Model Of Proximal Tubule Cells Research Articles

Albumin Expands Albumin Reabsorption Capacity in Proximal Tubule Epithelial Cells through a Positive Feedback Loop between AKT and Megalin.

Renal proximal tubule cells (PTECs) act as urine gatekeepers, constantly and efficiently avoiding urinary protein waste through receptor-mediated endocytosis. Despite its importance, little is known about how this process is modulated in physiologic conditions. Data suggest that the phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) pathway regulates PTEC protein reabsorption. Here, we worked on the hypothesis that the physiologic albumin concentration and PI3K/AKT pathway form a positive feedback loop to expand endocytic capacity. Using LLC-PK1 cells, a model of PTECs, we showed that the PI3K/AKT pathway is required for megalin recycling and surface expression, affecting albumin uptake. Inhibition of this pathway stalls megalin at EEA1+ endosomes. Physiologic albumin concentration (0.01 mg/mL) activated AKT; this depends on megalin-mediated albumin endocytosis and requires previous activation of PI3K/mTORC2. This effect is correlated to the increase in albumin endocytosis, a phenomenon that we refer to as “albumin-induced albumin endocytosis”. Mice treated with L-lysine present decreased albumin endocytosis leading to proteinuria and albuminuria associated with inhibition of AKT activity. Renal cortex explants obtained from control mice treated with MK-2206 decreased albumin uptake and promoted megalin internalization. Our data highlight the mechanism behind the capacity of PTECs to adapt albumin reabsorption to physiologic fluctuations in its filtration, avoiding urinary excretion.

Therapeutic concentrations of calcineurin inhibitors do not deregulate glutathione redox balance in human renal proximal tubule cells.

The calcineurin inhibitors (CNI) cyclosporine A and tacrolimus comprise the basis of immunosuppressive regimes in all solid organ transplantation. However, long-term or high exposure to CNI leads to histological and functional renal damage (CNI-associated nephrotoxicity). In the kidney, proximal tubule cells are the only cells that metabolize CNI and these cells are believed to play a central role in the origin of the toxicity for this class of drugs, although the underlying mechanisms are not clear. Several studies have reported oxidative stress as an important mediator of CNI-associated nephrotoxicity in response to CNI exposure in different available proximal tubule cell models. However, former models often made use of supra-therapeutic levels of tissue drug exposure. In addition, they were not shown to express the relevant enzymes (e.g., CYP3A5) and transporters (e.g., P-glycoprotein) for the metabolism of CNI in human proximal tubule cells. Moreover, the used methods for detecting ROS were potentially prone to false positive results. In this study, we used a novel proximal tubule cell model established from human allograft biopsies that demonstrated functional expression of relevant enzymes and transporters for the disposition of CNI. We exposed these cells to CNI concentrations as found in tissue of stable solid organ transplant recipients with therapeutic blood concentrations. We measured the glutathione redox balance in this cell model by using organelle-targeted variants of roGFP2, a highly sensitive green fluorescent reporter protein that dynamically equilibrates with the glutathione redox couple through the action of endogenous glutaredoxins. Our findings provide evidence that CNI, at concentrations commonly found in allograft biopsies, do not alter the glutathione redox balance in mitochondria, peroxisomes, and the cytosol. However, at supra-therapeutic concentrations, cyclosporine A but not tacrolimus increases the ratio of oxidized/reduced glutathione in the mitochondria, suggestive of imbalances in the redox environment.

PKB is a central molecule in the modulation of Na+-ATPase activity by albumin in renal proximal tubule cells

Evidence points to a possible role of tubular sodium reabsorption in worsening renal injury. Proximal tubule (PT) albumin overload is a critical process in the development of tubule-interstitial injury (TII), and consequently in progression of renal disease. We studied the possible correlation between changes in albumin concentration in the lumen of PT with modification of Na+-ATPase activity. An albumin overload animal model and LLC-PK1 cells as a model of PT cells were used. Albumin overload was induced by intraperitoneal injection of BSA in 14-week-old male Wistar rats. An increase in sodium clearance, fractional excretion of sodium, proteinuria, ratio between urinary protein and creatinine, and albuminuria were observed. These observations indicate that there could be a correlation between an increase in albumin in the lumen of PTs and renal sodium excretion. We observed that the activity of both Na+-ATPase and (Na++K+)ATPase decreased in the renal cortex of an albumin overload animal model. Using LLC-PK1 cells as a model of PT cells, inhibition of Na+-ATPase activity was observed with higher albumin concentrations, similar to that observed in the animal model. The inhibition of protein kinase B by higher albumin concentration was found to be a critical step in the inhibition of Na+-ATPase activity. Interestingly, activation of the ERK1/2/mTORC1/S6K pathway was required for protein kinase B inhibition. This mechanism leads to a decrease in protein kinase C activity and, consequently to inhibition of Na+-ATPase activity. Taken together, our results indicate that the molecular mechanism underlying the modulation of PT Na+-ATPase activity by albumin overload involves activation of the ERK1/2/mTORC1/S6K pathway, which leads to inhibition of the mTORC2/PKB/PKC pathway. Our findings contribute to better understanding regarding handing of renal Na+ induced by albumin overload in the lumen of PTs and, consequently, in the progression of renal disease.

High glucose reduces megalin-mediated albumin endocytosis in renal proximal tubule cells through protein kinase B O-GlcNAcylation

The role of albumin reabsorption in proximal tubule (PT) cells has emerged as an important factor in the genesis of albuminuria observed in the early stages of diabetes. Evidence has shown that a decrease in megalin expression could be the key mechanism in this process. In the present work, we investigated the molecular mechanism underlying the modulation of albumin endocytosis in LLC-PK1 cells, a model of PT cells. High glucose concentrations (HG) inhibited megalin expression and albumin endocytosis after 48 h of incubation. This inhibitory effect involves the entrance of glucose into PT cells through SGLT located at the luminal membrane. Once inside PT cells, glucose is diverted to the hexosamine biosynthetic pathway (HBP) increasing O-GlcNAcylation of several intracellular proteins, including PKB. This process promotes the inhibition of PKB activity measured by its phosphorylation at Thr-308 and Ser-473 and phosphorylation of specific substrates, glycogen synthase kinase 3β (GSK3β) and tuberous sclerosis complex 2. The decrease in PKB activity led to a decrease in megalin expression and, consequently, reducing albumin endocytosis in LLC-PK1 cells. HG did not change mammalian target of rapamycin (mTOR) C2 activity, responsible for phosphorylated PKB at Ser-473. In addition, HG activated the mTORC1/S6K pathway, but this effect was not correlated to the decrease in megalin expression or albumin endocytosis. Taken together, our data help to clarify the current understanding underlying the genesis of tubular albuminuria induced by hyperglycemia in the early stage of diabetes pathogenesis.

Calcium-sensing receptor (CaSR) modulates vacuolar H+-ATPase activity in a cell model of proximal tubule.

The calcium-sensing receptor (CaSR) is localized in the apical membrane of proximal tubules in close proximity to the transporters responsible for proton secretion. Therefore, the aim of the present study was to analyze the effects of CaSR stimulation on the biochemical activity of the vacuolar H+-ATPase in a cellular model of proximal tubule cells, OKP cells. Biochemical activity of H+-ATPase was performed using cell homogenates, and the inorganic phosphate released was determined by a colorimetric method. Changes in cytosolic ionized calcium [Ca2+]i were also determined using Fluo-4. A significant increase of vacuolar H+-ATPase activity was observed when the CaSR was stimulated with agonists such as Gd3+ (300µM) and neomycin (200µM). This activity was also stimulated in a dose-dependent fashion by changes in extracellular Ca2+ (Ca2+o) between 10-4 and 2mM. Gd3+ and neomycin produced a sustained rise of [Ca2+]i, an effect that disappears when extracellular calcium was removed in the presence of 0.1µM thapsigargin. Inhibition of phospholipase C (PLC) activity with U73122 (5 × 10-8M) reduced the increase in [Ca2+]i induced by neomycin. CaSR stimulation induces an increase in the vacuolar H+-ATPase activity of OKP cells, an effect that involves an increase in [Ca2+]i and require phospholipase C activity. The consequent decrease in intratubular pH could lead to increase ionization of luminal calcium, potentially enhancing its reabsorption in distal tubule segments and reducing the formation of calcium phosphate stones.

The lipid 5-phoshatase SHIP2 controls renal brush border ultrastructure and function by regulating the activation of ERM proteins

The microvillus brush border on the renal proximal tubule epithelium allows the controlled reabsorption of solutes that are filtered through the glomerulus and thus participates in general body homeostasis. Here, using the lipid 5-phosphatase Ship2 global knockout mice, proximal tubule-specific Ship2 knockout mice, and a proximal tubule cell model in which SHIP2 is inactivated, we show that SHIP2 is a negative regulator of microvilli formation, thereby controlling solute reabsorption by the proximal tubule. We found increased PtdIns(4,5)P2 substrate and decreased PtdIns4P product when SHIP2 was inactivated, associated with hyperactivated ezrin/radixin/moesin proteins and increased Rho-GTP. Thus, inactivation of SHIP2 leads to increased microvilli formation and solute reabsorption by the renal proximal tubule. This may represent an innovative therapeutic target for renal Fanconi syndrome characterized by decreased reabsorption of solutes by this nephron segment.

Missense mutations and proximal RTA. Have we reached a new threshold? Focus on “Missense mutation T485S alters NBCe1-A electrogenicity causing proximal renal tubular acidosis”

THE REABSORPTION OF BICARBONATE by the kidney requires parallel operation of apical transporters that secrete H and corresponding basolateral transporters that absorb bicarbonate. Approximately 80% to 90% of the filtered bicarbonate is reabsorbed in the proximal tubule, where secretion of H into HCO3 -rich glomerular filtrate by the Na/H exchanger, NHE-3, results in the formation of H2CO3 that is rapidly converted, within the tubule lumen, by membrane-bound carbonic anhydrase (type IV), to H2O and CO2, which can freely diffuse into the proximal tubule and, via intracellular carbonic anhydrase (type II), is converted to intracellular H2CO3 and HCO3 . Bicarbonate exits the cell via the electrogenic Na-3HCO3 symporter (NBCe1-A), with the negative cell potential serving as the primary driving force. This process is summarized in the proximal tubule cell model displayed in Fig. 1. Renal tubular acidosis (RTA) is a rare group of disorders whether inherited or acquired, although the incidence and prevalence are not well defined. The phenotype, when fully expressed, is recognized clinically as a chronic non-gap metabolic acidosis in the face of evidence of an inability of the kidney to excrete net acid appropriately, and is often associated with abnor

Generalized Epithelial Transport Modeling: Computational Physiology

We present an epithelial modeling tool facilitating the development and validation of models of individual transport processes and whole cell transport. Making use of established standards in computational physiology, our tool enables the user to assemble epithelial cell models from a library of reusable transport process descriptions, starting from either a blank canvas or pre‐existing templates and models from the library. We are developing a library of reusable renal epithelial transport models, ranging from models of individual transporters to whole cell models. Our tool makes use of biological information associated with these models, such as location within a cell membrane or identification of the ions being transported, to guide the user in the assembly of new models. This, combined with other associated information in our library, enables the user to determine a suitable starting point for their work. Such as adding a novel model of the SGLT2 to an existing proximal tubule cell model; or a generic epithelial cell ready for specific transporter models. We additionally associate experimental protocols with the mathematical models, which allow the user to compare their work against existing data. Users are able to contribute new models, protocols, and data to the library for easy dissemination to, and reuse by, the community. Supported by the Virtual Physiological Rat Project, NIH grant [P50‐GM094503].

Shank2 contributes to the apical retention and intracellular redistribution of NaPiIIa in OK cells

In renal proximal tubule (PT) cells, sodium-phosphate cotransporter IIa (NaPiIIa) is normally concentrated within the apical membrane where it reabsorbs ∼70% of luminal phosphate (Pi). NaPiIIa activity is acutely regulated by moderating its abundance within the apical membrane. Under low-Pi conditions, NaPiIIa is retained within the apical membrane. Under high-Pi conditions, NaPiIIa is retrieved from the apical membrane and trafficked to the lysosomes for degradation. The present study investigates the role of Shank2 in regulating the distribution of NaPiIIa. In opossum kidney cells, a PT cell model, knockdown of Shank2 in cells maintained in low-Pi media resulted in a marked decrease in NaPiIIa abundance. After being transferred into high-Pi media, live-cell imaging showed that mRFP-Shank2E and GFP-NaPiIIa underwent endocytosis and trafficked together through the subapical domain. Fluorescence cross-correlation spectroscopy demonstrated that GFP-NaPiIIa and mRFP-Shank2 have indistinguishable diffusion coefficients and migrated through the subapical domain in temporal synchrony. Raster image cross-correlation spectroscopy demonstrated these two proteins course through the subapical domain in temporal-spatial synchrony. In the microvilli of cells under low-Pi conditions and in the subapical domain of cells under high-Pi conditions, fluorescence lifetime imaging microscopy-Forster resonance energy transfer analysis of Cer-NaPiIIa and EYFP-Shank2E found these fluors reside within 10 nm of each other. Demonstrating a complexity of functions, in cells maintained under low-Pi conditions, Shank2 plays an essential role in the apical retention of NaPiIIa while under high-Pi conditions Shank2 remains associated with NaPiIIa and escorts NaPiIIa through the cell interior.

Does Albumin Prime the Pump?

Albumin modulates the abundance and activity of renal Na,K-ATPase.

The Parathyroid Hormone 1 Receptor Directly Binds to the FERM Domain of Ezrin, an Interaction that Supports Apical Receptor Localization and Signaling in LLC-PK1 Cells

PTH 1 receptor (PTH1R) regulates mineral ion homeostasis. Both apical and basolateral PTH1R subpopulations exist within the renal proximal tubule. The purpose of this research was to examine determinants within the PTH1R that direct apical localization. When expressed in LLC-PK1 cells, a proximal tubule cell model, the PTH1R localizes to both apical and basolateral membranes. The C terminus of the PTH1R contains a psd-95, discs large, ZO-1 domain interaction motif that binds the sodium-hydrogen exchanger regulatory factor 1 (NHERF-1), a renal tubule scaffold protein. Receptors lacking the psd-95, discs large, ZO-1 domain interaction motif (PTH1R-CDelta4) partly localize to apical membranes, suggesting that additional factors may be involved. Ezrin, a membrane-cytoskeleton linking protein, directly binds NHERF-1 and thus links assembled complexes to actin. In vitro, subdomain C of the ezrin band 4.1, ezrin, radixin, domain interacts with the C-terminal tail of the PTH1R on a site that is mutually exclusive from the NHERF-1 interaction domain, suggesting the presence of a ternary complex. Mutating the lysine-arginine-lysine motif within the juxtamembrane region of the PTH1R C-terminal tail to alanines markedly disrupts interactions with the band 4.1, ezrin, radixin, domain of ezrin both in vitro and within cells. Inclusion of these mutations in the context of the full-length PTH1R disrupts apical localization with no effect on basolateral expression. Expression of a dominant-negative ezrin selectively disrupts apical expression and signaling of the PTH1R. However, dominant-negative ezrin does not affect expression or signaling of the basolateral PTH1R subpopulation. These findings reveal that direct ezrin interactions promote PTH1R apical localization and signaling in LLC-PK1 cells.

Development of an AT2-deficient proximal tubule cell line for transport studies.

Angiotensin II is a major regulatory peptide for proximal tubule Na(+) reabsorption acting through two distinct receptor subtypes: AT(1) and AT(2). Physiological or pathological roles of AT(2) have been difficult to unravel because angiotensin II can affect Na(+) transport either directly via AT(2) on luminal or peritubular plasma membranes of proximal tubule cells or indirectly via the renal vasculature. Furthermore, separate systemic and intratubular renin-angiotensin systems impart considerable complexity to angiotensin's regulation. A transport-competent, proximal tubule cell model that lacks AT(2) is a potentially useful tool to assess cellular angiotensin II regulation. To this end, AT(2)-receptor-deficient mice were bred with an Immortomouse, which harbors the thermolabile immortalization gene SV40 large-T antigen (Tag), and AT(2)-receptor-deficient [AT(2) (-/-)], Tag heterozygous [Tag (+/-)] F(2) offspring were selected for cell line generation. S1 proximal tubule segments were microdissected, and epithelial cell outgrowth was expanded in culture. Cells that formed confluent, electrically resistive monolayers were selected for cryopreservation, and one isolate was extensively characterized for conductance (2 mS/cm(2)), short-circuit current (Isc; 0.2 microA/cm(2)), and proximal tubule-specific Na3(+) - succinate (DeltaIsc = 0.8 microA/cm(2) at 2 mM succinate) and Na3(+) - phosphate cotransport (DeltaIsc = 3 microA/cm(2) at 1 mM phosphate). Light microscopy showed a uniform, cobblestone-shaped monolayer with prominent cilia and brush borders. AT(2) receptor functionality, as demonstrated by angiotensin II inhibition of ANF-stimulated cGMP synthesis, was absent in AT(2)-deficient cells but prominent in wild-type cells. This transport competent cell line in conjunction with corresponding wild type and AT(1)-deficient lines should help explain angiotensin II signaling relevant to Na(+) transport.

Albumin endocytosis in proximal tubule cells is modulated by angiotensin II through an AT 2 receptor-mediated protein kinase B activation

Albumin endocytosis in renal proximal tubule cells is a clathrin- and receptor-mediated mechanism that, in several pathophysiological conditions, is involved in initiating or promoting tubule-interstitial disease. Although much work has been done on this pathway, the regulation of albumin endocytosis in proximal tubule cells is not well understood. Here, we study the modulation by angiotensin II (Ang II) of albumin endocytosis in LLC-PK1, a model of proximal tubule cells. We observed that Ang II increases albumin endocytosis by approximately 100% at 10(-9) M. This effect is completely reversed by 10(-9) M PD123319, a specific AT(2) receptor antagonist, but not by losartan, a specific AT(1) receptor antagonist, at concentrations up to 10(-7) M. The Ang II effect on albumin endocytosis is also reversed by: phosphoinositide 3-kinase inhibitors LY294002 (2.5 x 10(-6) M) or wortmannin (10(-7) M), the protein kinase B inhibitor (2 x 10(-5) M), and staurosporine (2 x 10(-6) M), an inhibitor of 3'-phosphoinositide-dependent kinase 1. Ang II induced the selective phosphorylation of protein kinase B (PKB) at the Thr-308 residue without a change in Ser-473 phosphorylation, a combination that leads to an increase in PKB activity. These effects were completely abolished by 3 x 10(-6) M staurosporine or 10(-8) M PD123319. Our experiments also showed that PKB is present in the membrane fraction in overnight-starved LLC-PK1 cells. Taken together, these data show that Ang II increases albumin endocytosis through an AT(2) receptor mediated by activation of PKB in the plasma membrane, which depends on the basal activity of the phosphatidyl-inositol 3-kinase.

Polarized rabbit type 1 angiotensin II receptors manifest differential rates of endocytosis and recycling.

Receptor-mediated endocytosis and recycling have been described for extrarenal angiotensin II (ANG II) receptors. In proximal tubule (PT) epithelia expressing polarized ANG II receptors, these processes have not been examined as thoroughly. We utilized a PT cell model, LLC-PKCl4 cells stably transfected with rabbit type 1 ANG II receptor (AT1R) cDNA, to investigate these properties. LLC-PK-AT1R cells expressed the rabbit AT1R transcript and displayed losartan-inhibitable specific 125I-labeled ANG II binding at apical (AP) and basolateral (BL) membranes when grown on permeable supports. AP AT1R internalized 125I-ANG II more rapidly than BL AT1R, and phenylarsine oxide treatment inhibited AP AT1R internalization without significantly affecting BL AT1R endocytosis. Pertussis toxin had no effect on AP or BL AT1R endocytosis. In addition, AP AT1R recovered specific 125I-ANG II binding after ANG II treatment (a measure of recycling). BL AT1R displayed minimal recovery of 125I-ANG II binding after ANG II pretreatment. These data suggested that AP AT1R enter endocytic/endosomal pathways. Phospholipase A2 (PLA2) activity has been linked to endosomal fusion in other systems, and PT brush-border membrane AT1R also have been associated with PLA2 activity. LLC-PK-AT1R cells were therefore treated with quinacrine, a nonspecific PLA2 inhibitor, or Compound I (CI), a selective Ca(2+)-independent PLA2 inhibitor, to determine if PLA2 activity was linked to AT1R recycling. Both quinacrine and CI decreased AP AT1R recycling without affecting BL AT1R recycling. Polarized AT1R in LLC-PKCl4 cells thus display differential rates of endocytosis and recycling.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucocorticoid and acid-base homeostasis: effects on glutamine metabolism and transport.

The amino acid glutamine serves as a major fuel, powering tubular transport processes and base generation in the kidneys of acidotic animals. Removing the adrenals limits the kidneys' response to an exogenous acid load and impairs the removal rate of glutamine from the blood. Administering glucocorticoid to adrenalectomized (ADX) rats reverses a small net release of glutamine to uptake; similarly, administering an exogenous acid load to ADX rats restores the kidneys to organs of net extraction. Glucocorticoid-induced uptake is substantially less than the filtered glutamine load in contrast to uptake across both the luminal and basolateral surfaces when filtered HCO3- is reduced. Furthermore, mitochondrial glutamine oxidation is glucocorticoid dependent with a distinct impairment exhibited by the ADX acidotic rat kidney. However, combining the effect of reduced filtered HCO3- with glucocorticoid supplementation results in a marked potentiation of glutamine uptake coupled to oxidation. These results are consistent with a proposed proximal tubule cell model in which basolateral glutamine uptake is inversely related to basolateral HCO3- efflux and coupled, in the presence of glucocorticoid, to mitochondrial oxidation. This model of glucocorticoid-induced ammoniagenesis predicts an increased renal work capacity when basolateral transport of a major metabolic fuel, glutamine, is closely coupled to its oxidative metabolism.