Proliferation capacity of the renal proximal tubule involves the bulk of differentiated epithelial cells

We investigated the proliferative capacity of renal proximal tubular cells in healthy rats. Previously, we observed that tubular cells originate from differentiated cells. We now found 1) by application of bromo-deoxyuridine (BrdU) for 14 days and costaining for BrdU, and the G(1)-phase marker cyclin D1 that the bulk of cells in the S3 segment of juvenile rats were involved in proliferation; 2) that although the proliferation rate was about 10-fold higher in juvenile rats compared with adult rats, roughly 40% of S3 cells were in G(1) in both groups; 3) that after a strong mitotic stimulus (lead acetate), proliferation was similar in juveniles and adults; 4) that there was a high incidence of cyclin D1-positive cells also in the healthy human kidney; and 5) by labeling dividing cells with BrdU for 2 days before the application of lead acetate and subsequent costaining for BrdU and cell cycle markers, that, although a strong mitotic stimulus does not abolish the period of quiescence following division, it shortens it markedly. Thus the capacity of the proximal tubule to rapidly recruit cells into division relies on a large reserve pool of cells in G(1) and on the shortening of the obligatory period of quiescence that follows division.     

Exit from the Mitotic Cycle in Root Meristems of Zea mays L.

The choice between two modes of exit from the mitotic cycleat the margins of meristems has been made easier by surveyingthe range of the numbers of cell contacts between contiguousfiles in root apices of Zea mays L. The range shows that somecells must go out of cycle while others remain in cycle forat least three further generations. The view that cycling endsby a fall in the proliferative fraction is supported by theexistence of pulse-labelled telophases in the proximal regionof the menstem. These are most likely due to acceleration ofthe mitotic cycle which has to be contrasted with decelerationof the overall rate of cell proliferation. The work is discussedin relation to patterns of cycling in the different tissuesof the apex. mitotic cycle, cell size, meristem, proliferative fraction, Zea mays L, maize     

Effect of head activator on proliferation, head-specific determination and differentiation of epithelial cells in hydra

In hydra the differentiation of head-specific ectodermal epithelial cells from multipotent stem cells is a multistep process in which cell cycle progression is regulated at three restriction points. Head activator acts as a positive signal at these restriction points. At the G2/mitosis boundary of epithelial stem cells head activator functions as a mitogen, being necessary for cell division. Subsequently, in or before S phase, head activator acts as determinant to ensure commitment of epithelial cells to head-specific determination. This effect of head activator requires hundredfold-higher concentrations, and may also require longer incubation times, than for cell proliferation. Epithelial cells thus committed to head-specific differentiation become arrested in G2 as a third and last restriction point in the cell cycle. They require disinhibition by decapitation and probably the presence of head activator for final differentiation, which then occurs in G2.     

Past stem cells and finally in transit: SLC1A3 instructs skin niche coupling

During postnatal growth of the skin and homeostatic hair cycling, different proliferative compartments contribute to tissue expansion and establishment of a functional epidermis. A new study by Reichenbach et al ( 2018 ) reports coordinated proliferation of three distinct epithelial stem cell niches and a role for glutamate transporter Slc1a3 in synchronisation of activated stem and progenitor cells.     

Cell cycle block in anergic T cells during tolerance induction

The induction of anergy, or T cell unresponsiveness to antigen, is preceded by T cell activation and cell division in response to fed antigens. These events parallel the activation observed in T cells following sensitization with antigen and adjuvant. The events that distinguish eventual sensitization versus tolerance remain unclear. Using a T lymphocyte transfer model specific to OVA, we demonstrated previously that oral encounter with antigen leads to functional anergy. Antigen-specific CD4+ T cells nevertheless become activated and cycle briefly after encounter with antigen. In this study, we measured the extent of cell cycling of antigen-specific T cells after oral encounter with their antigen. Whereas T cells cycle on the average of eight times in 4 days after conventional immunization, an abortive proliferation was observed in the draining LN T cells after oral encounter with antigen; OVA-specific T cells divided fewer times after exposure to fed OVA, compared to T cells in mice immunized with OVA. This abortive proliferation is antigen specific and not due to bystander suppression, as coadministration of an unrelated antigen that was previously used as a tolerogen does not alter the degree of abortive proliferation. Measurement of BrdU incorporation in mice that were previously fed ovalbumin indicates that up to 3 days following feeding, OVA-specific cells are actively cycling in vivo. However, by day 4, they have stopped cycling while identical T cells in OVA-sensitized mice continue to cycle. Our results indicate either that tolerance is a default pathway and a secondary stimulus is required at day 3 to progress to sensitization, or that elements that limit cell cycle progression are provided for tolerance induction.     

Redistribution of microvilli and membrane enzymes in isolated rat proximal tubule cells.

Isolated cells from the proximal tubule have previously been used to study specific renal physiological processes. Here we analysed the structural changes induced by mechanical isolation on rat proximal tubule cells. As major modifications we detected: 1) a redistribution of brush border microvilli, which spread out over all the cell surface immediately after isolation; 2) a rapid redistribution of the apical membrane enzyme markers aminopeptidase N and isomaltase; and 3) a striking decrease of membrane surface amplification factor. The results show that cells isolated from the proximal tubule rapidly loose their characteristic polarized membrane structure. Loss of epithelial polarity implies loss of several vectorial transports and should thus be cautiously taken into account when working with this model system.     

Dipeptide-induced Cl- secretion in proximal tubule cells

During a survey of dipeptides that might be transported by therenal PEPT2 transporter in proximal tubule cells, we discovered thatacidic dipeptides could stimulate transient secretory anion current andconductance increases in intact cell monolayers. The stimulatory effectof acidic dipeptides was observed in several proximal tubule cell linesthat have been recently developed by immortalization of early proximaltubule primary cultures from the Wistar-Kyoto and spontaneouslyhypertensive rat strains and humans, suggesting that this phenomenon isa characteristic of proximal tubule cells. The electrical currentinduced in intact monolayers by Ala-Asp, a representative of theseacidic dipeptides, must representCl secretion rather thanNa⁺ orH⁺ absorption, because1) it wasNa⁺ independent,2) it showed a pH dependencedifferent from that of the PEPT2 cotransporter, and3) it correlated with anAla-Asp-induced increase inCl conductance of theapical membrane in basolaterally amphotericin B-permeabilizedmonolayers. The secretory current could be inhibited by stilbenedisulfonates, but not diphenylamine-2-carboxylates, suggesting anon-cystic fibrosis transmembrane conductance regulator type ofCl conductance. The effectof Ala-Asp was dose dependent, with an apparent 50% effectiveconcentration of ~1 mM. Ala-Asp also produced intracellularacidification, suggesting that acidic dipeptides are also substratesfor an H⁺-peptide cotransporter.

     

Resident stem cells are not required for exercise-induced fiber-type switching and angiogenesis but are necessary for activity-dependent muscle growth

Skeletal muscle undergoes active remodeling in response to endurance exercise training, and the underlying mechanisms of this remodeling remain to be defined fully. We have recently obtained evidence that voluntary running induces cell cycle gene expression and cell proliferation in mouse plantaris muscles that undergo fast-to-slow fiber-type switching and angiogenesis after long-term exercise. To ascertain the functional role of cell proliferation in skeletal muscle adaptation, we performed in vivo 5-bromo-2'-deoxyuridine (BrdU) pulse labeling (a single intraperitoneal injection), which demonstrated a phasic increase (5- to 10-fold) in BrdU-positive cells in plantaris muscle between days 3 and 14 during 4 wk of voluntary running. Daily intraperitoneal injection of BrdU for 4 wk labeled 2.0% and 15.4% of the nuclei in plantaris muscle in sedentary and trained mice, respectively, and revealed the myogenic and angiogenic fates of the majority of proliferative cells. Ablation of resident stem cell activity by X-ray irradiation did not prevent voluntary running-induced increases of type IIa myofibers and CD31-positive endothelial cells but completely blocked the increase in muscle mass. These findings suggest that resident stem cell proliferation is not required for exercise-induced type IIb-to-IIa fiber-type switching and angiogenesis but is required for activity-dependent muscle growth. The origin of the angiogenic cells in this physiological exercise model remains to be determined. endurance exercise; adaptation     

Collective Cell Migration Drives Morphogenesis of the Kidney Nephron

Tissue organization in epithelial organs is achieved during development by the combined processes of cell differentiation and morphogenetic cell movements. In the kidney, the nephron is the functional organ unit. Each nephron is an epithelial tubule that is subdivided into discrete segments with specific transport functions. Little is known about how nephron segments are defined or how segments acquire their distinctive morphology and cell shape. Using live, in vivo cell imaging of the forming zebrafish pronephric nephron, we found that the migration of fully differentiated epithelial cells accounts for both the final position of nephron segment boundaries and the characteristic convolution of the proximal tubule. Pronephric cells maintain adherens junctions and polarized apical brush border membranes while they migrate collectively. Individual tubule cells exhibit basal membrane protrusions in the direction of movement and appear to establish transient, phosphorylated Focal Adhesion Kinase–positive adhesions to the basement membrane. Cell migration continued in the presence of camptothecin, indicating that cell division does not drive migration. Lengthening of the nephron was, however, accompanied by an increase in tubule cell number, specifically in the most distal, ret1-positive nephron segment. The initiation of cell migration coincided with the onset of fluid flow in the pronephros. Complete blockade of pronephric fluid flow prevented cell migration and proximal nephron convolution. Selective blockade of proximal, filtration-driven fluid flow shifted the position of tubule convolution distally and revealed a role for cilia-driven fluid flow in persistent migration of distal nephron cells. We conclude that nephron morphogenesis is driven by fluid flow–dependent, collective epithelial cell migration within the confines of the tubule basement membrane. Our results establish intimate links between nephron function, fluid flow, and morphogenesis.     

Morphological and functional characterization of isolated stem cells from the midgut of Chilo suppressalis larvae

The proliferation and differentiation of stem cell populations allow the midgut to grow/regenerate in lepidopteran insect. Basic epithelial regenerative functions can be assessed in vitro by purifying these stem and mature cell populations. Therefore, we isolated and purified stem and mature cells from the midgut of C. suppressalis larvae by density gradient centrifugation and observed the morphologies of these cells. A flow cytometry method was used to monitor C. suppressalis stem cell proliferation and differentiation under different cell culture conditions. We observed high proportions of the stem and differentiating cells in third- and fourth-instar larvae, respectively, indicating that, in larvae, stem cells rapidly proliferate early in development and are strongly differentiated at late stages. Incubation in medium supplemented with fat body extract and ecdysone resulted in a significantly increased proportion of stem cells, not of the differentiating cells, indicating that co-culture with fat body extract and ecdysone stimulates the proliferation of C. suppressalis stem cells. Viability bioassays showed that Cry1Ab displayed significant cytotoxic effects on the midgut cell culture of C. suppressalis. The proportion of differentiating cells was significantly increased after a 48-h exposure to sublethal doses of Cry1Ab toxin, and peaked at the Cry1Ab concentration of 0.3 μg/ml, demonstrating that epithelial cells with strong regenerative capacity via the differentiation of stem cells. These results improve our understanding of C. suppressalis stem cell biology and illustrate the potential role of the enhanced midgut regeneration induced by stem cell proliferation or differentiation as a reparation mechanism to Bt toxin.     

Cell Kinetic Studies Fail to Identify Sequentially Proliferating Progenitors as the Major Source of Epithelial Renewal in the Adult Murine Prostate

There is evidence that stem cells and their progeny play a role in the development of the prostate. Although stem cells are also considered to give rise to differentiated progeny in the adult prostate epithelium ex vivo, the cohort of adult prostate stem cells in vivo as well as the mechanisms by which the adult prostate epithelium is maintained and regenerated remain highly controversial. We have attempted to resolve this conundrum by performing in vivo tracing of serially replicating cells after the sequential administration of two thymidine analogues to mice. Our results show that, during normal prostate homeostasis, sequentially proliferating cells are detected at a rate that is consistent with a stochastic process. These findings indicate that in vivo, under steady-state conditions, most adult prostate epithelial cells do not represent the progeny of a small number of specialized progenitors that generate sequentially replicating transit-amplifying (TA) cells but are formed by stochastic cell division. Similarly, no rapidly cycling TA cells were detected during regeneration following one cycle of androgen-mediated involution/regeneration of the prostate epithelium. These findings greatly enhance our understanding of the mechanisms regulating prostate epithelial cell renewal and may have significant implications in defining the cell of origin of proliferative prostatic diseases.     

Aging and insulin signaling differentially control normal and tumorous germline stem cells

Aging influences stem cells, but the processes involved remain unclear. Insulin signaling, which controls cellular nutrient sensing and organismal aging, regulates the G2 phase of Drosophila female germ line stem cell (GSC) division cycle in response to diet; furthermore, this signaling pathway is attenuated with age. The role of insulin signaling in GSCs as organisms age, however, is also unclear. Here, we report that aging results in the accumulation of tumorous GSCs, accompanied by a decline in GSC number and proliferation rate. Intriguingly, GSC loss with age is hastened by either accelerating (through eliminating expression of Myt1, a cell cycle inhibitory regulator) or delaying (through mutation of insulin receptor (dinR) GSC division, implying that disrupted cell cycle progression and insulin signaling contribute to age‐dependent GSC loss. As flies age, DNA damage accumulates in GSCs, and the S phase of the GSC cell cycle is prolonged. In addition, GSC tumors (which escape the normal stem cell regulatory microenvironment, known as the niche) still respond to aging in a similar manner to normal GSCs, suggesting that niche signals are not required for GSCs to sense or respond to aging. Finally, we show that GSCs from mated and unmated females behave similarly, indicating that female GSC–male communication does not affect GSCs with age. Our results indicate the differential effects of aging and diet mediated by insulin signaling on the stem cell division cycle, highlight the complexity of the regulation of stem cell aging, and describe a link between ovarian cancer and aging.     

Selective depletion of mouse kidney proximal straight tubule cells causes acute kidney injury

The proximal straight tubule (S3 segment) of the kidney is highly susceptible to ischemia and toxic insults but has a remarkable capacity to repair its structure and function. In response to such injuries, complex processes take place to regenerate the epithelial cells of the S3 segment; however, the precise molecular mechanisms of this regeneration are still being investigated. By applying the ??toxin receptor mediated cell knockout?? method under the control of the S3 segment-specific promoter/enhancer, Gsl5, which drives core 2 ??-1,6-N-acetylglucosaminyltransferase gene expression, we established a transgenic mouse line expressing the human diphtheria toxin (DT) receptor only in the S3 segment. The administration of DT to these transgenic mice caused the selective ablation of S3 segment cells in a dose-dependent manner, and transgenic mice exhibited polyuria containing serum albumin and subsequently developed oliguria. An increase in the concentration of blood urea nitrogen was also observed, and the peak BUN levels occurred 3?C7?days after DT administration. Histological analysis revealed that the most severe injury occurred in the S3 segments of the proximal tubule, in which tubular cells were exfoliated into the tubular lumen. In addition, aquaporin 7, which is localized exclusively to the S3 segment, was diminished. These results indicate that this transgenic mouse can suffer acute kidney injury (AKI) caused by S3 segment-specific damage after DT administration. This transgenic line offers an excellent model to uncover the mechanisms of AKI and its rapid recovery.     

Growth of human renal cortical tissue on collagen gel

Summary A model system for 3-dimensional “native-state” culture of tissues on collagen gels (Proc. Natl. Acad. Sci. USA 86:2013–2017; 1989) has been applied in this study to histologically normal human renal cortical tissue from 11 patients undergoing nephrectomy for renal cell carcinoma elsewhere in the kidney. Microbial contamination occurred in 12/90 cultures, the rest (78) were studied by visual inspection, histology, immunohistochemical analysis for pankeratin (epithelial cell origin), vimentin (mesenchymal cell origin), andp-glycoprotein (associated with proximal tubules), transmission electron microscopy (EM), incorporation of tritiated thymidine (³HTdR). In the first 10 days, explants showed³HTdR-labeled cells in tubule structures. The surrounding gel was invaded by cells forming tubule structures, sometimes with basement membrane. Some of these cells showed labeling by³HTdR and immunostaining positive for pankeratin andp-glycoprotein. EM showed well-polarized epithelial cells in tubule structures with tight junctions, interdigitating lateral processes, and microvilli characteristic of proximal and distal convoluted tubules.³HTdR-labeled cells in tubule structures were observed even 2 mo. after Passage 1, 6 mo. after the initial explantation. Tubule growth was most active and fibroblast proliferation was negligible from 2 to 4 wk postexplantation. The proliferation of tubulelike cells and formation of tubulelike structures in this system represents an opportunity to study human renal cortical tissue in vitro, under conditions more closely resembling in vivo circumstances than are present in other in vitro systems suitable for long-term study. This model has potential use for in vitro toxicology studies and studies of renal physiology.     

Endogenous expression of the renal high-affinity H+-peptide cotransporter in LLC-PK1 cells

The reabsorption of filtered di- andtripeptides as well as certain peptide mimetics from the tubular lumeninto renal epithelial cells is mediated by anH⁺-coupledhigh-affinity transport process. Here we demonstrate for the first timeH⁺-coupled uptake of dipeptidesinto the renal proximal tubule cell lineLLC-PK₁. Transport was assessed1) by uptake studies using theradiolabeled dipeptideD-[³H]Phe-L-Ala,2) by cellular accumulation of the fluorescent dipeptide D-Ala-Lys-AMCA, and3) by measurement of intracellularpH (pH_i) changes as aconsequence of H⁺-coupleddipeptide transport. Uptake ofD-Phe-L-Alaincreased linearly over 11 days postconfluency and showed all thecharacteristics of the kidney cortex high-affinity peptide transporter,e.g., a pH optimum for transport ofD-Phe-L-Alaof 6.0, an apparent K_m value forinflux of 25.8 ± 3.6 µM, and affinities of differently chargeddipeptides or the -lactam antibiotic cefadroxil to the binding sitein the range of 20-80 µM.pH_i measurements established thepeptide transporter to induce pronounced intracellular acidification inLLC-PK₁ cells and confirm itspostulated role as a cellular acid loader.

     

The human NBCe1-A mutant R881C, associated with proximal renal tubular acidosis, retains function but is mistargeted in polarized renal epithelia

The human electrogenic renal Na-HCO₃ cotransporter (NBCe1-A; SLC4A4) is localized to the basolateral membrane of proximal tubule cells. Mutations in the SLC4A4 gene cause an autosomal recessive proximal renal tubular acidosis (pRTA), a disease characterized by impaired ability of the proximal tubule to reabsorb HCO₃^– from the glomerular filtrate. Other symptoms can include mental retardation and ocular abnormalities. Recently, a novel homozygous missense mutant (R881C) of NBCe1-A was reported from a patient with a severe pRTA phenotype. The mutant protein was described as having a lower than normal activity when expressed in Xenopus oocytes, despite having normal Na⁺ affinity. However, without trafficking data, it is impossible to determine the molecular basis for the phenotype. In the present study, we expressed wild-type NBCe1-A (WT) and mutant NBCe1-A (R881C), tagged at the COOH terminus with enhanced green fluorescent protein (EGFP). This approach permitted semiquantification of surface expression in individual Xenopus oocytes before assay by two-electrode voltage clamp or measurements of intracellular pH. These data show that the mutation reduces the surface expression rather than the activity of the individual protein molecules. Confocal microscopy on polarized mammalian epithelial kidney cells [Madin-Darby canine kidney (MDCK)I] expressing nontagged WT or R881C demonstrates that WT is expressed at the basolateral membrane of these cells, whereas R881C is retained in the endoplasmic reticulum. In summary, the pathophysiology of pRTA caused by the R881C mutation is likely due to a deficit of NBCe1-A at the proximal tubule basolateral membrane, rather than a defect in the transport activity of individual molecules. bicarbonate; intracellular pH; acidbase; SLC4A4; Na⁺-HCO₃ cotransporter 1