Direct androgen receptor control of sexually dimorphic gene expression in the mammalian kidney.

Mammalian organs exhibit distinct physiology, disease susceptibility, and injury responses between the sexes. In the mouse kidney, sexually dimorphic gene activity maps predominantly to proximal tubule (PT) segments. Bulk RNA sequencing (RNA-seq) data demonstrated that sex differences were established from 4 and 8 weeks after birth under gonadal control. Hormone injection studies and genetic removal of androgen and estrogen receptors demonstrated androgen receptor (AR)-mediated regulation of gene activity in PT cells as the regulatory mechanism. Interestingly, caloric restriction feminizes the male kidney. Single-nuclear multiomic analysis identified putative cis-regulatory regions and cooperating factors mediating PT responses to AR activity in the mouse kidney. In the human kidney, a limited set of genes showed conserved sex-linked regulation, whereas analysis of the mouse liver underscored organ-specific differences in the regulation of sexually dimorphic gene expression. These findings raise interesting questions on the evolution, physiological significance, disease, and metabolic linkage of sexually dimorphic gene activity.

Direct androgen receptor regulation of sexually dimorphic gene expression in the mammalian kidney.

Mammalian organs exhibit distinct physiology, disease susceptibility and injury responses between the sexes. In the mouse kidney, sexually dimorphic gene activity maps predominantly to proximal tubule (PT) segments. Bulk RNA-seq data demonstrated sex differences were established from 4 and 8 weeks after birth under gonadal control. Hormone injection studies and genetic removal of androgen and estrogen receptors demonstrated androgen receptor (AR) mediated regulation of gene activity in PT cells as the regulatory mechanism. Interestingly, caloric restriction feminizes the male kidney. Single-nuclear multiomic analysis identified putative cis-regulatory regions and cooperating factors mediating PT responses to AR activity in the mouse kidney. In the human kidney, a limited set of genes showed conserved sex-linked regulation while analysis of the mouse liver underscored organ-specific differences in the regulation of sexually dimorphic gene expression. These findings raise interesting questions on the evolution, physiological significance, and disease and metabolic linkage, of sexually dimorphic gene activity.

A novel distal convoluted tubule-specific Cre-recombinase driven by the NaCl cotransporter gene.

Cre-lox technology has revolutionized research in renal physiology by allowing site-specific genetic recombination in individual nephron segments. The distal convoluted tubule (DCT), consisting of distinct early (DCT1) and late (DCT2) segments, plays a central role in Na+ and K+ homeostasis. The only established Cre line targeting the DCT is Pvalb-Cre, which is limited by noninducibility, activity along DCT1 only, and activity in neurons. Here, we report the characterization of the first Cre line specific to the entire DCT. CRISPR/Cas9 targeting was used to introduce a tamoxifen-inducible IRES-Cre-ERT2 cassette downstream of the coding region of the Slc12a3 gene encoding the NaCl cotransporter (NCC). The resulting Slc12a3-Cre-ERT2 mice were crossed with R26R-YFP reporter mice, which revealed minimal leakiness with 6.3% of NCC-positive cells expressing yellow fluorescent protein (YFP) in the absence of tamoxifen. After tamoxifen injection, YFP expression was observed in 91.2% of NCC-positive cells and only in NCC-positive cells, revealing high recombination efficiency and DCT specificity. Crossing to R26R-TdTomato mice revealed higher leakiness (64.5%), suggesting differential sensitivity of the floxed site. Western blot analysis revealed no differences in abundances of total NCC or the active phosphorylated form of NCC in Slc12a3-Cre-ERT2 mice of either sex compared with controls. Plasma K+ and Mg2+ concentrations and thiazide-sensitive Na+ and K+ excretion did not differ in Slc12a3-Cre-ERT2 mice compared with controls when sex matched. These data suggest genetic modification had no obvious effect on NCC function. Slc12a3-Cre-ERT2 mice are the first line generated demonstrating inducible Cre recombinase activity along the entire DCT and will be a useful tool to study DCT function.

Single-Cell Profiling Reveals Sex, Lineage, and Regional Diversity in the Mouse Kidney.

Chronic kidney disease affects 10% of the population with notable differences in ethnic and sex-related susceptibility to kidney injury and disease. Kidney dysfunction leads to significant morbidity and mortality and chronic disease in other organ systems. A mouse-organ-centered understanding underlies rapid progress in human disease modeling and cellular approaches to repair damaged systems. To enhance an understanding of the mammalian kidney, we combined anatomy-guided single-cell RNA sequencing of the adult male and female mouse kidney with in situ expression studies and cell lineage tracing. These studies reveal cell diversity and marked sex differences, distinct organization and cell composition of nephrons dependent on the time of nephron specification, and lineage convergence, in which contiguous functionally related cell types are specified from nephron and collecting system progenitor populations. A searchable database, Kidney Cell Explorer (https://cello.shinyapps.io/kidneycellexplorer/), enables gene-cell relationships to be viewed in the anatomical framework of the kidney.

[Methods for Determining and Applications of High-Resolution Vehicle Emission Inventory at County Scale].

This study proposes a method for high-resolution vehicle emission inventories at county scale based on field investigations and Google Earth satellite images, using the Tongzhou district of Beijing as an example for data analysis. VKT and the corresponding emissions of each pollutant were calculated using the number of registered vehicles and the real-time traffic volumes, respectively. The results showed that the mileage calculated based on the method using registered vehicles is about 37% less than that based on real-time traffic volumes, with the latter method providing data closer to the actual situation. The mileage for small passenger cars, large passenger vehicles, and medium-duty trucks were underestimated, and that for light trucks, heavy trucks, low-speed trucks were overestimated based on the registered vehicle method. Emissions from small passenger cars were also underestimated by about 51%, using the registered vehicle method. For emissions of large passenger vehicles, light trucks, and medium-duty trucks, there was less difference between the two methods. Based on the registered vehicle method, emissions of heavy trucks, low-speed trucks, and motorcycles were overestimated by about 41%, 30%, and 30%, respectively.

[Motor Vehicle Pollution Control Scenarios of Beijing Subsidiary Administrative Center Based on Road Traffic Flow].

In this study, the scenario analysis method was used to establish motor vehicle exhaust emission inventories based on road-traffic-flow information of the Beijing subsidiary administrative center under different scenarios, with a base year of 2015. The pollutant emissions in 2020 and 2025 were forecast by calculating the motor vehicle pollutant emission inventories of future road-traffic-flow information and various scenarios. The results showed that the kilometers travelled via motor vehicles and the road network density of the Beijing subsidiary administrative center both will increase continuously over the next 10 years. Compared with the baseline scenario, each control scenario had a certain degree of reduction in pollutant emissions, and the reduction rates increased with the strengthening of the measures. The emission reductions of pollutants were significant in the new energy vehicle promotion scenario, especially for NOx and PM. The effect of emission reduction of every pollutant was significant in the outgoing vehicle restriction scenario. Elimination of highly polluting vehicles had a significant effect in the short term, but the effect of long-term reduction was weak. The combined scenario achieved the best reduction rate of pollutant emissions, with CO, NOx, HC, and PM decreasing by 39.0%, 58.7%, 49.2%, and 55.5%, respectively.

[Evaluation on the Effectiveness of Vehicle Exhaust Emission Control Measures During the APEC Conference in Beijing].

Vehicle emission is one of the primary factors affecting the quality of atmospheric environment in Beijing. In order to improve the air quality during APEC conference, strict control measures including vehicle emission control were taken in Beijing during APEC meeting. Based on the activity level data of traffic volume, vehicle speed and vehicle types, the inventory of motor vehicle emissions in Beijing was developed following bottom-up methodology to assess the effectiveness of the control measures. The results showed that the traffic volume of Beijing road network during the APEC meeting decreased significantly, the vehicle speed increased obviously, and the largest decline of traffic volume was car. CO, NOx, HC and PM emissions of vehicle exhaust were reduced by 15.1%, 22.4%, 18.4% and 21.8% for freeways, 29.9%, 36.4%, 32.7% and 35.8% for major arterial, 35.7%, 41.7%, 38.4% and 41.2% for minor arterial, 40.8%, 46.5%, 43.1% and 46.0% for collectors, respectively. The vehicles exhaust emissions inventory before and during APEC conference was developed based on bottom-up emissions inventory method. The results indicated that CO, NOx, HC and PM emissions of vehicle exhaust were reduced by 37.5%, 43.4%, 39.9% and 42.9% in the study area, respectively.

Multichannel optical filters with an ultranarrow bandwidth based on sampled Brillouin dynamic gratings.

We first propose a multichannel optical filter with an ultra-narrow 3-dB bandwidth based on sampled Brillouin dynamic gratings (SBDGs). The multichannel optical filter is generated when an optical pulse interfaces with an optical pulse train based on an ordinary stimulated Brillouin scattering (SBS) process in a birefringent optical fiber. Multichannel optical filter based on SBDG is generated with a 3-dB bandwidth from 12.5 MHz to 1 GHz. In addition, a linearly chirped SBDG is proposed to generate multichannel dispersion compensator with a 3-dB bandwidth of 300 MHz and an extremely high dispersion value of 432 ns/nm. The proposed multichannel optical filters have important potential applications in the optical filtering, multichannel dispersion compensation and optical signal processing.

Retinoid signaling in progenitors controls specification and regeneration of the urothelium.

The urothelium is a multilayered epithelium that serves as a barrier between the urinary tract and blood, preventing the exchange of water and toxic substances. It consists of superficial cells specialized for synthesis and transport of uroplakins that assemble into a tough apical plaque, one or more layers of intermediate cells, and keratin 5-expressing basal cells (K5-BCs), which are considered to be progenitors in the urothelium and other specialized epithelia. Fate mapping, however, reveals that intermediate cells rather than K5-BCs are progenitors in the adult regenerating urothelium, that P cells, a transient population, are progenitors in the embryo, and that retinoids are critical in P cells and intermediate cells, respectively, for their specification during development and regeneration. These observations have important implications for tissue engineering and repair and, ultimately, may lead to treatments that prevent loss of the urothelial barrier, a major cause of voiding dysfunction and bladder pain syndrome.

Six2 and Wnt regulate self-renewal and commitment of nephron progenitors through shared gene regulatory networks.

A balance between Six2-dependent self-renewal and canonical Wnt signaling-directed commitment regulates mammalian nephrogenesis. Intersectional studies using chromatin immunoprecipitation and transcriptional profiling identified direct target genes shared by each pathway within nephron progenitors. Wnt4 and Fgf8 are essential for progenitor commitment; cis-regulatory modules flanking each gene are cobound by Six2 and ß-catenin and are dependent on conserved Lef/Tcf binding sites for activity. In vitro and in vivo analyses suggest that Six2 and Lef/Tcf factors form a regulatory complex that promotes progenitor maintenance while entry of ß-catenin into this complex promotes nephrogenesis. Alternative transcriptional responses associated with Six2 and ß-catenin cobinding events occur through non-Lef/Tcf DNA binding mechanisms, highlighting the regulatory complexity downstream of Wnt signaling in the developing mammalian kidney.

Identification of molecular compartments and genetic circuitry in the developing mammalian kidney.

Lengthy developmental programs generate cell diversity within an organotypic framework, enabling the later physiological actions of each organ system. Cell identity, cell diversity and cell function are determined by cell type-specific transcriptional programs; consequently, transcriptional regulatory factors are useful markers of emerging cellular complexity, and their expression patterns provide insights into the regulatory mechanisms at play. We performed a comprehensive genome-scale in situ expression screen of 921 transcriptional regulators in the developing mammalian urogenital system. Focusing on the kidney, analysis of regional-specific expression patterns identified novel markers and cell types associated with development and patterning of the urinary system. Furthermore, promoter analysis of synexpressed genes predicts transcriptional control mechanisms that regulate cell differentiation. The annotated informational resource (www.gudmap.org) will facilitate functional analysis of the mammalian kidney and provides useful information for the generation of novel genetic tools to manipulate emerging cell populations.

Temporal differences in granulosa cell specification in the ovary reflect distinct follicle fates in mice.

The embryonic origins of ovarian granulosa cells have been a subject of debate for decades. By tamoxifen-induced lineage tracing of Foxl2-expressing cells, we show that descendants of the bipotential supporting cell precursors in the early gonad contribute granulosa cells to a specific population of follicles in the medulla of the ovary that begin to grow immediately after birth. These precursor cells arise from the proliferative ovarian surface epithelium and enter mitotic arrest prior to upregulating Foxl2. Granulosa cells that populate the cortical primordial follicles activated in adult life derive from the surface epithelium perinatally, and enter mitotic arrest at that stage. Ingression from the surface epithelium dropped to undetectable levels by Postnatal Day 7, when most surviving oocytes were individually encapsulated by granulosa cells. These findings add complexity to the standard model of sex determination in which the Sertoli and granulosa cells of the adult testis and ovary directly stem from the supporting cell precursors of the bipotential gonad.

Sequence analysis of human cytomegalovirus US28 gene in low-passage clinical isolates from children and AIDS patients.

Human cytomegalovirus (HCMV) is often a dangerous opportunistic pathogen that causes significant morbidity and mortality in newborn children and immunocompromised patients. The different symptoms and tissue tropisms of HCMV infection may result from genetic polymorphism. This study investigated the sequence variability of the HCMV US28 ORF, which shows sequence homology to the G protein-coupled receptor. HCMV isolated from suspected pediatric cases and isolates from AIDS patients were compared in order to examine the possible associations between polymorphisms and pathogenesis. Seventy children with suspected congenital HCMV infection, who suffered from jaundice (47), megacolon (10), and microcephaly (13), and 17 AIDS patients, were studied. Mutation was prevalent among the sequences of US28, with a focus on the two ends of US28. The important functional groups of US28 are highly conserved. An unrooted tree showed that all sequences from suspected congenitally infected infants and AIDS patients were divided into three groups. Comparison showed that most of the sequences (12/17) from pediatric patients were included in the first group (G1), whereas most of the sequences (11/17) from AIDS patients were included in the third group (G3). The specific high mutation sites in US28 from children were located at the C terminus of the protein, whereas those from AIDS patients were located at the N terminus. We demonstrated the existence of polymorphisms among the US28 genes of clinical isolates of HCMV from infants with suspected congenital infection. Comparison of US28 sequences from AIDS patients with those from children showed that both sequences have their own specific high mutation points.

Polymorphisms of human cytomegalovirus UL148A, UL148B, UL148C, UL148D genes in clinical strains.

BACKGROUND: Clinical isolates of human cytomegalovirus (HCMV) display polymorphisms in multiple genes. Some authors have suggested that polymorphisms are implicated in HCMV-induced immunopathogenesis, as well as in strain-specific behaviors, such as tissue-tropism and the ability to establish persistent or latent infections. OBJECTIVE: To describe the features of HCMV UL148A, UL148B, UL148C and UL148D open reading frames (ORFs) and the variable sites within the frames in clinical strains. STUDY DESIGN: PCR was performed to amplify these ORFs in 22 clinical strains. PCR amplification products were sequenced directly and analyzed. RESULTS: The nucleotide diversity of UL148A, UL148B, UL148C and UL148D ORFs in studied strains is 0.5-8.3%, 0.5-4.6%, 0.5-3% and 1.7-8.1%, respectively; the amino acid diversity of their putative proteins is 1.3-6.3%, 1.3-5.0%, 1.3-3.9% and 1.7-8.1%, respectively, related to the Merlin strain. The modification sites of UL148A, UL148B, UL148C and UL148D predicted proteins from strains in unpassaged urine samples were conserved, except for strain U96, compared with that of the Merlin strain. By phylogenetic and statistical analysis, the UL148A and UL148D sequences of clinical strains were classified into three groups. CONCLUSION: Compared to the UL148A, UL148B and UL148D ORFs, the UL148C ORF was relatively conserved, as was the amino acid sequence of the UL148C putative protein. Isolates that have been passaged several times in human embryonic lung fibroblasts (HELF) showed some changes of modification sites, however. A discrete linkage was found between the groups of UL148A gene and those of UL148D gene.

[Polymorphisms of HCMV US28 gene of the clinical isolates from children].

BACKGROUND: To study the polymorphism of human cytomegalovirus US28 gene in children and investigate the relationship between the polymorphism and pathogenesis. METHODS: The FQ-PCR was carried out to determine the DNA quantity of clinical isolate and then the segmental PCR and HMA-SSCP were performed to test the mutation of US28 gene. The typical isolates from different diseases were selected to clone and sequence, then the results were analyzed. RESULTS: The nucleic acid mutation is frequent among the sequence of US28, those mutations focus on the two ends of US28, but most of them are sense mutation. The important functional groups of US28 are highly conserved. The amino acid mutation of some isolates resulted in the change of secondary structure, but the phylogenetic tree analysis did not show any clear association between the pathogenesis and the distribution of clinical isolates. The comparison of US28 sequences from AIDS patients with the sequences from children in our study showed that both sequences have their own specific high mutation points. CONCLUSION: There is polymorphism among the HCMV-US28 gene of clinical isolates from children. There observed no clear relationship was between the pathogenesis and the distribution of clinical isolates.