Cell Volume Regulation in the Proximal Tubule of Rat Kidney

We developed a dynamic model of a rat proximal convoluted tubule cell in order to investigate cell volume regulation mechanisms in this nephron segment. We examined whether regulatory volume decrease (RVD), which follows exposure to a hyposmotic peritubular solution, can be achieved solely via stimulation of basolateral K\(^+\) and \(\hbox {Cl}^-\) channels and \(\hbox {Na}^+\)–\(\hbox {HCO}_3^-\) cotransporters. We also determined whether regulatory volume increase (RVI), which follows exposure to a hyperosmotic peritubular solution under certain conditions, may be accomplished by activating basolateral \(\hbox {Na}^+\)/H\(^+\) exchangers. Model predictions were in good agreement with experimental observations in mouse proximal tubule cells assuming that a 10% increase in cell volume induces a fourfold increase in the expression of basolateral K\(^+\) and \(\hbox {Cl}^-\) channels and \(\hbox {Na}^+\)–\(\hbox {HCO}_3^-\) cotransporters. Our results also suggest that in response to a hyposmotic challenge and subsequent cell swelling, \(\hbox {Na}^+\)–\(\hbox {HCO}^-_3\) cotransporters are more efficient than basolateral K\(^+\) and \(\hbox {Cl}^-\) channels at lowering intracellular osmolality and reducing cell volume. Moreover, both RVD and RVI are predicted to stabilize net transcellular \(\hbox {Na}^+\) reabsorption, that is, to limit the net \(\hbox {Na}^+\) flux decrease during a hyposmotic challenge or the net \(\hbox {Na}^+\) flux increase during a hyperosmotic challenge.     

Exosomal GAPDH from Proximal Tubule Cells Regulate ENaC Activity

Exosomes are nanometer-scale, cell-derived vesicles that contain various molecules including nucleic acids, proteins, and lipids. These vesicles can release their cargo into adjacent or distant cells and mediate intercellular communication and cellular function. Here we examined the regulation of epithelial sodium channels in mpkCCD cells and distal tubule Xenopus 2F3 cells by exosomes isolated from proximal tubule LLC-PK1 cells. Cultured mpkCCD cells were stained with CTX coupled to a green fluorophore in order to label the cell membranes and freshly isolated exosomes from LLC-PK1 cells were labeled with the red lipophilic dye PKH26 in order to visualize uptake of exosomes into the cells. Single-channel patch clamp recordings showed the open probability of ENaC in Xenopus 2F3 cells and in freshly isolated split-open tubules decreased in response to exogenous application of exosomes derived from LLC-PK1 proximal tubule cells. Active GAPDH was identified within exosomes derived from proximal tubule LLC-PK1 cells. The effect on ENaC activity in Xenopus 2F3 cells was blunted after application of exosomes transfected with the GAPDH inhibitor heptelidic acid. Also, we show GAPDH and ENaC subunits associate in mpkCCD cells. These studies examine a potential role for exosomes in the regulation of ENaC activity and examine a possible mechanism for communication from proximal tubule cells to distal tubule and collecting duct cells.     

Kidney Proximal Tubule Cells Originate from Approximately Four Progenitor Cells and Make Distinct Patches in Mouse Aggregation Chimeras

Giant lysosomal granules of bg^J/bg^J mutant mice were used as a marker to investigate the histological composition of kidney proximal tubule cells. Embryos of the bg^J/bg^J genotype and those of the +/+ genotype were aggregated, and lysosomes of their proximal tubule cells were histochemically stained with the β-glucuronidase activity. Tubules composed of bg^J/bg^J -type epithelial cells alone, tubules composed of +/+-type epithelial cells alone and tubules containing both types of epithelial cells were observed in cross sections. When the long straight portion of proximal tubules was reconstructed from serial sections, most of the tubules were of the mixed type, and distinct patches of bg^J/bg^J -type or +/+-type epithelial cells were detectable. These patches appeared to extend to the longitudinal rather than the circumferential direction. This suggests the clonal proliferation followed the mixing of embryonic progenitor cells for proximal tubule cells. Estimation from proportions of bg^J/bg^J -type proximal tubules in total examined proximal tubules gave a pool size of approximately four primordial precursor cells for proximal tubules in both right and left kindeys. The fact that the proportion of bg^J/bg^J -type components was comparable between the right and left kidneys of each chimera suggests that all proximal tubule cells in both right and left kidneys may originate from common primordial precursor cells.     

Isolation of Cell Surface Membranes from Cultured C6 Glioblastoma Cells

Abstract: Plasma membranes were isolated from C6 glioblastoma cells by two methods. In the first method cells were treated with concanavalin A and lysed in hypotonic medium. After partial separation of plasma membranes from other cell material, the lectin was displaced with a-methyl-D-mannoside. In the second method untreated cells or cells iodinated in a lactoperoxidase-catalyzed reaction were homogenized in isotonic medium. Membrane fractions obtaincd by either homogenization procedure were further purified by rate zonal and equilibrium centrifugations into linear density gradients. Disruption of the glioblastoma cell membrane gives rise to heterogeneous assemblies of mem- brane fragments. Two populations of plasma membranes were isolated from untreated and from iodinated cells: a "lighter")membrane fraction characterized by relatively lower sedimentation velocity and buoyant density, and a "heavier" membrane fraction of relatively faster sedimentation velocity and higher buoyant density. Both fractions showed electrophoretic patterns similar to those of ¹²⁵I-labeled cell surface proteins. Their specific (Na⁺+ K⁺)-ATPase activity was seven- to eightfold the homogenate activity (recovery, 13.1%). Both fractions were, however, still contaminated by smooth endo- plasmic reticulum, as judged from the activity 0: NADPH-dependent cytochrome c reductase (recovery, 2.4%). It is suggested that plasma membrane fragments present in the two fractions might differ in the organization of their structures, e.g., membrane vesicle intactness and membrane orientation.     

Pharmacological Properties and Physiological Function of a P2X-Like Current in Single Proximal Tubule Cells Isolated from Frog Kidney

Although previous studies have provided evidence for the expression of P2X receptors in renal proximal tubule, only one cell line study has provided functional evidence. The current study investigated the pharmacological properties and physiological role of native P2X-like currents in single frog proximal tubule cells using the whole-cell patch-clamp technique. Extracellular ATP activated a cation conductance (P2X(f)) that was also Ca2+-permeable. The agonist sequence for activation was ATP = αβ-MeATP > BzATP = 2-MeSATP, and P2X(f) was inhibited by suramin, PPADS and TNP-ATP. Activation of P2X(f) attenuated the rundown of a quinidine-sensitive K+ conductance, suggesting that P2X(f) plays a role in K+ channel regulation. In addition, ATP/ADP apyrase and inhibitors of P2X(f) inhibited regulatory volume decrease (RVD). These data are consistent with the presence of a P2X receptor that plays a role in the regulation of cell volume and K+ channels in frog renal proximal tubule cells.     

Insulin-induced Stimulation of Na+,K+-ATPase Activity in Kidney Proximal Tubule Cells Depends on Phosphorylation of the α-Subunit at Tyr-10

Phosphorylation of the α-subunit of Na⁺,K⁺-ATPase plays an important role in the regulation of this pump. Recent studies suggest that insulin, known to increase solute and fluid reabsorption in mammalian proximal convoluted tubule (PCT), is stimulating Na⁺,K⁺-ATPase activity through the tyrosine phosphorylation process. This study was therefore undertaken to evaluate the role of tyrosine phosphorylation of the Na⁺,K⁺-ATPase α-subunit in the action of insulin. In rat PCT, insulin and orthovanadate (a tyrosine phosphatase inhibitor) increased tyrosine phosphorylation level of the α-subunit more than twofold. Their effects were not additive, suggesting a common mechanism of action. Insulin-induced tyrosine phosphorylation was prevented by genistein, a tyrosine kinase inhibitor. The site of tyrosine phosphorylation was identified on Tyr-10 by controlled trypsinolysis in rat PCTs and by site-directed mutagenesis in opossum kidney cells transfected with rat α-subunit. The functional relevance of Tyr-10 phosphorylation was assessed by 1) the abolition of insulin-induced stimulation of the ouabain-sensitive ⁸⁶Rb uptake in opossum kidney cells expressing mutant rat α1-subunits wherein tyrosine was replaced by alanine or glutamine; and 2) the similarity of the time course and dose dependency of the insulin-induced increase in ouabain-sensitive ⁸⁶Rb uptake and tyrosine phosphorylation. These findings indicate that phosphorylation of the Na⁺,K⁺-ATPase α-subunit at Tyr-10 likely participates in the physiological control of sodium reabsorption in PCT.     

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.     

肾脏干细胞

肾脏千细胞是成体干细胞研究中最晚最新的,目前肾脏干细胞的研究也限定在发育中的胚胎肾,对胚胎肾干细胞的来源和定位的研究处于探索起步阶段。大量研究证明,胚性肾脏干细胞来源于输尿管芽诱导后的后肾间充质。胚性肾发育中主要的基因和转录因子可提供鉴定肾脏干细胞的标志分子。胚胎肾干细胞发育时,渗透压、氧压等多种因素组成的微环境提示髓质部可能是成体干细胞存在区域,肾乳头部为千细胞的壁龛。实验证明肾外组织对成体肾有修补作用。通过对胚胎肾发育和成体肾修复的细胞和分子机制进一步了解肾脏干细胞。     

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.     

Activity of Hydra Cells in vitro and in Regenerating Cell Reaggregates

Hydra were disaggregated into single cells and maintained ina complex medium for a few days. The cells are capable of macromolecularsynthesis, of processing RNA, and of carrying out the formationof a nematocyst capsule. Single cells can be centrifuged intoaggregates which will develop into normal hydra. Changes inmorphology and cell composition are described. Interstitialcells after a 1-day delay resume their normal mitotic and differentiationactivity in the aggregates. The method provides new means forstudying problems of cell differentiation in hydra.     

Water Permeability of Brush Border Membrane Vesicles from Kidney Proximal Tubule

Brush border membrane vesicles (BBMV) maintain an initial hydrostatic pressure difference between the intra- and extravesicular medium, which causes membrane strain and surface area expansion (Soveral, Macey & Moura, 1997). This has not been taken into account in prior osmotic water permeability P _f evaluations. In this paper, we find further evidence for the pressure in the variation of stopped-flow light scattering traces with different vesicle preparations. Response to osmotic shock is used to estimate water permeability in BBMV prepared with buffers of different osmolarities (18 and 85 mosM). Data analysis includes the dissipation of both osmotic and hydrostatic pressure gradients. P _f values were of the order of 4 × 10⁻³ cm sec⁻¹ independent of the osmolarity of the preparation buffer. Arrhenius plots of P _f vs. 1/T were linear, showing a single activation energy of 4.6 kcal mol⁻¹. The initial osmotic response which is significantly retarded is correlated with the period of elevated hydrostatic pressure. We interpret this as an inhibition of P _f caused by membrane strain and suggest how this inhibition may play a role in cell volume regulation in the proximal tubule. Received: 8 August 1996/Revised: 4 March 1997     

Golgi Tubule Traffic and the Effects of Brefeldin A Visualized in Living Cells

The Golgi complex is a dynamic organelle engaged in both secretory and retrograde membrane traffic. Here, we use green fluorescent protein–Golgi protein chimeras to study Golgi morphology in vivo. In untreated cells, membrane tubules were a ubiquitous, prominent feature of the Golgi complex, serving both to interconnect adjacent Golgi elements and to carry membrane outward along microtubules after detaching from stable Golgi structures. Brefeldin A treatment, which reversibly disassembles the Golgi complex, accentuated tubule formation without tubule detachment. A tubule network extending throughout the cytoplasm was quickly generated and persisted for 5–10 min until rapidly emptying Golgi contents into the ER within 15–30 s. Both lipid and protein emptied from the Golgi at similar rapid rates, leaving no Golgi structure behind, indicating that Golgi membranes do not simply mix but are absorbed into the ER in BFA-treated cells. The directionality of redistribution implied Golgi membranes are at a higher free energy state than ER membranes. Analysis of its kinetics suggested a mechanism that is analogous to wetting or adsorptive phenomena in which a tension-driven membrane flow supplements diffusive transfer of Golgi membrane into the ER. Such nonselective, flow-assisted transport of Golgi membranes into ER suggests that mechanisms that regulate retrograde tubule formation and detachment from the Golgi complex are integral to the existence and maintenance of this organelle.     

Sertoli Cells Maintain Leydig Cell Number and Peritubular Myoid Cell Activity in the Adult Mouse Testis

The Sertoli cells are critical regulators of testis differentiation and development. In the adult, however, their known function is restricted largely to maintenance of spermatogenesis. To determine whether the Sertoli cells regulate other aspects of adult testis biology we have used a novel transgenic mouse model in which Amh-Cre induces expression of the receptor for Diphtheria toxin (iDTR) specifically within Sertoli cells. This causes controlled, cell-specific and acute ablation of the Sertoli cell population in the adult animal following Diphtheria toxin injection. Results show that Sertoli cell ablation leads to rapid loss of all germ cell populations. In addition, adult Leydig cell numbers decline by 75% with the remaining cells concentrated around the rete and in the sub-capsular region. In the absence of Sertoli cells, peritubular myoid cell activity is reduced but the cells retain an ability to exclude immune cells from the seminiferous tubules. These data demonstrate that, in addition to support of spermatogenesis, Sertoli cells are required in the adult testis both for retention of the normal adult Leydig cell population and for support of normal peritubular myoid cell function. This has implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health.     

Mapping Goblet Cells in Mucous Membranes

A method is described for determining the number of goblet cells of the villi and crypts of Lieberkuhn in the small intestine with an accuracy far exceeding that which appears to be possible by counting on tissue sections. In groups of intact villi or crypts, previously isolated by microdissection, the goblet cells are stained, with as little staining as possible of the other tissue elements; thereafter the preparations are made transparent by embedding in a medium possessing a refractive index similar to that of the tissue. The staining is performed by the McManus-Hotchkiss periodic-leucofuchsin method (1948) with the modification that SchifPs reagent is diluted with 3 parts of water, the staining period cut down to 2V4-3 minutes, and the rinsing with bisulfite solution to 4-6 minutes. The embedding medium consists of colophonium and quinine hydrochloride in anise oil (Aurell, 1938). By this procedure, all the stained cells of the preparation may be visualized by manipulating the fine adjustment of the microscope. Counting of the goblet cells of the villi may be performed with great accuracy by projecting the picture of the preparation from the microscope on sectional paper and placing dots in the positions of the stained cells. The degree of magnification is determined by a corresponding projection of the scale of a micrometer disc.     

The Physics of Cell Membranes

An overview is given of the fundamental physics underlying the self-assembly, molecular organisation and electrical properties of the membranes that envelop living cells. These ultra thin (∼ 6 nm) membranes act as a diffusion barrier between the cell interior (cytoplasm) and the external medium. They consist basically of a bi-molecular film of lipid molecules in which are embedded functional proteins that perform a variety of functions, including energy transduction, signalling, transport of ions (and othermolecules), etc. Some examples are also presented of the fascinating and socially and commercially important applications of membrane biophysics. This revised version was published online in July 2006 with corrections to the Cover Date.