Identification of three distinct cell populations for urate excretion in human kidneys.

In humans, uric acid is an end-product of purine metabolism. Urate excretion from the human kidney is tightly regulated by reabsorption and secretion. At least eleven genes have been identified as human renal urate transporters. However, it remains unclear whether all renal tubular cells express the same set of urate transporters. Here, we show renal tubular cells are divided into three distinct cell populations for urate handling. Analysis of healthy human kidneys at single-cell resolution revealed that not all tubular cells expressed the same set of urate transporters. Only 32% of tubular cells were related to both reabsorption and secretion, while the remaining tubular cells were related to either reabsorption or secretion at 5% and 63%, respectively. These results provide physiological insight into the molecular function of the transporters and renal urate handling on single-cell units. Our findings suggest that three different cell populations cooperate to regulate urate excretion from the human kidney, and our proposed framework is a step forward in broadening the view from the molecular to the cellular level of transport capacity.

Inducing human retinal pigment epithelium-like cells from somatic tissue.

Regenerative medicine relies on basic research outcomes that are only practical when cost effective. The human eyeball requires the retinal pigment epithelium (RPE) to interface the neural retina and the choroid at large. Millions of people suffer from age-related macular degeneration (AMD), a blinding multifactor genetic disease among RPE degradation pathologies. Recently, autologous pluripotent stem-cell-derived RPE cells were prohibitively expensive due to time; therefore, we developed a faster reprogramming system. We stably induced RPE-like cells (iRPE) from human fibroblasts (Fibs) by conditional overexpression of both broad plasticity and lineage-specific transcription factors (TFs). iRPE cells displayed critical RPE benchmarks and significant in vivo integration in transplanted retinas. Herein, we detail the iRPE system with comprehensive single-cell RNA sequencing (scRNA-seq) profiling to interpret and characterize its best cells. We anticipate that our system may enable robust retinal cell induction for basic research and affordable autologous human RPE tissue for regenerative cell therapy.

Reprogramming epiblast stem cells into pre-implantation blastocyst cell-like cells.

Recently, a new wave of synthetic embryo systems (SESs) has been established from cultured cells for efficient and ethical embryonic development research. We recently reported our epiblast stem cell (EPISC) reprogramming SES that generates numerous blastocyst (BC)-like hemispheres (BCLH) with pluripotent and extraembryonic cell features detected by microscopy. Here, we further explored the system over key time points with single-cell RNA-sequencing analysis. We found broad induction of the 2C-like reporter MERVL and RNA velocities diverging to three major cell populations with gene expression profiles resembling those of pluripotent epiblast, primitive endoderm, and trophectoderm. Enrichment of those three induced BC-like cell fates involved key gene-regulatory networks, zygotic genome activation-related genes, and specific RNA splicing, and many cells closely resembled in silico models. This analysis confirms the induction of extraembryonic cell populations during EPISC reprogramming. We anticipate that our unique BCLH SES and rich dataset may uncover new facets of cell potency, improve developmental biology, and advance biomedicine.

27-Hydroxycholesterol regulates human SLC22A12 gene expression through estrogen receptor action.

The excretion and reabsorption of uric acid both to and from urine are tightly regulated by uric acid transporters. Metabolic syndrome conditions, such as obesity, hypercholesterolemia, and insulin resistance, are believed to regulate the expression of uric acid transporters and decrease the excretion of uric acid. However, the mechanisms driving cholesterol impacts on uric acid transporters have been unknown. Here, we show that cholesterol metabolite 27-hydroxycholesterol (27HC) upregulates the uric acid reabsorption transporter URAT1 encoded by SLC22A12 via estrogen receptors (ER). Transcriptional motif analysis showed that the SLC22A12 gene promoter has more estrogen response elements (EREs) than other uric acid reabsorption transporters such as SLC22A11 and SLC22A13, and 27HC-activated SLC22A12 gene promoter via ER through EREs. Furthermore, 27HC increased SLC22A12 gene expression in human kidney organoids. Our results suggest that in hypercholesterolemic conditions, elevated levels of 27HC derived from cholesterol induce URAT1/SLC22A12 expression to increase uric acid reabsorption, and thereby, could increase serum uric acid levels.

Involvement of nitric oxide production in the impairment of skin blood flow response to local cooling in diabetic db/db mice.

An enhanced vasocontrictor activity of cutaneous vessels participates in the reduction of skin blood flow induced by cooling. The present study investigated changes in the local response to cooling in hyperglycemic conditions. Male diabetic db/db and control C57BL/6J mice, anaesthetized with pentobarbitone, were treated with tetrodotoxin for eliminating the sympathetic nerve tone and artificially ventilated. The plantar skin blood flow (PSBF) was measured by laser Doppler flowmetry. Cooling the air temperature around the foot reduced PSBF in a temperature-dependent manner in control and db/db mice. The PSBF reduction was significantly smaller in db/db mice than in control mice. Phentolamine, a non-selective α-antagonist, bunazosin, a selective α1-antagonist, MK-912, a selective α2C-antagonist, and Y-27632, a Rho-kinase inhibitor, significantly inhibited the PSBF reduction induced by cooling to 15 °C in both mice and the inhibitory effects were comparable between these mice. The cooling-induced PSBF reduction was also significantly inhibited by N(ω)-nitro-L-arginine, an inhibitor of nitric oxide synthase, in control mice; however, the inhibitory effect of N(ω)-nitro-L-arginine was not observed in db/db mice. The reduction of PSBF induced by the intraarterial administration of adrenaline was comparable between control and db/db mice both before and after the treatment with N(ω)-nitro-L-arginine. It is thus likely that the reduction of skin blood flow induced by local cooling might be partly mediated by a decrease in endothelium-derived nitric oxide production, and that an impairment of the nitric oxide production might be related to reduced vasocontrictor response to cooling in db/db mice.

Differential involvement of alpha1-adrenoceptors in vasoconstrictor responses to cooling in mouse plantar arteries in vitro and in vivo.

Cooling-induced reduction of skin blood flow results from a reflex increase in sympathetic output and an enhanced vasoconstrictor activity of skin vessels. The latter has been proposed to be mediated by increased reactivity of alpha(2C)-adrenoceptors during cooling in studies with isolated cutaneous vessels in vitro. We have previously shown in studies with tetrodotoxin-treated mice in vivo that reduction of plantar skin blood flow (PSBF) induced by local cooling results primarily from increased reactivity of alpha(2C)-adrenoceptors. In addition, we showed that part of the cooling-induced response was also mediated by alpha(1)-adrenoceptors. However, the mechanisms involved in the cooling-induced responses mediated by alpha(1)-adrenoceptors have not been elucidated. The present study is an investigation seeking to clarify the mechanisms involving alpha(1)-adrenoceptors. Medial plantar arteries were isolated from male ddY mice and changes in vessel diameter were measured in vitro using pressurized arteriography. In vivo measurements of PSBF were performed on artificially ventilated tetrodotoxin treated mice, anaesthetized with pentobarbital sodium, using laser Doppler flowmetry, with the probe positioned above the medial plantar artery. In the in vitro studies with isolated plantar arteries, cooling from 37 to 28 degrees C did not affect the constrictor potency of phenylephrine, an alpha(1)-adrenoceptor agonist, and the threshold concentration to evoke constriction was rather higher at 28 degrees C than it was at 37 degrees C. The cooling also suppressed the constrictor efficacy of UK14,304, an alpha(2)-adrenoceptor agonist. In contrast, cooling the air temperature around the foot from 25 to 10 degrees C in vivo decreased PSBF, which was significantly inhibited by phentolamine, an alpha-adrenoceptor antagonist, although MK-912, an alpha(2C)-adrenoceptor antagonist, had no effect on it. These results suggest that although alpha(1)-adrenoceptors are involved in cooling-induced reduction of PSBF in mice, the response is unlikely to result from an enhancement of alpha(1)-adrenoceptor-mediated vasoconstriction of plantar arteries during cooling.