Opossum Kidney Cells Research Articles

Sex-dependent impairment of kidney function in megalin knockout mice

Megalin ( Lrp2) is a multiligand receptor that drives endocytic flux in the kidney proximal tubule (PT) and is necessary for the recovery of albumin and other filtered proteins that escape the glomerular filtration barrier. We previously found that knocking out Lrp2 in the opossum kidney (OK) PT cell line leads to differential expression of genes involved in mitochondrial and metabolic function. Additionally, we observed large reductions in sodium glucose co-transporter 2 (Sglt2) transcript (>80%) and protein (~40%) expression levels. While Sglt2 transcript and protein levels in Lrp2 KO in mice were more modestly reduced, male and female KO mice fed regular chow or a Western diet (WD) had increased tolerance to a high glucose challenge compared with control mice. Surprisingly, male Lrp2 KO mice fed a WD developed substantial kidney injury, while female KO mice had significantly less injury. In metabolic studies, Lrp2 KO mice preferentially utilized fatty acid oxidation compared to control mice. We hypothesize that compromised endocytic flux in Lrp2 KO mice impairs mitochondrial and metabolic function to worsen kidney damage in a sex-dependent manner. Current studies are focused on a variety of approaches to test how reduced endocytic capacity impacts mitochondrial function and metabolism in Lrp2 KO cells and control OK cells. We will confirm our findings in age-matched male and female control and Lrp2 KO mice. These studies will help to determine the role of megalin expression and function on PT metabolism, function, and overall health. National Institutes of Health: T32DK061296, T32AG021885-20, RO1 DK125049, RO1 DK118726, S10 OD028596, U54 DK137329. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sglt2-independent effects of gliflozins on proximal tubule function

Background: Beyond glycemic control, Sglt2 inhibitors (Sglt2i) have protective effects on renal function. Sglt2i-mediated reductions in Na+ reabsorption have been suggested to protect PT cells from damage. The mechanisms responsible for renoprotection are unknown and have been suggested to involve inhibition of NHE3 leading to reduced ATP-dependent tubular workload and mitochondrial oxygen consumption. NHE3 activity is also important for regulation of endosomal pH, and the effects of Sglt2i on endocytic function are unknown. Aim of this study: to dissect the direct and immediate effects of SGLT2i on PT cell functions using opossum kidney (OK) PT cells, which represents a powerful physiologically-relevant system model. Methods: We tested the direct effects of Sglt2i on Na+-dependent fluid transport and endocytic uptake in a highly differentiated cell culture model that recapitulates the morphology, metabolism, and high endocytic and transport capacity of PT cells in vivo. The effect of gliflozins on albumin uptake and on fluid transport was quantified in OK PT cells. The effect of canagliflozin vs the NHE3 inhibitor S3226 on early endosome pH in OK cells was measured using fluorescence ratio imaging. Effects of gliflozins, NHE3 inhibitors, and AMPK pathway perturbants were determined by immunoblotting. 10 week-old male C57BL/6 mice were given canagliflozin or empagliflozin by oral gavage daily following vehicle gavage. Urine was collected via metabolic cages at baseline and after 24 h and 48h. Creatinine and albumin were measured by ELISA. Results: Canagliflozin but not empagliflozin reduced fluid transport across cell monolayers and inhibited endocytic uptake of albumin. These effects were independent of glucose and occurred at clinically relevant concentrations of drug. Canagliflozin did not affect endosomal pH, and albumin uptake was further reduced when added together with the selective NHE3 inhibitor S3226. Additionally, canagliflozin did not alter NHE3 phosphorylation when added alone, but prevented the reduction in phosphorylation caused by the selective NHE3 inhibitor S3226. Mice given a single dose of canagliflozin excreted twice as much urine over 24 h compared with empagliflozin-treated animals, suggesting that off-target effects of canagliflozin contribute significantly to reduced Na+-dependent fluid transport in vivo. Conclusions: Canagliflozin reduces fluid transport and albumin uptake in PT cells via effects on mitochondrial function upstream of the AMPK/mTOR axis. Sglt2i-mediated reductions in Na+-dependent fluid transport in the PT are secondary to reduced mitochondrial function and are not NHE3-specific. ASN Pre-doctoral fellowship Award, National Institutes of Health: R01 DK118726, R01 DK125049, S10 OD021627, Dialysis Clinic Inc. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Megalin-mediated siRNA uptake in kidney proximal tubule cells

Small interfering RNA (siRNA) is a clinically validated therapeutic modality, which silences gene expression via RNA interference (RNAi). Systemically delivered siRNA molecules are excreted primarily via the kidneys, where they accumulate in the renal cortex. Proximal epithelial cells within the tubule are thus an attractive target cell for utilizing RNAi, where the primary mode of entry is likely endocytic uptake. Here we report using a well-differentiated, opossum kidney (OK) proximal tubule cell (PTEC) line as a model system to mimic proximal tubule biology and investigate siRNA uptake. Fluorescently tagged siRNA were employed to observed uptake, which was observed in both a time and concentration dependent manner occurring predominantly from the apical surface of OK cells. Live cell imaging revealed significant colocalization of siRNAs with LysoTracker, consistent with endocytic uptake and delivery to lysosomes. We investigated the role of key PTEC solute recovery receptors, megalin and cubilin. siRNA uptake was inhibited by the megalin-binding protein RAP; and was independently reduced in Lrp2 knock-out (KO) cell lines. This work suggests that PTECs employ megalin to facilitate uptake of siRNA via endocytosis. Additionally, we observed colocalization of siRNA molecules and megalin in mouse kidneys from in vivo studies. Elucidating the mechanism(s) for siRNA uptake in the proximal tubule aids in the development of RNAi renal therapeutics and, more broadly, the understanding of class effects associated with siRNA nephrotoxicity. Additional work is focused on intracellular traffcking and megalin mediated siRNA uptake in mice. Project funded by Judo Bio. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Ip6k1 and Ip6k2 Kinases Are Critical for Normal Renal Tubular Function.

Kidneys are gatekeepers of systemic inorganic phosphate balance because they control urinary phosphate excretion. In yeast and plants, inositol hexakisphosphate kinases (IP6Ks) are central to regulate phosphate metabolism, whereas their role in mammalian phosphate homeostasis is mostly unknown. We demonstrate in a renal cell line and in mice that Ip6k1 and Ip6k2 are critical for normal expression and function of the major renal Na + /Pi transporters NaPi-IIa and NaPi-IIc. Moreover, Ip6k1/2-/- mice also show symptoms of more generalized kidney dysfunction. Thus, our results suggest that IP6Ks are essential for phosphate metabolism and proper kidney function in mammals. Inorganic phosphate is an essential mineral, and its plasma levels are tightly regulated. In mammals, kidneys are critical for maintaining phosphate homeostasis through mechanisms that ultimately regulate the expression of the Na + /Pi cotransporters NaPi-IIa and NaPi-IIc in proximal tubules. Inositol pyrophosphate 5-IP 7 , generated by IP6Ks, is a main regulator of phosphate metabolism in yeast and plants. IP6Ks are conserved in mammals, but their role in phosphate metabolism in vivo remains unexplored. We used in vitro (opossum kidney cells) and in vivo (renal tubular-specific Ip6k1/2-/- mice) models to analyze the role of IP6K1/2 in phosphate homeostasis in mammals. In both systems, Ip6k1 and Ip6k2 are responsible for synthesis of 5-IP 7 . Depletion of Ip6k1/2 in vitro reduced phosphate transport and mRNA expression of Na + /Pi cotransporters, and it blunts phosphate transport adaptation to changes in ambient phosphate. Renal ablation of both kinases in mice also downregulates the expression of NaPi-IIa and NaPi-IIc and lowered the uptake of phosphate into proximal renal brush border membranes. In addition, the absence of Ip6k1 and Ip6k2 reduced the plasma concentration of fibroblast growth factor 23 and increased bone resorption, despite of which homozygous males develop hypophosphatemia. Ip6k1/2-/- mice also show increased diuresis, albuminuria, and hypercalciuria, although the morphology of glomeruli and proximal brush border membrane seemed unaffected. Depletion of renal Ip6k1/2 in mice not only altered phosphate homeostasis but also dysregulated other kidney functions.

Receptor-associated protein impairs ligand binding to megalin and megalin-dependent endocytic flux in proximal tubule cells.

Proximal tubule (PT) cells retrieve albumin and a broad array of other ligands from the glomerular ultrafiltrate. Efficient uptake of albumin requires PT expression of both megalin and cubilin receptors. Although most proteins engage cubilin selectively, megalin is required to maintain robust flux through the apical endocytic pathway. Receptor-associated protein (RAP) is a chaperone that directs megalin to the cell surface, and recombinant RAP dramatically inhibits the uptake of numerous megalin and cubilin ligands. The mechanism by which this occurs has been suggested to involve competitive inhibition of ligand binding and/or conformational changes in megalin that prevent interaction with ligands and/or with cubilin. To discriminate between these possibilities, we determined the effect of RAP on endocytosis of albumin, which binds to cubilin and megalin receptors with high and low affinity, respectively. Uptake was quantified in opossum kidney (OK) cells and in megalin or cubilin (Cubn) knockout (KO) clones. Surprisingly, RAP inhibited fluid-phase uptake in addition to receptor-mediated uptake in OK cells and Cubn KO cells but had no effect on endocytosis when megalin was absent. The apparent Ki for RAP inhibition of albumin uptake was 10-fold higher in Cubn KO cells compared with parental OK cells. We conclude that in addition to its predicted high-affinity competition for ligand binding to megalin, the primary effect of RAP on PT cell endocytosis is to globally dampen megalin-dependent endocytic flux. Our data explain the complex effects of RAP on binding and uptake of filtered proteins and reveal a novel role in modulating endocytosis in PT cells.NEW & NOTEWORTHY Receptor-associated protein inhibits binding and uptake of all known endogenous ligands by megalin and cubilin receptors via unknown mechanism(s). Here, we took advantage of recently generated knockout cell lines to dissect the effect of this protein on megalin- and cubilin-mediated endocytosis. Our study reveals a novel role for receptor-associated protein in blocking megalin-stimulated endocytic uptake of fluid-phase markers and receptor-bound ligands in proximal tubule cells in addition to its direct effect on ligand binding to megalin receptors.

Ang II acutely stimulates Na,K-pump in cells from proximal tubules by increasing its phosphorylation at S938 via a PI3K/AKT pathway.

Angiotensin II (Ang II)-dependent stimulation of the AT1 receptor in proximal tubules increases sodium reabsorption and blood pressure. Reabsorption is driven by the Na,K-pump that is acutely stimulated by Ang II, which requires phosphorylation of serine-938 (S938). This site is present in humans and only known to phosphorylated by PKA. Yet, activation of AT1 decreases cAMP required to activate PKA and inhibiting PKA does not block Ang II-dependent phosphorylation of S938. We tested the hypothesis that Ang II-dependent activation is mediated via increased phosphorylation at S938 through a PI3K/AKT-dependent pathway. Experiments were conducted using opossum kidney cells, a proximal tubule cell line, stably co-expressing the AT1 receptor and either the wild-type (α-1.wild-type) or an alanine substituted (α-1.S938A) form of rat kidney Na,K-pump. A 5-min exposure to 10pM Ang II significantly activated Na,K-pump activity (56%) measured as short-circuit current across polarized α-1.wild-type cells. Wortmannin, at a concentration that selectively inhibits PI3K, blocked that Ang II-dependent activation. Ang II did not stimulate Na,K-pump activity in α-1.S938A cells. Ang II at 10 and 100 pM increased phosphorylation at S938 in α-1.wild-type cells measured in whole cell lysates. The increase was inhibited by wortmannin plus H-89, an inhibitor of PKA, not by either alone. Ang II activated AKT inhibited by wortmannin, not H-89. These data support our hypothesis and show that Ang II-dependent phosphorylation at S938 stimulates Na,K-pump activity and transcellular sodium transport.

An Adaptable Physiological Model of Endocytic Megalin Trafficking in Opossum Kidney Cells and Mouse Kidney Proximal Tubule.

The cells that comprise the proximal tubule (PT) are specialized for high-capacity apical endocytosis necessary to maintain a protein-free urine. Filtered proteins are reclaimed via receptor-mediated endocytosis facilitated by the multiligand receptors megalin and cubilin. Despite the importance of this pathway, we lack a detailed understanding of megalin trafficking kinetics and how they are regulated. Here, we utilized biochemical and quantitative imaging methods in a highly differentiated model of opossum kidney (OK) cells and in mouse kidney in vivo to develop mathematical models of megalin traffic. A preliminary model based on biochemically quantified kinetic parameters was refined by colocalization of megalin with individual apical endocytic compartment markers. Our model predicts that megalin is rapidly internalized, resulting in primarily intracellular distribution of the receptor at steady state. Moreover, our data show that early endosomes mature rapidly in PT cells and suggest that Rab11 is the primary mediator of apical recycling of megalin from maturing endocytic compartments. Apical recycling represents the rate-limiting component of endocytic traffic, suggesting that this step has the largest impact in determining the endocytic capacity of PT cells. Adaptation of our model to the S1 segment of mouse PT using colocalization data obtained in kidney sections confirms basic aspects of our model and suggests that our OK cell model largely recapitulates in vivo membrane trafficking kinetics. We provide a downloadable application that can be used to adapt our working parameters to further study how endocytic capacity of PT cells may be altered under normal and disease conditions.

SGLT2 Inhibitor Effects on Proximal Tubule Cell Function

Sodium glucose transporter inhibitors (SGLTis) have been shown to improve cardiac and kidney health outcomes in patients with type 2 diabetes mellitus as well as in nondiabetic individuals. Both renal and extrarenal targets of SGLTis have been implicated in these protective effects, and it is unclear whether SGLTi protection against kidney disease occurs via the same mechanisms under diabetic vs nondiabetic conditions. To begin to address these questions, we examined the effects of SGLTis on proximal tubule (PT) transport and endocytosis in a well‐differentiated opossum kidney (OK) cell model that recapitulates essential morphological, metabolic, and functional characteristics of the PT in vivo. Treatment with canagliflozin, empagliflozin, or tofogliflozin reduced endocytic uptake of albumin by these cells in a dose‐dependent manner. Similarly, treatment with SGLTis inhibited fluid transport across OK cell monolayers. SGLTis have been shown to impair NHE3 transport, and as predicted, inhibition of NHE3 had similar effects on endocytic uptake and fluid transport. We recently discovered that knockout (KO) of megalin in OK cells results in a dramatic reduction in SGLT2 transcription with no change in NHE3 expression. Megalin KO cells had considerably lower rates of fluid transport that were not further affected by SGLT2i treatment. Current studies are directed towards understanding the interplay between megalin, SGLTs, and NHE3 expression and activity at the plasma membrane and in endosomal compartments in the modulation of PT endocytic function and transport.

A‐Kinase anchoring protein 2 (AKAP2) and Pendrin; cross‐talk between cell signaling and renal physiology

Kidney tubules play a pivotal role in the maintenance of body fluid homeostasis and pH regulation. Accordingly, kidney disorders are known to be strongly associated with hypertension and acid‐base imbalance. In the kidney, the intercalated cells of the nephron are the main site of acid‐base balance: type A are acid‐secreting whereas type B are base‐secreting. The chloride bicarbonate exchanger Pendrin is present in type B intercalated cells and participates actively in regulating blood pressure and NaCl balance. It is still not entirely deciphered how Pendrin activity is regulated; nevertheless, it was previously demonstrated that Pendrin is regulated by c‐AMP/Protein Kinase A (PKA) signaling pathway. Consequently, in this study, our hypothesis is that the A‐Kinase anchoring protein 2 (AKAP2), strongly expressed in the kidney, regulates in time and space the intracellular signal transduction.In order to investigate AKAP2 involvement in Pendrin regulation and trafficking to the apical plasma membrane, we generated an inducible and nephron‐specific Akap2Pax8/LC1 mice knockout model. By confocal microscopy, fluorescent immunostaining on kidney tissue sections showed that AKAP2 is present in the tubules and most importantly colocalizes with Pendrin at the apical plasma membrane of the intercalated cells. To show that Pendrin and AKAP2 are interacting, we used the Proximity Ligation Assay (PLA) and, positively, we could reveal the association between the two proteins. The latter finding was also confirmed in vitro taking advantage of the Opossum Kidney Cell line (OKP) stably expressing Pendrin and co‐transfected with AKAP2, we could co‐immunoprecipitate Pendrin and AKAP2 after crosslinking on live cells. Fluorescent immunostaining in Akap2Pax8/LC1 show that Pendrin tends to be shifted from the apical membrane (seen in control mice) to intracellular compartments.In conclusion, our data suggest that AKAP2 and Pendrin are interacting, and AKAP2 may play a key role in Pendrin trafficking to the apical plasma membrane, hence in regulating its activity.

Inhibition of phosphate transport by NAD+/NADH brush border membrane vesicles.

Nicotinamide is an important regulator of Pi homeostasis after conversion into NAD+/NADH. In this work, we have studied the classical inhibition of Pi transport by these compounds in the brush border membrane vesicles (BBMV) of rat kidney and rat intestine, and we examined the effects in opossum kidney (OK) cells and in phosphate transporter-expressing Xenopus laevis oocytes. In BBMV, NAD+ required preincubation at either room temperature or on ice to inhibit Pi uptake in BBMV. However, no effects were observed in the known Slc34 or Slc20 Pi transporters expressed in Xenopus oocytes, in OK cells, or in isolated rat cortical nephron segments. In BBMV from jejunum or kidney cortex, the inhibition of Pi transport was specific, dose-related, and followed a competitive inhibition pattern, as shown by linear transformation and nonlinear regression analyses. A Ki value of 538 µM NAD+ in kidney BBMV was obtained. Ribosylation inhibitors and ribosylation assays revealed no evidence that this reaction was responsible for inhibiting Pi transport. An analysis of the persistence of NAD+/NADH revealed a half-life of just 2 min during preincubation. Out of several metabolites of NAD degradation, only ADP-ribose was able to inhibit Pi uptake. Pi concentration also increased during 30 min of preincubation, up to 0.67 mM, most likely as a metabolic end product. In conclusion, the classical inhibition of Pi transport by NAD+/NADH in BBMV seems to be caused by the degradation metabolites of these compounds during the preincubation time.

Inositol hexakisphosphate kinases 1 and 2 (IP6K1/2) are involved in renal control of phosphate homeostasis

Phosphate (Pi) is an indispensable component of living organisms. In the body, Pi homeostasis is tightly regulated by four main tissues: intestine, kidney, bone and parathyroid glands. There are multiple complex mechanisms underlying the regulation of serum Pi including hormones and transporters. Although plenty of studies have analysed Pi regulation, the mechanism with which Pi levels are sensed by mammalian cells remains elusive. Molecular mechanisms of Pi-sensing have been elucidated in detail in yeast and bacteria. However, most of the Pi-sensing proteins identified in yeast and bacteria are encoded by genes that are not conserved in mammals. As a major exception, inositol polyphosphates—which respond to changes in ambient Pi in yeast and, in turn, regulate the expression of Pi transporters—are also found in mammals. Here, we addressed the role of inositol polyphosphates and their biogenic enzymes, the IP6Ks (especially IP6K1 and 2), in cellular Pi sensing in mammals. Human and mouse kidneys express the IP6K1 and IP6K2 isoforms. Likewise, Opossum Kidney (OK) cells, a model for renal proximal tubules express IP6K1 and 2. Pharmacological inhibition of IP6K1/2 or CRISPR/Cas9 mediated knockout in OK cells downregulates Pi transporter mRNA, particularly NaPi-IIa/c. Ip6k1-/-2-/- KO cells also exhibited decreased Pi uptake (by 70%) and an abrogated adaptation to ambient Pi. To study the role of IP6K1 and 2 in vivo, we generated renal tubule-specific inducible ip6k1-/-2-/- KO mice: the mice had strongly reduced expression of renal Pi transporters, at the protein and mRNA levels, and lower serum Pi (by 20%) together with the transcriptional downregulation of many other transporters. Serum intact fibroblast growth factor 23—a principal phosphaturic hormone—was also substantially reduced (10-fold) in these mice. Our data indicate that the deletion of IP6K1 and 2 decreases transcription of renal Pi transporters both in vivo and in vitro, and that Ip6k1-/-2-/- cells are unable to adapt to ambient Pi. Therefore, IP6K1 and 2 play a critical role in renal Pi metabolism and, potentially, sensing.

Lack of ER Stress in NHERF1‐Deficient Proximal Tubule Cells Exhibiting Aberrant Npt2a Localization

Background NHERF1 (Na+-hydrogen exchanger regulatory factor isoform 1) anchors Npt2a (Na+-dependent phosphate cotransporter type IIa) in the brush border membrane (BBM) of proximal tubules through its cross-linking of Npt2a with the actin-filament binding protein, Ezrin. In the absence of NHERF1, Npt2a accumulates aberrantly in a subapical compartment. We have demonstrated that Npt2a associates with NHERF1 in the ER/Golgi as well as the BBM, suggesting a critical role for NHERF1 in the forward trafficking of Npt2a from the ER/Golgi to the BBM. Because increased levels of ER stress have been associated with intracellular accumulation of mis-localized proteins, we hypothesized that NHERF1 deficient proximal tubule cells may experience chronic ER stress associated with mis-localization of Npt2a. Methods We measured protein expression of four ER stress markers (phosphorylated eIF2α, GRP78, GRP94, and p58IPK) in kidney cortex isolated from C57BL/6J wild type (WT) mice and their NHERF1 knock-out (KO) littermates by immunoblot and mRNA levels by RNAseq. We expressed a fluorescently labeled WT Npt2a and NHERF1 or a Npt2a/NHERF1 chimera substituting the C terminal PDZ binding motif of Npt2a with the ERM (Ezrin-Radixin-Moesin) binding domain of NHERF1 in human embryonic kidney (HEK), opossum kidney (OK), and NHERF1-deficient opossum kidney (OKH) cells. We compared WT vs chimera protein expression by confocal microscopy and protein function by radiolabeled phosphate uptake. Results We found no differences in kidney cortex protein or mRNA expression of these ER stress markers between WT and KO animals. Confocal microscopy of the Npt2a chimera expressed in HEK, OK, and OKH cells exhibited enhanced apical membrane localization when compared to WT Npt2a in all three cell types. However, no increase in radiolabeled phosphate uptake was observed between cells expressing the Npt2a chimera or wild type Npt2a. Conclusions The lack of an ER stress response suggests that despite association between NHERF1 and Npt2a in the ER/Golgi, NHERF1 is not required for normal Npt2a folding. Enhanced apical expression of the Npt2a chimera confirms the essential role of Ezrin in BBM localization of Npt2a, however, appropriate Npt2a function requires the presence of more than the ERM binding domain of NHERF1, possibly involving the association of other unidentified proteins.

Characterization of Sodium‐Dependent Pi Transport Inhibition by NAD

Hyperphosphatemia during end-stage renal disease causes many adverse outcomes. To prevent them, several Pi intestinal absorption inhibitors are necessary for targeting either the paracellular (diffusion) or the transcellular (transport) absorption pathway. Nicotinamide adenine dinucleotide (NADH/NAD+) is a classical inhibitor of intestinal and renal Pi transport and is a circadian modulator of phosphatemia. The mechanism of NAD transporter inhibition remains unclear, although a competitive inhibition mechanism has been suggested. In this work, we sought to determine if this mechanism has a direct effect on Pi transporters. The five known rat Na/Pi cotransporters (NaPi2a, NaPi2b, NaPi2c, PiT1, and PiT2) were expressed in Xenopus laevis oocytes. Uptake of 32P-Pi was performed for three days post-injection of 5 ng of the corresponding cRNAs per oocyte and after 30 minutes of preincubation with 0.5 mM NAD, NADH, or NAM. None of the compounds affected the Pi uptake of any of the expressed Pi transporters in oocytes. Pi transport in the brush border membrane vesicles (BBMV) of either rat kidney cortex or jejunum epithelium was also assayed. As expected, it was significantly inhibited by NADH and NAD when BBMV were preincubated for 30 min at room or ice-cold temperature. NAM had no effect, and the effect was specific for Na-dependent Pi uptake (D-glucose uptake was not affected). Dose-response analyses on jejunum or kidney BBMV revealed IC50 values in the micromolar range for 50 µM of Pi uptake. Several Michaelis-Menten kinetics were performed in the presence of different concentrations of NAD, and a Lineweaver-Burk plot suggested changes in affinity (i.e., competitive inhibition), which contrasts with the lack of an effect on the transporters expressed in oocytes. The competitive model was confirmed by non-linear regression of a global fit with shared parameters, providing a Ki value of 538 µM NAD. When the effect of NAD was assayed in Opossum Kidney cells, a proximal tubule cell line model expressing a regulated NaPi2a, no evidence of Pi transport inhibition was observed. To check whether NAD-mediated ribosylation was involved in Pi transport inhibition, a classical ribosylation assay was performed with 32P-NAD, using jejunum and renal BBMV. No evidence of ribosylation was found for NaPi2b or NaPi2a, even after one month of exposure to an X-ray film. This was confirmed using several ribosylation inhibitors (meta-iodobenzylguanidine, novobiocin, vitamin K1, vitamin K3, or 3-methoxybenzamide), which failed to prevent NAD inhibition of Pi transport in BBMV. In conclusion, despite the kinetic findings in BBMV, NAD inhibition of Na/Pi-cotransporters is, most likely, not mediated through a direct interaction with these transporters. Instead, inhibition seems to be mediated through an indirect mechanism acting on components that are absent in Xenopus oocytes and in OK cells, which ends in the modification of Pi transport affinity (Km) and occurs at ice-cold temperature. Nevertheless, the unlikely inhibition of a novel, unknown Pi transporter cannot be completely discarded.

Transcriptional Programs Driving Shear Stress-Induced Differentiation of Kidney Proximal Tubule Cells in Culture.

Cultured cell models are an essential complement to dissecting kidney proximal tubule (PT) function in health and disease but do not fully recapitulate key features of this nephron segment. We recently determined that culture of opossum kidney (OK) cells under continuous orbital shear stress (OSS) significantly augments their morphological and functional resemblance to PTs in vivo. Here we used RNASeq to identify temporal transcriptional changes upon cell culture under static or shear stress conditions. Comparison of gene expression in cells cultured under static or OSS conditions with a database of rat nephron segment gene expression confirms that OK cells cultured under OSS are more similar to the PT in vivo compared with cells maintained under static conditions. Both improved oxygenation and mechanosensitive stimuli contribute to the enhanced differentiation in these cells, and we identified temporal changes in gene expression of known mechanosensitive targets. We observed changes in mRNA and protein levels of membrane trafficking components that may contribute to the enhanced endocytic capacity of cells cultured under OSS. Our data reveal pathways that may be critical for PT differentiation in vivo and validate the utility of this improved cell culture model as a tool to study PT function.

Transcriptomes of Major Proximal Tubule Cell Culture Models.

Cultured cell lines are widely used for research in the physiology, pathophysiology, toxicology, and pharmacology of the renal proximal tubule. The lines that are most appropriate for a given use depend upon the genes expressed. New tools for transcriptomic profiling using RNA sequencing (RNA-Seq) make it possible to catalog expressed genes in each cell line. Fourteen different proximal tubule cell lines, representing six species, were grown on permeable supports under conditions specific for the respective lines. RNA-Seq followed standard procedures. Transcripts expressed in cell lines variably matched transcripts selectively expressed in native proximal tubule. Opossum kidney (OK) cells displayed the highest percentage match (45% of proximal marker genes [TPM threshold =15]), with pig kidney cells (LLC-PK1) close behind (39%). Lower-percentage matches were seen for various human lines, including HK-2 (26%), and lines from rodent kidneys, such as NRK-52E (23%). Nominally, identical OK cells from different sources differed substantially in expression of proximal tubule markers. Mapping cell line transcriptomes to gene sets for various proximal tubule functions (sodium and water transport, protein transport, metabolic functions, endocrine functions) showed that different lines may be optimal for experimentally modeling each function. An online resource (https://esbl.nhlbi.nih.gov/JBrowse/KCT/) has been created to interrogate cell line transcriptome data. Proteomic analysis of NRK-52E cells confirmed low expression of many proximal tubule marker proteins. No cell line fully matched the transcriptome of native proximal tubule cells. However, some of the lines tested are suitable for the study of particular metabolic and transport processes seen in the proximal tubule.

PF-06869206 is a selective inhibitor of renal Pi transport: evidence from in vitro and in vivo studies.

Plasma phosphate (Pi) levels are tightly controlled, and elevated plasma Pi levels are associated with an increased risk of cardiovascular complications and death. Two renal transport proteins mediate the majority of Pi reabsorption: Na+-phosphate cotransporters Npt2a and Npt2c, with Npt2a accounting for 70-80% of Pi reabsorption. The aim of the present study was to determine the in vitro effects of a novel Npt2a inhibitor (PF-06869206) in opossum kidney (OK) cells as well as determine its selectivity in vivo in Npt2a knockout (Npt2a-/-) mice. In OK cells, Npt2a inhibitor caused dose-dependent reductions of Na+-dependent Pi uptake (IC50: ~1.4 μmol/L), whereas the unselective Npt2 inhibitor phosphonoformic acid (PFA) resulted in an ~20% stronger inhibition of Pi uptake. The dose-dependent inhibitory effects were present after 24 h of incubation with both low- and high-Pi media. Michaelis-Menten kinetics in OK cells identified an ~2.4-fold higher Km for Pi in response to Npt2a inhibition with no significant change in apparent Vmax. Higher parathyroid hormone concentrations decreased Pi uptake equivalent to the maximal inhibitory effect of Npt2a inhibitor. In vivo, the Npt2a inhibitor induced a dose-dependent increase in urinary Pi excretion in wild-type mice (ED50: ~23 mg/kg), which was completely absent in Npt2a-/- mice, alongside a lack of decrease in plasma Pi. Of note, the Npt2a inhibitor-induced dose-dependent increase in urinary Na+ excretion was still present in Npt2a-/- mice, a response possibly mediated by an off-target acute inhibitory effect of the Npt2a inhibitor on open probability of the epithelial Na+ channel in the cortical collecting duct.

In vitro effects of Npt2a inhibition in renal proximal tubule cells

Elevated plasma phosphate levels are associated with an increased risk of cardiovascular complications and death. The kidneys play an important role in maintaining plasma phosphate levels and patients with chronic kidney disease (CKD) develop hyperphosphatemia. Two renal transport proteins mediate phosphate reabsorption, the sodium‐phosphate cotransporters Npt2a and Npt2c, with Npt2a accounting for 70 to 80% of phosphate reabsorption and Npt2c for the remainder. We recently described that pharmacological inhibition of Npt2a by a selective Npt2a inhibitor (Npt2a‐I, PF‐06869206) induced phosphaturia and reduced plasma phosphate in a mouse models of normal and reduced kidney function (J Am Soc Nephrol. 2019;30(11):2128–2139). The aim of the current study was to determine the in vitro effects of this Npt2a‐I in opossum kidney (OK) cells, a cellular model of renal epithelia where Npt2a is endogenously expressed. 32P radiotracer was used to study the inhibitory effects of Npt2a‐I on phosphate transport. Npt2a‐I dose‐response (0.01 – 100 μM) studies were performed in the cells by incubating different concentrations of Npt2a‐I with 32P for 10 minutes. Npt2a inhibition caused a dose‐dependent decrease in phosphate transport (from 107±9 pmol/well/min in response to control (DMSO, 0.1%) to a maximum inhibitory effect of 33±1 pmol/well/min at 100 μM Npt2a‐I) showing a half maximal inhibitory concentration (IC50) of ~3 μM. Approximately 70% of phosphate transport in OK cells was inhibited by the highest dose of Npt2a‐I (100 μM); as comparison, the nonselective Npt2 inhibitor phosphonoformate (PFA, 30 mM) inhibited ~90% of phosphate transport. The kinetics of transport inhibition by Npt2a‐I were studied using phosphate concentrations varying from 50 – 400 μM with or without Npt2a‐I (3 μM). Npt2a‐I increased the Michaelis‐Menten constant, Km (506±124 vs 210±22 mM) for phosphate; in contrast, Vmax was unaffected (348±31 vs 300±54 pmol/min/well), indicating competitive inhibition. Preincubation of OK cells with 3 μM Npt2a‐I for 1, 2 or 3 hours caused a significant reduction in phosphate transport compared to control (29±1 vs 40±1, 28±1 vs 37±1 and 30±1 vs 37±1 pmol/min/mg protein, respectively); whereas 24 hour preincubation increased phosphate transport significantly (49±3 vs 39±2 pmol/min/mg protein). Twenty‐four hour of incubation with vehicle or Npt2a‐I (30 μM) did not show cytotoxic effects (5.1±0.9 and 4.7±0.3% dead cells). In summary, our studies demonstrate that in vitro Npt2a inhibition causes a dose‐dependent reduction in phosphate transport in OK cells without affecting their viability. Our kinetic data imply the compound acts as a competitive Npt2a inhibitor.Support or Funding InformationThis work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases grant 1R01DK110621 (to Dr. Rieg) and American Heart Association Transformational Research Award 19TPA34850116 (to Dr. Rieg). Dr. Thomas was supported by an American Heart Association postdoctoral fellowship (19POST34400026) and Mr. Xue by a predoctoral fellowship (18PRE33990236).

Loss of CLC‐5 reduces endocytic capacity and alters cholesterol distribution in the proximal tubule

Dent disease is a progressive X‐linked disorder affecting the kidney proximal tubule (PT) caused by loss of function of the Cl−/H+ exchanger, CLC‐5. Early symptoms include low molecular weight (LMW) proteinuria resulting from inefficient reabsorption of filtered proteins by the PT. However, the role of CLC‐5 in this process in unclear. Knockout of Clc‐5 in mice recapitulates the LMW proteinuria observed in human disease and causes decreased PT levels of megalin and cubilin, the receptors that bind to and recover filtered proteins. How loss of CLC‐5 leads to reduced receptor expression remains unknown. We used a previously developed opossum kidney (OK) cell culture model that recapitulates morphologic and functional features of the PT in vivo to study the role of Clc‐5 in the endocytic pathway. In Clc‐5 siRNA knockdown (KD) and CRISPR/Cas9 mediated Clc‐5 knockout (KO) cells, there was a significant decrease in endocytic capacity that was rescued by heterologous expression of wild‐type human CLC‐5. Additionally, we used CRISPR/Cas9 technology to generate Clc‐5 knockout mice and confirmed LMW proteinuria in the KO. Heterozygous females also have reduced PT albumin uptake and megalin expression. Previous gene expression studies in Clcn5 KO mice suggest there are alterations in cholesterol and lipid metabolic enzymes, and elevated cholesterol levels have been shown to alter recycling endosome organization and impair fast recycling. We hypothesize that loss of CLC‐5 disrupts cholesterol homeostasis, resulting in reduced recycling and concomitantly increased degradation of megalin. Consistent with this, we observed an accumulation and redistribution of cholesterol in Clc‐5 KD OK cells. We are currently working to quantify cholesterol in Clc‐5 KO OK cells. In female mice with only one functional copy of Clc‐5, we observed a dramatic a change in the distribution of cholesterol in the PT when compared to WT mice. Current studies are focused on quantifying the trafficking kinetics and distribution of megalin in the PT in the absence of CLC‐5 and determining whether altered cholesterol metabolism contributes to the reduced endocytic capacity of the PT and resulting LMW proteinuria in Dent disease.Support or Funding InformationNIH F31 DK121394, R01 DK101484, R01 DK100357, P30 DK079307, and ASN Pre‐Doctoral Fellowship

Distinct functions of megalin and cubilin receptors in recovery of normal and nephrotic levels of filtered albumin.

Proximal tubule (PT) cells express a single saturable albumin-binding site whose affinity matches the estimated tubular concentration of albumin; however, albumin uptake capacity is greatly increased under nephrotic conditions. Deciphering the individual contributions of megalin and cubilin to the uptake of normal and nephrotic levels of albumin is impossible in vivo, as knockout of megalin in mice globally disrupts PT endocytic uptake. We quantified concentration-dependent albumin uptake in an optimized opossum kidney cell culture model and fit the kinetic profiles to identify albumin-binding affinities and uptake capacities. Mathematical deconvolution fit best to a three-component model that included saturable high- and low-affinity uptake sites for albumin and underlying nonsaturable uptake consistent with passive uptake of albumin in the fluid phase. Knockdown of cubilin or its chaperone amnionless selectively reduced the binding capacity of the high-affinity site, whereas knockdown of megalin impacted the low-affinity site. Knockdown of disabled-2 decreased the capacities of both binding sites. Additionally, knockdown of megalin or disabled-2 profoundly inhibited the uptake of a fluid phase marker, with cubilin knockdown having a more modest effect. We propose a novel model for albumin retrieval along the PT in which cubilin and megalin receptors have different functions in recovering filtered albumin in proximal tubule cells. Cubilin binding to albumin is tuned to capture normally filtered levels of the protein. In contrast, megalin binding to albumin is of lower affinity, and its expression is also essential for enabling the recovery of high concentrations of albumin in the fluid phase.

Estrogen receptor α (ERα) indirectly induces transcription of human renal organic anion transporter 1 (OAT1).

Organic anion transporter 1 (OAT1) is a polyspecific transport protein located in the basolateral membrane of renal proximal tubule cells. OAT1 plays a pivotal role in drug clearance. Adverse drug reactions (ADR) are observed more frequently in women than in men, especially ADR are higher in women for drugs which are known interactors of OAT1. Sex‐dependent expression of Oat1 has been observed in rodents with a tendency to male‐dominant expression. This study aims at elucidating the transcriptional regulation of human OAT1 and tests the effect of estrogen receptor α (ERα). Promoter activation of OAT1 was assessed by luciferase assays carried out by Opossum kidney (OK) cells, transiently transfected with promoter constructs of human OAT1 and expression vectors for ERα and exposed to 100 nmol/L 17β‐estradiol. Furthermore, a transcription factor array and proteomic analysis was performed to identify estrogen‐induced transcription factors. Human OAT1 was significantly activated by ligand activated ERα. However, activation occurred without a direct interaction of ERα with the OAT1 promoter. Our data rather show an activation of the transcription factors CCAAT‐box‐binding transcription factor (CBF) and heterogeneous nuclear ribonucleoprotein K (HNRNPK) by ERα, which in turn bind and initiate OAT1 promoter activity. Herewith, we provide novel evidence of estrogen‐dependent, transcriptional regulation of polyspecific drug transporters including the estrogen‐induced transcription factors CBF and HNRNPK.