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

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.

Effect of luminal flow on doming of mpkCCD cells in a 3D perfusable kidney cortical collecting duct model.

The cortical collecting duct (CCD) of the mammalian kidney plays a major role in the maintenance of total body electrolyte, acid/base, and fluid homeostasis by tubular reabsorption and excretion. The mammalian CCD is heterogeneous, composed of Na+-absorbing principal cells (PCs) and acid-base-transporting intercalated cells (ICs). Perturbations in luminal flow rate alter hydrodynamic forces to which these cells in the cylindrical tubules are exposed. However, most studies of tubular ion transport have been performed in cell monolayers grown on or epithelial sheets affixed to a flat support, since analysis of transepithelial transport in native tubules by in vitro microperfusion requires considerable expertise. Here, we report on the generation and characterization of an in vitro, perfusable three-dimensional kidney CCD model (3D CCD), in which immortalized mouse PC-like mpkCCD cells are seeded within a cylindrical channel embedded within an engineered extracellular matrix and subjected to luminal fluid flow. We find that a tight epithelial barrier composed of differentiated and polarized PCs forms within 1 wk. Immunofluorescence microscopy reveals the apical epithelial Na+ channel ENaC and basolateral Na+/K+-ATPase. On cessation of luminal flow, benzamil-inhibitable cell doming is observed within these 3D CCDs consistent with the presence of ENaC-mediated Na+ absorption. Our 3D CCD provides a geometrically and microphysiologically relevant platform for studying the development and physiology of renal tubule segments.

Intercalated cell BKα subunit is required for flow-induced K+ secretion.

BK channels are expressed in intercalated cells (ICs) and principal cells (PCs) in the cortical collecting duct (CCD) of the mammalian kidney and have been proposed to be responsible for flow-induced K+ secretion (FIKS) and K+ adaptation. To examine the IC-specific role of BK channels, we generated a mouse with targeted disruption of the pore-forming BK α subunit (BKα) in ICs (IC-BKα-KO). Whole cell charybdotoxin-sensitive (ChTX-sensitive) K+ currents were readily detected in control ICs but largely absent in ICs of IC-BKα-KO mice. When placed on a high K+ (HK) diet for 13 days, blood [K+] was significantly greater in IC-BKα-KO mice versus controls in males only, although urinary K+ excretion rates following isotonic volume expansion were similar in males and females. FIKS was present in microperfused CCDs isolated from controls but was absent in IC-BKα-KO CCDs of both sexes. Also, flow-stimulated epithelial Na+ channel-mediated (ENaC-mediated) Na+ absorption was greater in CCDs from female IC-BKα-KO mice than in CCDs from males. Our results confirm a critical role of IC BK channels in FIKS. Sex contributes to the capacity for adaptation to a HK diet in IC-BKα-KO mice.

Correction: Integrative approach to sporadic Alzheimer's disease: deficiency of TYROBP in cerebral Aß amyloidosis mouse normalizes clinical phenotype and complement subnetwork molecular pathology without reducing Aß burden.

This article was originally published under standard licence, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the paper have been modified accordingly.

Integrative approach to sporadic Alzheimer's disease: deficiency of TYROBP in cerebral Aß amyloidosis mouse normalizes clinical phenotype and complement subnetwork molecular pathology without reducing Aß burden.

Integrative gene network approaches enable new avenues of exploration that implicate causal genes in sporadic late-onset Alzheimer's disease (LOAD) pathogenesis, thereby offering novel insights for drug-discovery programs. We previously constructed a probabilistic causal network model of sporadic LOAD and identified TYROBP/DAP12, encoding a microglial transmembrane signaling polypeptide and direct adapter of TREM2, as the most robust key driver gene in the network. Here, we show that absence of TYROBP/DAP12 in a mouse model of AD-type cerebral Aß amyloidosis (APPKM670/671NL/PSEN1Δexon9) recapitulates the expected network characteristics by normalizing the transcriptome of APP/PSEN1 mice and repressing the induction of genes involved in the switch from homeostatic microglia to disease-associated microglia (DAM), including Trem2, complement (C1qa, C1qb, C1qc, and Itgax), Clec7a and Cst7. Importantly, we show that constitutive absence of TYROBP/DAP12 in the amyloidosis mouse model prevented appearance of the electrophysiological and learning behavior alterations associated with the phenotype of APPKM670/671NL/PSEN1Δexon9 mice. Our results suggest that TYROBP/DAP12 could represent a novel therapeutic target to slow, arrest, or prevent the development of sporadic LOAD. These data establish that the network pathology observed in postmortem human LOAD brain can be faithfully recapitulated in the brain of a genetically manipulated mouse. These data also validate our multiscale gene networks by demonstrating how the networks intersect with the standard neuropathological features of LOAD.