already under physiological conditions, Atg5-null tubule cells display a significant accumulation of p62 and oxidative stress markers. Strikingly, tubular cell Atg5-deficiency dramatically sensitizes the kidneys to ischemic injury, resulting in impaired kidney function, accumulation of damaged mitochondria as well as increased tubular cell apoptosis and proliferation, highlighting the critical role that autophagy plays in maintaining tubular cell integrity during stress conditions.     

Differentiation of the epithelium of the pronephros and the primary kidney in frogs]

The kidneys of tadpoles of different developmental stages were examined in preparations processed histologically and histochemically. It was found that differentiation of the provisory excretory organ tubules in frogs was "shortened" or "accelerated" after P. P. Ivanov's terminology, and developed differently as compared with differentiation of tubules of the definitive organ of excretion -- the primary kidney. When differentiating the epithelium of the proximal portion of the primary kidney nephron passes the stage of the high prismatic false-stratified epithelium. The pronephros tubules do not pass this stage and the epithelium becomes a strict monolayer from the very beginning. No mitoses are observed in the pronephros tubule epithelium even at the earliest differentiation stages. Later on, the beginning of tubule functioning, and with the reduction, and later disappearance of yolk granules in the epithelium solitary mitoses make their appearance. The mitotic activity of the primary kidney tubule epithelium is very high (70%) at the early stage of differentiation. Then its mitotic activity decreases (30%), and after the beginning of the tubule functioning mitoses in its epithelium become solitary.     

Ultrastructure of maxillary gland of Antrobathynella stammeri (Syncarida,Malacostraca)

The maxillary gland of the highly adapted stygobiont species, Antrobathynella stammeri (Bathynellacea, Syncarida), consists of an end sac, an excretory tubule, and a terminal duct. No valve was found. The excretory tubule forms a loop extending back into the fourth thoracic segment. The end sac is composed of five typical podocytes. Ultrastructurally and functionally, two cell types characterize, respectively, proximal and distal sections of the excretory tubule. Epithelial cells are covered with extremely broad (up to 0.4 μm) microvilli. A basal labyrinth was not seen. Therefore, it is unlikely that the maxillary gland is able to produce a hypoosmotic urine necessary for freshwater animals. Tubule cells can be surrounded by parenchymal cells that produce numerous vesicles, suggesting possible physiological interactions between tubule cells and parenchyma. The ectodermal terminal duct is lined with cuticle and is differentiated into a pulsatile body consisting of two interconnected ampules. The first functions as a bladder. The second ampule, under muscular control, excretes the urine. Pulsatile body, looping tubule, and broad microvilli appear to be distinctive features of the bathynellacid excretory organ. © 1996 Wiley-Liss, Inc.     

Vairimorpha necatrix in Adipose Cells of Trichoplusia ni

Vairimorpha necatrix infected adipose ceiis of the fat body organ of Trichoplusia ni larvae 3–31/2 days after exposure of the larvae to infective spores. During the subsequent 4–6 days, the parasitized adipose cells were hypertrophied in part due to the rapid propagation of V. necatrix schizonts. A calcium-sensitive tubule network developed at the interface of the schizonts and the adipose ceil cytoplasm. The paired nuclei of V. necatrix have pores at the nuclear interface. The pores for each nucleus at this interface are spatially positioned so that they are in conjunction; hence, there is the potential for a channel system between the 2 nuclei.     

Cytotoxic effects of FK506 on human renal proximal tubule cells in culture

Summary FK506 has been used as the primary immunosuppressive agent administered after a variety of organ transplants, with less reported nephrotoxicity than that of cyclosporine. This study examined in vitro cytotoxicity of FK506 on normal human renal proximal tubule cells. Cytotoxicity was assessed by neutral red inclusion and trypan blue exclusion; morphology was assessed by light and transmission electron microscopy. Neutral red inclusion decreased to less than 10% of the control after 3 days exposure to 200μg/ml FK506. Forty microgram per milliliter FK506 caused a decrease in neutral red inclusion to 61% of the control on Day 7, with recovery to 86% on Day 12. Similarly, trypan blue exclusion decreased to 66% of the control following 7 days exposure to 40μg/ml FK506, and confluency of the monolayer was reduced to 50% as evidenced by phase contrast microscopy. After a 12-day exposure, treated monolayers became more confluent. On ultrastructural examination, FK506-treated cells exhibited increased cytoplasmic vacuolation and lipid inclusion. These data suggest that FK506 is reversibly and mildly toxic to monolayers of human renal proximal tubule cells and are consistent with clinical reports of reversible nephrotoxicity.     

Olfactory and vomeronasal systems of caecilians (Amphibia: Gymnophiona)

The morphology of both the main nasal cavity and the vomeronasal organ differs among species representing six families of caecilians. The main nasal cavity is either divided or undivided. The vomeronasal organ differs in position (mediolateral, lateral), size (large vomeronasal organ in the aquatic species), and shape (mediolateral extension, vomeronasal organ with a lateral rostral projection). The great amount of respiratory epithelium of the main nasal cavity, the large vomeronasal organ, and its extensive innervation in typhlonectids may reflect both phylogeny and habitat adaptation, for these taxa are secondarily aquatic or semiaquatic and have several concomitant morphological and physiological modifications. The vomeronasal organ is associated with the caecilian tentacle as the tentacular ducts open into it. This association is further evidence for the involvement of the caecilian tentacle in vomeronasal chemoperception and may represent the mechanism by which these animals smell though the main nasal cavity is closed during burrowing or swimming. Labelings of primary olfactory and vomeronasal projections by means of horseradish peroxidase reaction reveal that the pattern of vomeronasal projections is similar in Ichthyophis kohtaoensis, Dermophis mexicanus, and Typhlonectes natans, even though T. natans possess stronger vomeronasal projections relative to olfactory projections than I. kohtaoensis and D. mexicanus. However, there are differences with respect to the patterns of olfactory projections. The olfactory projection of I. kohtaoensis is characterized by many displaced glomeruli. T. natans has the smallest olfactory projection. The nervus terminalis is associated with the olfactory system as shown by selective labelings of olfactory projections. Six characters potentially useful for phylogenetic analysis emerge from this study of comparative morphology. The characters were subjected to analysis using PAUP to see (1) if any resolution occurred and (2) if any groups were distinguished, whether they corresponded to phylogenetic arrangements based on other morphological characters. The characters are too few to produce nested dichotomous sets for all cases, but they do support the two typhlonectid genera examined and Dermophis and Gymnopis as sister taxa discrete from other groups, and they show that species within genera cluster together.     

Long Term Culture of Human Kidney Proximal Tubule Epithelial Cells Maintains Lineage Functions and Serves as an Ex vivo Model for Coronavirus Associated Kidney Injury

The mechanism of how SARS-CoV-2 causes severe multi-organ failure is largely unknown. Acute kidney injury(AKI) is one of the frequent organ damage in severe COVID-19 patients. Previous studies have shown that human renal tubule cells could be the potential host cells targeted by SARS-CoV-2. Traditional cancer cell lines or immortalized cell lines are genetically and phenotypically different from host cells. Animal models are widely used, but often fail to reflect a physiological and pathogenic status because of species tropisms. There is an unmet need for normal human epithelial cells for disease modeling. In this study, we successfully established long term cultures of normal human kidney proximal tubule epithelial cells(KPTECs) in 2 D and 3 D culture systems using conditional reprogramming(CR) and organoids techniques.These cells had the ability to differentiate and repair DNA damage, and showed no transforming property. Importantly, the CR KPTECs maintained lineage function with expression of specific transporters(SLC34 A3 and cubilin). They also expressed angiotensin-converting enzyme 2(ACE2), a receptor for SARS-CoV and SARS-CoV-2. In contrast, cancer cell line did not express endogenous SLC34 A3, cubilin and ACE2. Very interestingly, ACE2 expression was around twofold higher in 3 D organoids culture compared to that in 2 D CR culture condition. Pseudovirion assays demonstrated that SARS-CoV spike(S) protein was able to enter CR cells with luciferase reporter. This integrated 2 D CR and 3 D organoid cultures provide a physiological ex vivo model to study kidney functions, innate immune response of kidney cells to viruses, and a novel platform for drug discovery and safety evaluation.     

Ultrastructural organization of the transition from the distal nephron to the collecting duct in the desert rodent Psammomys obesus

Summary The transition from the nephron to the collecting duct is formed by three tubular segments (convoluted part of the distal tubule, connecting tubule, cortical collecting duct), which in the desert rodent, Psammomys obesus, transform gradually from one segment to the next, due to intermingling of their different cell types.The convoluted part of the distal tubule (DTC) starts abruptly, shortly beyond the macula densa and initially is homogeneously composed of characteristic DTC-cells. Subsequently, the DTC-cells intermingle with intercalated cells. The first appearance of the connecting-tubule cell, which gradually replaces the DTC-cell, is regarded as the beginning of the connecting tubule. The major portion of the connecting tubule is lined by connecting-tubule cells and intercalated cells. The first appearance of the principal cell between them defines the beginning of the cortical collecting duct, which in the medullary ray is lined by principal and intercalated cells only.Each cell type is described in detail and discussed in relation to the assumed function of the tubular segments.Interspecies differences in the cellular composition of the transitional zone from the nephron to the collecting duct are discussed in relation to the different organization of the collecting duct system.     

Generation of easily accessible human kidney tubules on two‐dimensional surfaces in vitro

The generation of tissue‐like structures in vitro is of major interest for various fields of research including in vitro toxicology, regenerative therapies and tissue engineering. Usually 3D matrices are used to engineer tissue‐like structures in vitro, and for the generation of kidney tubules, 3D gels are employed. Kidney tubules embedded within 3D gels are difficult to access for manipulations and imaging. Here we show how large and functional human kidney tubules can be generated in vitro on 2D surfaces, without the use of 3D matrices. The mechanism used by human primary renal proximal tubule cells for tubulogenesis on 2D surfaces appears to be distinct from the mechanism employed in 3D gels, and tubulogenesis on 2D surfaces involves interactions between epithelial and mesenchymal cells. The process is induced by transforming growth factor‐β₁, and enhanced by a 3D substrate architecture. However, after triggering the process, the formation of renal tubules occurs with remarkable independence from the substrate architecture. Human proximal tubules generated on 2D surfaces typically have a length of several millimetres, and are easily accessible for manipulations and imaging, which makes them attractive for basic research and in vitro nephrotoxicology. The experimental system described also allows for in vitro studies on how primary human kidney cells regenerate renal structures after organ disruption. The finding that human kidney cells organize tissue‐like structures independently from the substrate architecture has important consequences for kidney tissue engineering, and it will be important, for instance, to inhibit the process of tubulogenesis on 2D surfaces in bioartificial kidneys.     

人多能干细胞来源的肾定向分化平台的构建

肾是一种重要的人体器官,具有多种生理功能。然而,全球范围内约有10%的人口患有肾疾病。因此,建立一种接近人体肾的结构与功能的模型进行肾疾病的研究是十分必要的。多能干细胞体外定向诱导分化技术的兴起,为再生医学和精准医学领域注入了新的动力。本研究通过在体外条件下模拟体内肾发育的过程,将人多能干细胞包括胚胎干细胞和诱导多能干细胞,通过体外定向诱导分化形成肾的祖细胞,进而建立肾的结构与功能单位:肾元。该研究通过激活WNT信号通路,同时抑制TGF-β信号通路,将人多能干细胞从多能态定向诱导至原条阶段。之后通过细胞自分化的能力使其发育至中间中胚层,再通过激活FGF信号通路,将其分化至肾祖细胞阶段。流式细胞检测结果显示,肾祖细胞占总细胞数的51.5%~61.9%。通过免疫荧光检测发现:分化得到的结构中包含肾小球足细胞、近端小管、远端小管等肾组织结构。该研究建立的肾体外分化方法,具有稳定性好、分化效率高、重复性好的特点。为研究人类肾的早期发育机制,肾疾病模型构建,以及药物筛选提供了一种新的方法。     

Our perception of developmental plasticity: esse est percipi (to be is to be perceived)?

The continuing interest in the biology of stem cells is enhanced by new discoveries surrounding developmental plasticity of both embryonic and adult stem cells. Adoptive transfer of concepts and definitions from the hematopoietic system to other tissue stem cells suggests inclusion of characteristics such as ability to self-renew and differentiate to functionally reconstitute a tissue/organ of origin. How adequate and accurate are these definitions? Within the great unknown of how these cells function, modulate their gene expression patterns and respond to extrinsic signals, it is apparent that there are numerous levels of stemness. We may envision a scale of developmental flexibility. At one end of the scale are positioned the embryonic stem cells, and at the other end are positioned partially-differentiated, differentiation restricted (committed) tissue/organ stem cells. There is evidence that some stem cells in the adult are pluripotent, thus positioned close to the embryonic end of the stem scale. It is uncertain yet to what extent stem cells can move back and forth along the stem scale.     

The control of Malpighian tubule developmental physiology by 20-hydroxyecdysone and juvenile hormone

The control of developmental changes in Malpighian tubule cell structure and fluid secretion by 20-hydroxyecdysone and juvenile hormone in the skipper butterfly Calpodes ethlius were studied using (1) in vitro tissue culture, (2) in vivo injection and topical application and (3) tubule transplantation experiments. At pupation, 20-hydroxyecdysone initiates cell remodelling and switches off fluid secretion in the Malpighian tubules. Juvenile hormone inhibits these alterations provided that treatment is begun on the first day of the last larval stage. In the pupal stage, 20-hydroxyecdysone triggers the differentiation of adult cell structure which culminates in the renewal of fluid secretion. The results show that 20-hydroxyecdysone and juvenile hormone regulate Malpighian tubule function by altering cell structure and are discussed with respect to the hormonal reprogramming of the Malpighian tubule cells during development.     

Structural and functional changes in the malpighian tubules of Carausius morosus during dehydration and starvation

Summary Carausius morosus starved and deprived of water lose about 30% of their body weight in 4 days, mainly due to water loss. Isolated inferior tubules from starved dehydrated insects secrete urine at 0.041 nl·mm^–1·min^–1 compared with 0.118 nl·mm^–1·min^–1 in those from fed hydrated insects. This difference is due partly to the level of a diuretic (and perhaps also an antidiuretic) hormone in the haemolymph acting directly on the urine-secreting mechanism and partly to changes in the intrinsic capacity of the tubule cells for urine secretion. This latter change is accompanied by structural changes in the tubules. During starvation and dehydration the lumen becomes packed with white granules, the height of the type 1 cells is reduced, their basal infoldings and brush border become shorter and their mitochondrial volume is reduced.     

Aldosterone induces chloride transport in the cortical collecting tubule

The ontogenetic differentiation of transepithelial chloride transport was evaluated in the cortical collecting tubule of the rabbit kidney. Tubules from four control groups (I-IV) were studied during in vitro perfusion. I: body weight 150-280 g; II: 330-480 g; III: 530-880 g; IV: 980-1610 g. In each group, aldosterone (100 micrograms/100 g body weight/day) was given subcutaneously in three doses daily, for 6 days (IA-IVA). Transepithelial net chloride flux (pmol cm2 s1) increased by a factor of almost 3 from group I to group IV (p less than 0.01). Aldosterone induces net chloride flux by 103% (P = 0.03) in IA and by 78% (P = 0.01) in IIA; changes in groups III (21%) and IV (27%) were small. Therefore, the mineralocorticoid induces transepithelial chloride transport in cortical collecting tubule during early transport differentiation. The inducing action decreases with natural differentiation. Moreover, aldosterone alone suffices to induce the complete expression of transepithelial chloride transport in the cortical collecting tubule.