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.

Oral administration of drugs with hypersensitivity potential induces germinal center hyperplasia in secondary lymphoid organ/tissue in Brown Norway rats, and this histological lesion is a promising candidate as a predictive biomarker for drug hypersensitivity occurrence in humans.

It is important to evaluate the potential of drug hypersensitivity as well as other adverse effects during the preclinical stage of the drug development process, but validated methods are not available yet. In the present study we examined whether it would be possible to develop a new predictive model of drug hypersensitivity using Brown Norway (BN) rats. As representative drugs with hypersensitivity potential in humans, phenytoin (PHT), carbamazepine (CBZ), amoxicillin (AMX), and sulfamethoxazole (SMX) were orally administered to BN rats for 28days to investigate their effects on these animals by examinations including observation of clinical signs, hematology, determination of serum IgE levels, histology, and flow cytometric analysis. Skin rashes were not observed in any animals treated with these drugs. Increases in the number of circulating inflammatory cells and serum IgE level did not necessarily occur in the animals treated with these drugs. However, histological examination revealed that germinal center hyperplasia was commonly induced in secondary lymphoid organs/tissues in the animals treated with these drugs. In cytometric analysis, changes in proportions of lymphocyte subsets were noted in the spleen of the animals treated with PHT or CBZ during the early period of administration. The results indicated that the potential of drug hypersensitivity was identified in BN rat by performing histological examination of secondary lymphoid organs/tissues. Data obtained herein suggested that drugs with hypersensitivity potential in humans gained immune reactivity in BN rat, and the germinal center hyperplasia induced by administration of these drugs may serve as a predictive biomarker for drug hypersensitivity occurrence.

The effects of clobazam treatment in rats on the expression of genes and proteins encoding glucronosyltransferase 1A/2B (UGT1A/2B) and multidrug resistance-associated protein-2 (MRP2), and development of thyroid follicular cell hypertrophy.

Clobazam (CLB) is known to increase hepatobiliary thyroxine (T4) clearance in Sprague-Dawley (SD) rats, which results in hypothyroidism followed by thyroid follicular cell hypertrophy. However, the mechanism of the acceleration of T4-clearance has not been fully investigated. In the present study, we tried to clarify the roles of hepatic UDP-glucronosyltransferase (UGT) isoenzymes (UGT1A and UGT2B) and efflux transporter (multidrug resistance-associated protein-2; MRP2) in the CLB-induced acceleration of T4-clearance using two mutant rat strains, UGT1A-deficient mutant (Gunn) and MRP2-deficient mutant (EHBR) rats, especially focusing on thyroid morphology, levels of circulating hormones (T4 and triiodothyronine (T3)) and thyroid-stimulating hormone (TSH), and mRNA or protein expressions of UGTs (Ugt1a1, Ugt1a6, and Ugt2b1/2) and MRP2 (Mrp). CLB induced thyroid morphological changes with increases in TSH in SD and Gunn rats, but not in EHBR rats. T4 was slightly decreased in SD and Gunn rats, and T3 was decreased in Gunn rats, whereas these hormones were maintained in EHBR rats. Hepatic Ugt1a1, Ugt1a6, Ugt2b1/2, and Mrp2 mRNAs were upregulated in SD rats. In Gunn rats, UGT1A mRNAs (Ugt1a1/6) and protein levels were quite low, but UGT2B mRNAs (Ugt2b1/2) and protein were prominently upregulated. In SD and Gunn rats, MRP2 mRNA and protein were upregulated to the same degree. These results suggest that MRP2 is an important contributor in development of the thyroid cellular hypertrophy in CLB-treated rats, and that UGT1A and UGT2B work in concert with MRP2 in the presence of MRP2 function to enable the effective elimination of thyroid hormones.

Increased c-Myc activity and DNA damage in hematopoietic progenitors precede myeloproliferative disease in Spa-1-deficiency.

Mice deficient for Spa-1 encoding Rap GTPase-activating protein develop myeloproliferative disorder (MPD) of late onset with frequent blast crises. The mechanisms for MPD development as well as the reasons for long latency, however, remain elusive. We demonstrate here that preleukemic, disease-free Spa-1(-/-) mice show reduced steady-state hematopoiesis and attenuated resistance to whole body γ-ray irradiation, which are attributable to the sustained p53 response in hematopoietic progenitor cells (HPCs). Preleukemic Spa-1(-/-) HPCs show c-Myc overexpression with increased p19Arf as well as enhanced γH2AX expression with activation of Atm/Chk pathway. We also show that deregulated Rap signaling in the absence of Spa-1 enhances post-transcriptional c-Myc stability and induces DNA damage in a p38MAPK-dependent manner, leading to p53 activation. Genetic studies indicate that the introduction of p53(+/-) and p53(-/-) mutations in Spa-1(-/-) mice results in the acceleration of typical MPD and rapid development of blastic leukemia, respectively. These results suggest that increased c-Myc expression and DNA damage in HPCs precede MPD development in Spa-1(-/-) mice, and the resulting p53 response functions as a barrier for the onset of MPD and blast crises progression.

Rap1 signal controls B cell receptor repertoire and generation of self-reactive B1a cells.

We previously reported that the mice deficient for SPA-1, a Rap1 GTPase-activating protein, developed hematopoietic stem cell disorders. Here, we demonstrate that SPA-1(-/-) mice show an age-dependent increase in B220(high) B1a cells producing anti-dsDNA antibody and lupus-like nephritis. SPA-1(-/-) peritoneal B1 cells revealed the altered Vkappa gene repertoire, including skewed Vkappa4 usage and the significant Igkappa/Iglambda isotype inclusion indicative of extensive receptor editing. Rap1GTP induced OcaB gene activation via p38MAPK-dependent Creb phosphorylation, and consistently, SPA-1(-/-) immature BM B cells showing high Rap1GTP exhibited the augmented expression of OcaB and Vkappa4 genes. SPA-1(-/-) BM cells could transfer the autoimmunity in association with the generation of peritoneal B220(high) B1a cells in Rag-2(-/-) recipients. Finally, a portion of SPA-1(-/-) mice developed B1 cell leukemia with hemolytic autoantibody. Present results suggest that the regulated Rap1 signal in the immature B cells plays a role in modifying the B cell receptor repertoire and in maintaining the self-tolerance.