Functional maturation of kidney organoid tubules: PIEZO1-mediated Ca2+ signaling.

Kidney organoids cultured on adherent matrices in the presence of superfusate flow generate vascular networks and exhibit more mature podocyte and tubular compartments compared with static controls (Homan KA, Gupta N, Kroll KT, Kolesky DB, Skylar-Scott M, Miyoshi T, Mau D, Valerius MT, Ferrante T, Bonventre JV, Lewis JA, Morizane R. Nat Methods 16: 255-262, 2019; Takasato M, Er PX, Chiu HS, Maier B, Baillie GJ, Ferguson C, Parton RG, Wolvetang EJ, Roost MS, Chuva de Sousa Lopes SM, Little MH. Nature 526: 564-568, 2015.). However, their physiological function has yet to be systematically investigated. Here, we measured mechano-induced changes in intracellular Ca2+ concentration ([Ca2+]i) in tubules isolated from organoids cultured for 21-64 days, microperfused in vitro or affixed to the base of a specimen chamber, and loaded with fura-2 to measure [Ca2+]i. A rapid >2.5-fold increase in [Ca2+]i from a baseline of 195.0 ± 22.1 nM (n = 9; P ≤ 0.001) was observed when microperfused tubules from organoids >40 days in culture were subjected to luminal flow. In contrast, no response was detected in tubules isolated from organoids <30 days in culture. Nonperfused tubules (41 days) subjected to a 10-fold increase in bath flow rate also exhibited a threefold increase in [Ca2+]i from baseline (P < 0.001). Mechanosensitive PIEZO1 channels contribute to the flow-induced [Ca2+]i response in mouse distal tubule (Carrisoza-Gaytan R, Dalghi MG, Apodaca GL, Kleyman TR, Satlin LM. The FASEB J 33: 824.25, 2019.). Immunodetectable apical and basolateral PIEZO1 was identified in tubular structures by 21 days in culture. Basolateral PIEZO1 appeared to be functional as basolateral exposure of nonperfused tubules to the PIEZO1 activator Yoda 1 increased [Ca2+]i (P ≤ 0.001) in segments from organoids cultured for >30 days, with peak [Ca2+]i increasing with advancing days in culture. These results are consistent with a maturational increase in number and/or activity of flow/stretch-sensitive Ca2+ channels, including PIEZO1, in tubules of static organoids in culture.

Bioavailability, Efficacy and Safety of Generic Immunosuppressive Drugs for Kidney Transplantation: A Systematic Review and Meta-Analysis.

BACKGROUND: Concerns exist over the extrapolation of bioavailability studies of generic immunosuppressive drugs in healthy volunteers, regarding their efficacy and safety in kidney transplant recipients. We conducted a meta-analysis of trials examining the bioavailability of generic (test) immunosuppressive drugs relative to their brand (reference) counterparts in healthy volunteers, based on the US Food and Drug Administration requirements for approval of generics, and their efficacy and safety in kidney transplant recipients. METHODS: Eligible studies were identified in PubMed, Cochrane Central Register of Controlled Trials, Scopus, ClinicalTrials.gov, and conference abstracts. RESULTS: Twenty crossover trials of healthy volunteers (n = 641) and 6 parallel-arm randomized controlled trials of kidney transplant recipients (n = 594) were identified. The 90% CI of the pooled test-to-reference drug ratio for maximum or peak plasma concentration (Cmax) and area under the plasma concentration time-curve from time 0 to time of last determinable concentration (AUC(0-t)) fell within the required range (0.80-1.25) for cyclosporine (Cmax 0.91; 90% CI 0.86-0.95; and AUC(0-t) 0.97; 90% CI 0.94-1.00), tacrolimus (Cmax 1.17; 90% CI 1.09-1.24; and AUC(0-t) 1.00; 90% CI 0.97-1.03) and mycophenolate mofetil (Cmax 0.98; 90% CI 0.96-1.01; and AUC(0-t) 1.00; 90% CI 0.99-1.01). In subgroup analyses, some generic cyclosporine formulations did not meet criteria for bioequivalence. No significant differences were observed in the time to maximum plasma concentration and terminal plasma half-life between generic and brand drugs. In parallel-arm trials, generic cyclosporine was non-inferior to brand counterpart in terms of acute allograft rejection, infections, and death. CONCLUSIONS: Not all generic immunosuppressive drugs have similar relative bioavailability to their brand name counterparts. Evidence on their efficacy and safety is inconclusive. Tighter regulatory requirement for approval of generic drugs with narrow therapeutic index is needed.

ATP/ADP biosensor organoids for drug nephrotoxicity assessment.

Drug nephrotoxicity is a common healthcare problem in hospitalized patients and a major limitation during drug development. Multi-segmented kidney organoids derived from human pluripotent stem cells may complement traditional cell culture and animal experiments for nephrotoxicity assessment. Here we evaluate the capability of kidney organoids to investigate drug toxicity in vitro. Kidney organoids express renal drug transporters, OAT1, OAT3, and OCT2, while a human proximal tubular cell line shows the absence of OAT1 and OAT3. Tenofovir and aristolochic acid (AA) induce proximal tubular injury in organoids which is ameliorated by an OAT inhibitor, probenecid, without damage to podocytes. Similarly, cisplatin causes proximal tubular damage that can be relieved by an OCT inhibitor, cimetidine, collectively suggesting the presence of functional OATs and OCTs in organoid proximal tubules. Puromycin aminonucleoside (PAN) induced segment-specific injury in glomerular podocytes in kidney organoids in the absence of tubular injury. Reporter organoids were generated with an ATP/ADP biosensor, which may be applicable to high-throughput screening in the future. In conclusion, the kidney organoid is a useful tool for toxicity assessment in the multicellular context and may contribute to nephrotoxicity assessment during drug development.

Organoid-on-a-chip model of human ARPKD reveals mechanosensing pathomechanisms for drug discovery.

Organoids serve as a novel tool for disease modeling in three-dimensional multicellular contexts. Static organoids, however, lack the requisite biophysical microenvironment such as fluid flow, limiting their ability to faithfully recapitulate disease pathology. Here, we unite organoids with organ-on-a-chip technology to unravel disease pathology and develop therapies for autosomal recessive polycystic kidney disease. PKHD1-mutant organoids-on-a-chip are subjected to flow that induces clinically relevant phenotypes of distal nephron dilatation. Transcriptomics discover 229 signal pathways that are not identified by static models. Mechanosensing molecules, RAC1 and FOS, are identified as potential therapeutic targets and validated by patient kidney samples. On the basis of this insight, we tested two U.S. Food and Drug Administration-approved and one investigational new drugs that target RAC1 and FOS in our organoid-on-a-chip model, which suppressed cyst formation. Our observations highlight the vast potential of organoid-on-a-chip models to elucidate complex disease mechanisms for therapeutic testing and discovery.

A Role for 3D Printing in Kidney-on-a-Chip Platforms.

The advancement of "kidney-on-a-chip" platforms - submillimeter-scale fluidic systems designed to recapitulate renal functions in vitro - directly impacts a wide range of biomedical fields, including drug screening, cell and tissue engineering, toxicity testing, and disease modelling. To fabricate kidney-on-a-chip technologies, researchers have primarily adapted traditional micromachining techniques that are rooted in the integrated circuit industry; hence the term, "chip." A significant challenge, however, is that such methods are inherently monolithic, which limits one's ability to accurately recreate the geometric and architectural complexity of the kidney in vivo. Better reproduction of the anatomical complexity of the kidney will allow for more instructive modelling of physiological and pathophysiological events. Emerging additive manufacturing or "three-dimensional (3D) printing" techniques could provide a promising alternative to conventional methodologies. In this article, we discuss recent progress in the development of both kidney-on-a-chip platforms and state-of-the-art submillimeter-scale 3D printing methods, with a focus on biophysical and architectural capabilities. Lastly, we examine the potential for 3D printing-based approaches to extend the efficacy of kidney-on-a-chip systems.