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A 3D Renal Proximal Tubule on Chip Model Phenocopies Lowe Syndrome and Dent II Disease Tubulopathy.

ABSTRACT: Lowe syndrome and Dent II disease are X-linked monogenetic diseases characterised by a renal reabsorption defect in the proximal tubules and caused by mutations in the OCRL gene, which codes for an inositol-5-phosphatase. The life expectancy of patients suffering from Lowe syndrome is largely reduced because of the development of chronic kidney disease and related complications. There is a need for physiological human in vitro models for Lowe syndrome/Dent II disease to study the underpinning disease mechanisms and to identify and characterise potential drugs and drug targets. Here, we describe a proximal tubule organ on chip model combining a 3D tubule architecture with fluid flow shear stress that phenocopies hallmarks of Lowe syndrome/Dent II disease. We demonstrate the high suitability of our in vitro model for drug target validation. Furthermore, using this model, we demonstrate that proximal tubule cells lacking OCRL expression upregulate markers typical for epithelial-mesenchymal transition (EMT), including the transcription factor SNAI2/Slug, and show increased collagen expression and deposition, which potentially contributes to interstitial fibrosis and disease progression as observed in Lowe syndrome and Dent II disease.

SUBMITTER: Naik S

PROVIDER: S-EPMC8161077 | biostudies-literature |

REPOSITORIES: biostudies-literature

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A 3D renal proximal tubule on chip model phenocopies Lowe syndrome and Dent II disease tubulopathy.

Project description:Lowe syndrome and Dent II disease are X-linked monogenetic diseases characterised by a renal reabsorption defect in the proximal tubules and caused by mutations in the OCRL gene, which codes for an inositol-5-phosphatase. The life expectancy of patients suffering from Lowe syndrome is largely reduced because of the development of chronic kidney disease and related complications. There is a need for physiological human in-vitro models for Lowe syndrome/Dent II disease to study underpinning disease mechanisms and to identify and characterise potential drugs and drug targets. Here we describe a proximal tubule organ on chip model combining 3D tubule architecture with fluid flow shear stress, which phenocopies hallmarks of Lowe syndrome/Dent II disease. We demonstrate the principal suitability of our in-vitro model for drug target validation. Furthermore, using this model we demonstrate that proximal tubule cells lacking OCRL expression upregulate markers typical for epithelial-mesenchymal transition (EMT) including the transcription factor SNAI2/Slug and show increased collagen expression and deposition, which potentially contributes to interstitial fibrosis and disease progression as observed in Lowe syndrome and Dent II disease.

2021-05-21 | GSE171848 | GEO

A 3D renal proximal tubule on chip model phenocopies Lowe syndrome and Dent II disease tubulopathy.

Project description:A 3D renal proximal tubule on chip model phenocopies Lowe syndrome and Dent II disease tubulopathy.

| PRJNA721193 | ENA

Mouse model for Lowe syndrome/Dent Disease 2 renal tubulopathy.

Project description:The Lowe oculocerebrorenal syndrome is an X-linked disorder characterized by congenital cataracts, cognitive disability, and proximal tubular dysfunction. Both this syndrome and Dent Disease 2 result from loss-of-function mutations in the OCRL gene, which encodes a type II phosphatidylinositol bisphosphate 5-phosphatase. Ocrl-deficient mice are unaffected, however, which we believe reflects a difference in how humans and mice cope with the enzyme deficiency. Inpp5b and INPP5B, paralogous autosomal genes that encode another type II phosphoinositide 5-phosphatase in mice and humans, respectively, might explain the distinct phenotype in the two species because they are the closest paralogs to Ocrl and OCRL in their respective genomes yet differ between the two species with regard to expression and splicing. Here, we generated Ocrl(-/-) mice that express INPP5B but not Inpp5b. Similar to the human syndromes, all showed reduced postnatal growth, low molecular weight proteinuria, and aminoaciduria. Thus, we created an animal model for OCRL and Dent Disease 2 tubulopathy by humanizing a modifier paralog in mice already carrying the mutant disease gene.

| S-EPMC3060438 | biostudies-literature

Kidney Tubular Ablation of <i>Ocrl</i>/<i>Inpp5b</i> Phenocopies Lowe Syndrome Tubulopathy.

Project description:Lowe syndrome and Dent disease are two conditions that result from mutations of the inositol 5-phosphatase oculocerebrorenal syndrome of Lowe (OCRL) and share the feature of impaired kidney proximal tubule function. Genetic ablation of Ocrl in mice failed to recapitulate the human phenotypes, possibly because of the redundant functions of OCRL and its paralog type 2 inositol polyphosphate-5-phosphatase (INPP5B). Germline knockout of both paralogs in mice results in early embryonic lethality. We report that kidney tubule-specific inactivation of Inpp5b on a global Ocrl-knockout mouse background resulted in low molecular weight proteinuria, phosphaturia, and acidemia. At the cellular level, we observed a striking impairment of clathrin-dependent and -independent endocytosis in proximal tubules, phenocopying what has been reported for Dent disease caused by mutations in the gene encoding endosomal proton-chloride exchange transporter 5. These results suggest that the functions of OCRL/INPP5B and proton-chloride exchange transporter 5 converge on shared mechanisms, the impairment of which has a dramatic effect on proximal tubule endocytosis.

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Renal reabsorption in 3D vascularized proximal tubule models.

Project description:Three-dimensional renal tissues that emulate the cellular composition, geometry, and function of native kidney tissue would enable fundamental studies of filtration and reabsorption. Here, we have created 3D vascularized proximal tubule models composed of adjacent conduits that are lined with confluent epithelium and endothelium, embedded in a permeable ECM, and independently addressed using a closed-loop perfusion system to investigate renal reabsorption. Our 3D kidney tissue allows for coculture of proximal tubule epithelium and vascular endothelium that exhibits active reabsorption via tubular-vascular exchange of solutes akin to native kidney tissue. Using this model, both albumin uptake and glucose reabsorption are quantified as a function of time. Epithelium-endothelium cross-talk is further studied by exposing proximal tubule cells to hyperglycemic conditions and monitoring endothelial cell dysfunction. This diseased state can be rescued by administering a glucose transport inhibitor. Our 3D kidney tissue provides a platform for in vitro studies of kidney function, disease modeling, and pharmacology.

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3D proximal tubule-on-chip model derived from kidney organoids with improved drug uptake.

Project description:Three-dimensional, organ-on-chip models that recapitulate kidney tissue are needed for drug screening and disease modeling. Here, we report a method for creating a perfusable 3D proximal tubule model composed of epithelial cells isolated from kidney organoids matured under static conditions. These organoid-derived proximal tubule epithelial cells (OPTECs) are seeded in cylindrical channels fully embedded within an extracellular matrix, where they form a confluent monolayer. A second perfusable channel is placed adjacent to each proximal tubule within these reusable multiplexed chips to mimic basolateral drug transport and uptake. Our 3D OPTEC-on-chip model exhibits significant upregulation of organic cation (OCT2) and organic anion (OAT1/3) transporters, which leads to improved drug uptake, compared to control chips based on immortalized proximal tubule epithelial cells. Hence, OPTEC tubules exhibit a higher normalized lactate dehydrogenase (LDH) release, when exposed to known nephrotoxins, cisplatin and aristolochic acid, which are diminished upon adding OCT2 and OAT1/3 transport inhibitors. Our integrated multifluidic platform paves the way for personalized kidney-on-chip models for drug screening and disease modeling.

| S-EPMC9440090 | biostudies-literature

The phosphoinositide 3-kinase inhibitor alpelisib restores actin organization and improves proximal tubule dysfunction in vitro and in a mouse model of Lowe syndrome and Dent disease.

Project description:Loss-of-function mutations in the OCRL gene, which encodes the phosphatidylinositol [PI] 4,5-bisphosphate [PI(4,5)P2] 5-phosphatase OCRL, cause defective endocytosis and proximal tubule dysfunction in Lowe syndrome and Dent disease 2. The defect is due to increased levels of PI(4,5)P2 and aberrant actin polymerization, blocking endosomal trafficking. PI 3-phosphate [PI(3)P] has been recently identified as a coactivator with PI(4,5)P2 in the actin pathway. Here, we tested the hypothesis that phosphoinositide 3-kinase (PI3K) inhibitors may rescue the endocytic defect imparted by OCRL loss, by rebalancing phosphoinositide signals to the actin machinery. The broad-range PI3K inhibitor copanlisib and class IA p110α PI3K inhibitor alpelisib reduced aberrant actin polymerization in OCRL-deficient human kidney cells in vitro. Levels of PI 3,4,5-trisphosphate, PI(4,5)P2 and PI(3)P were all reduced with alpelisib treatment, and siRNA knockdown of the PI3K catalytic subunit p110α phenocopied the actin phenotype. In a humanized OcrlY/- mouse model, alpelisib reduced endosomal actin staining while restoring stress fiber architecture and levels of megalin at the plasma membrane of proximal tubule cells, reflected by improved endocytic uptake of low molecular weight proteins in vivo. Thus, our findings support the link between phosphoinositide lipids, actin polymerization and endocytic trafficking in the proximal tubule and represent a proof-of-concept for repurposing alpelisib in Lowe syndrome/Dent disease 2.

| S-EPMC7550850 | biostudies-literature

Renal Deletion of LRRC8/VRAC Channels Induces Proximal Tubulopathy.

Project description:BackgroundVolume-regulated anion channels (VRACs) are heterohexamers of LRRC8A with LRRC8B, -C, -D, or -E in various combinations. Depending on the subunit composition, these swelling-activated channels conduct chloride, amino acids, organic osmolytes, and drugs. Despite VRACs' role in cell volume regulation, and large osmolarity changes in the kidney, neither the localization nor the function of VRACs in the kidney is known.MethodsMice expressing epitope-tagged LRRC8 subunits were used to determine the renal localization of all VRAC subunits. Mice carrying constitutive deletions of Lrrc8b-e, or with inducible or cell-specific ablation of Lrrc8a, were analyzed to assess renal functions of VRACs. Analysis included histology, urine and serum parameters in different diuresis states, and metabolomics.ResultsThe kidney expresses all five VRAC subunits with strikingly distinct localization. Whereas LRRC8C is exclusively found in vascular endothelium, all other subunits are found in the nephron. LRRC8E is specific for intercalated cells, whereas LRRC8A, LRRC8B, and LRRC8D are prominent in basolateral membranes of proximal tubules. Conditional deletion of LRRC8A in proximal but not distal tubules and constitutive deletion of LRRC8D cause proximal tubular injury, increased diuresis, and mild Fanconi-like symptoms.ConclusionsVRAC/LRRC8 channels are crucial for the function and integrity of proximal tubules, but not for more distal nephron segments despite their larger need for volume regulation. LRRC8A/D channels may be required for the basolateral exit of many organic compounds, including cellular metabolites, in proximal tubules. Proximal tubular injury likely results from combined accumulation of several transported molecules in the absence of VRAC channels.

| S-EPMC9342636 | biostudies-literature

Effects of Proximal Tubule Shortening on Protein Excretion in a Lowe Syndrome Model.

Project description:BackgroundLowe syndrome (LS) is an X-linked recessive disorder caused by mutations in OCRL, which encodes the enzyme OCRL. Symptoms of LS include proximal tubule (PT) dysfunction typically characterized by low molecular weight proteinuria, renal tubular acidosis (RTA), aminoaciduria, and hypercalciuria. How mutant OCRL causes these symptoms isn't clear.MethodsWe examined the effect of deleting OCRL on endocytic traffic and cell division in newly created human PT CRISPR/Cas9 OCRL knockout cells, multiple PT cell lines treated with OCRL-targeting siRNA, and in orcl-mutant zebrafish.ResultsOCRL-depleted human cells proliferated more slowly and about 10% of them were multinucleated compared with fewer than 2% of matched control cells. Heterologous expression of wild-type, but not phosphatase-deficient, OCRL prevented the accumulation of multinucleated cells after acute knockdown of OCRL but could not rescue the phenotype in stably edited knockout cell lines. Mathematic modeling confirmed that reduced PT length can account for the urinary excretion profile in LS. Both ocrl mutant zebrafish and zebrafish injected with ocrl morpholino showed truncated expression of megalin along the pronephric kidney, consistent with a shortened S1 segment.ConclusionsOur data suggest a unifying model to explain how loss of OCRL results in tubular proteinuria as well as the other commonly observed renal manifestations of LS. We hypothesize that defective cell division during kidney development and/or repair compromises PT length and impairs kidney function in LS patients.

| S-EPMC6934997 | biostudies-literature

3D Proximal Tubule Tissues Recapitulate Key Aspects of Renal Physiology to Enable Nephrotoxicity Testing.

Project description:Due to its exposure to high concentrations of xenobiotics, the kidney proximal tubule is a primary site of nephrotoxicity and resulting attrition in the drug development pipeline. Current pre-clinical methods using 2D cell cultures and animal models are unable to fully recapitulate clinical drug responses due to limited in vitro functional lifespan, or species-specific differences. Using Organovo's proprietary 3D bioprinting platform, we have developed a fully cellular human in vitro model of the proximal tubule interstitial interface comprising renal fibroblasts, endothelial cells, and primary human renal proximal tubule epithelial cells to enable more accurate prediction of tissue-level clinical outcomes. Histological characterization demonstrated formation of extensive microvascular networks supported by endogenous extracellular matrix deposition. The epithelial cells of the 3D proximal tubule tissues demonstrated tight junction formation and expression of renal uptake and efflux transporters; the polarized localization and function of P-gp and SGLT2 were confirmed. Treatment of 3D proximal tubule tissues with the nephrotoxin cisplatin induced loss of tissue viability and epithelial cells in a dose-dependent fashion, and cimetidine rescued these effects, confirming the role of the OCT2 transporter in cisplatin-induced nephrotoxicity. The tissues also demonstrated a fibrotic response to TGFβ as assessed by an increase in gene expression associated with human fibrosis and histological verification of excess extracellular matrix deposition. Together, these results suggest that the bioprinted 3D proximal tubule model can serve as a test bed for the mechanistic assessment of human nephrotoxicity and the development of pathogenic states involving epithelial-interstitial interactions, making them an important adjunct to animal studies.

| S-EPMC5340751 | biostudies-literature

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