Efficient Messenger RNA Delivery to the Kidney Using Renal Pelvis Injection in Mice

Renal dysfunction is often associated with the inflammatory cascade, leading to non-reversible nephrofibrosis. Gene therapy has the ability to treat the pathology. However, the difficulty in introducing genes into the kidney, via either viral vectors or plasmid DNA (pDNA), has hampered its extensive clinical use. Messenger RNA (mRNA) therapeutics has recently attracted attention as alternative gene therapies. mRNA allows protein production into post-mitotic cells without the need for transport to the nuclei in the target cells. However, few studies have reported the delivery of mRNA to the kidney. In this study, we attempted to deliver mRNA to the kidney based on the principle of pressure stimulation, by administering mRNA-loaded polyplex nanomicelles via a renal pelvis injection, directly into the kidney. Compared with the administration of naked plasmid DNA (pDNA) and naked mRNA, the mRNA-loaded nanomicelles diffusely induced protein expression in a greater number of cells at the tubular epithelium for some days. The plasma creatinine (Cre) and blood urea nitrogen (BUN) levels after the administration remained similar to those of the sham-operated controls, without marked changes in histological sections. The safety and efficacy of mRNA-loaded nanomicelles would make distinct contributions to the development of mRNA therapeutics for the kidney

Vasopressin gene products are colocalised with corticotrophin‐releasing factor within neurosecretory vesicles in the external zone of the median eminence of the Japanese macaque monkey (Macaca fuscata)

Arginine vasopressin (AVP), when released into portal capillaries with corticotrophin‐releasing factor (CRF) from terminals of parvocellular neurones of the hypothalamic paraventricular nucleus (PVH), facilitates the secretion of adrenocorticotrophic hormone (ACTH) in stressed rodents. The AVP gene encodes a propeptide precursor containing AVP, AVP‐associated neurophysin II (NPII), and a glycopeptide copeptin, although it is currently unclear whether copeptin is always cleaved from the neurophysin and whether the NPII and/or copeptin have any functional role in the pituitary. Furthermore, for primates, it is unknown whether CRF, AVP, NPII and copeptin are all colocalised in neurosecretory vesicles in the terminal region of the paraventricular CRF neurone axons. Therefore, we investigated, by fluorescence and immunogold immunocytochemistry, the cellular and subcellular relationships of these peptides in the CRF‐ and AVP‐producing cells in unstressed Japanese macaque monkeys (Macaca fuscata). Reverse transcription‐polymerase chain reaction analysis showed the expression of both CRF and AVP mRNAs in the monkey PVH. As expected, in the magnocellular neurones of the PVH and supraoptic nucleus, essentially no CRF immunoreactivity could be detected in NPII‐immunoreactive (AVP‐producing) neurones. Immunofluorescence showed that, in the parvocellular part of the PVH, NPII was detectable in a subpopulation (approximately 39%) of the numerous CRF‐immunoreactive neuronal perikarya, whereas, in the outer median eminence, NPII was more prominent (approximately 52%) in the CRF varicosities. Triple immunoelectron microscopy in the median eminence demonstrated the presence of both NPII and copeptin immunoreactivity in dense‐cored vesicles of CRF‐containing axons. The results are consistent with an idea that the AVP propeptide is processed and NPII and copeptin are colocalised in hypothalamic‐pituitary CRF axons in the median eminence of a primate. The CRF, AVP and copeptin are all co‐packaged in neurosecretory vesicles in monkeys and are thus likely to be co‐released into the portal capillary blood to amplify ACTH release from the primate anterior pituitary

Structure of the far-red light utilizing photosystem I of Acaryochloris marina

赤外光駆動型光合成をクライオ電顕で捉えることに成功 --低いエネルギーで通常の光化学反応が駆動される仕組み--. 京都大学プレスリリース. 2021-04-21.Acaryochloris marina is one of the cyanobacterial species that can use far-red light to drive photochemical reactions for oxygenic photosynthesis. Here, we report the structure of A. marina photosystem I (PSI) reaction center, determined by cryo-electron microscopy at 2.58 Å resolution. The structure reveals an arrangement of electron carriers and light-harvesting pigments distinct from other type I reaction centers. The paired chlorophyll, or special pair (also referred to as P740 in this case), is a dimer of chlorophyll d and its epimer chlorophyll d′. The primary electron acceptor is pheophytin a, a metal-less chlorin. We show the architecture of this PSI reaction center is composed of 11 subunits and we identify key components that help explain how the low energy yield from far-red light is efficiently utilized for driving oxygenic photosynthesis

Variation of pro‐vasopressin processing in parvocellular and magnocellular neurons in the paraventricular nucleus of the hypothalamus: Evidence from the vasopressin‐related glycopeptide copeptin

Arginine vasopressin (AVP) is synthesized in parvocellular‐ and magnocellular neuroendocrine neurons in the paraventricular nucleus (PVN) of the hypothalamus. Whereas magnocellular AVP neurons project primarily to the posterior pituitary, parvocellular AVP neurons project to the median eminence (ME) and to extrahypothalamic areas. The AVP gene encodes pre‐pro‐AVP that comprises the signal peptide, AVP, neurophysin (NPII), and a copeptin glycopeptide. In the present study, we used an N‐terminal copeptin antiserum to examine copeptin expression in magnocellular and parvocellular neurons in the hypothalamus in the mouse, rat, and macaque monkey. Although magnocellular NPII‐expressing neurons exhibited strong N‐terminal copeptin immunoreactivity in all three species, a great majority (~90%) of parvocellular neurons that expressed NPII was devoid of copeptin immunoreactivity in the mouse, and in approximately half (~53%) of them in the rat, whereas in monkey hypothalamus, virtually all NPII‐immunoreactive parvocellular neurons contained strong copeptin immunoreactivity. Immunoelectron microscopy in the mouse clearly showed copeptin‐immunoreactivity co‐localized with NPII‐immunoreactivity in neurosecretory vesicles in the internal layer of the ME and posterior pituitary, but not in the external layer of the ME. Intracerebroventricular administration of a prohormone convertase inhibitor, hexa‐d‐arginine amide resulted in a marked reduction of copeptin‐immunoreactivity in the NPII‐immunoreactive magnocellular PVN neurons in the mouse, suggesting that low protease activity and incomplete processing of pro‐AVP could explain the disproportionally low levels of N‐terminal copeptin expression in rodent AVP (NPII)‐expressing parvocellular neurons. Physiologic and phylogenetic aspects of copeptin expression among neuroendocrine neurons require further exploration

Characterization of transgene expression and pDNA distribution of the suctioned kidney in mice

We have previously developed an efficient and safe transfection method for the kidney in mice: renal suction-mediated transfection. In this study, we verified the detailed characteristics of transgene expression and plasmid DNA (pDNA) in mice to develop therapeutic strategies and application to gene function analysis in the kidney. After naked pDNA was administered intravenously, the right kidney was immediately suctioned by a tissue suction device. We examined the spatial distribution of transgene expression and pDNA in the suctioned kidney using tissue clearing by CUBIC, ClearT2, and Scale SQ reagents. Spatial distribution analysis showed that pDNA was transfected into extravascular cells and sufficiently delivered to the deep renal cortex. In addition, we revealed that transgene expression occurred mainly in peritubular fibroblasts of the suctioned kidney by tissue clearing and immunohistochemistry. Next, we confirmed the periods of pDNA uptake and activation of transcription factors nuclear factor-κB and activator protein 1 by luciferase assays. Moreover, the use of a pCpG-free plasmid enabled sustained transgene expression in the suctioned kidney. In conclusion, analyses of the spatial distribution and immunostaining of the section suggest that pDNA and transgene expression occurs mainly in peritubular fibroblasts of the suctioned kidney. In addition, we clarified some factors for efficient and/or sustained transgene expression in the suctioned kidney

Effects of Tissue Pressure on Transgene Expression Characteristics via Renal Local Administration Routes from Ureter or Renal Artery in the Rat Kidney

We previously developed a renal pressure-mediated transfection method (renal pressure method) as a kidney-specific in vivo gene delivery system. However, additional information on selecting other injection routes and applicable animals remains unclear. In this study, we selected renal arterial and ureteral injections as local administration routes and evaluated the characteristics of gene delivery such as efficacy, safety, and distribution in pressured kidney of rat. Immediately after the naked pDNA injection, via renal artery or ureter, the left kidney of the rat was pressured using a pressure controlling device. Transfection efficiency of the pressured kidney was about 100-fold higher than that of the injection only group in both administration routes. The optimal pressure intensity in the rat kidney was 1.2 N/cm2 for renal arterial injection and 0.9 N/cm2 for ureteral injection. We found that transgene expression site differs according to administration route: cortical fibroblasts and renal tubule in renal arterial injection and cortical and medullary tubule and medullary collecting duct in ureteral injection. This is the first report to demonstrate that the renal pressure method can also be effective, after renal arterial and ureteral injections, in rat kidney

Degradation of Mutant Protein Aggregates within the Endoplasmic Reticulum of Vasopressin Neurons

Misfolded or unfolded proteins in the ER are said to be degraded only after translocation or isolation from the ER. Here, we describe a mechanism by which mutant proteins are degraded within the ER. Aggregates of mutant arginine vasopressin (AVP) precursor were confined to ER-associated compartments (ERACs) connected to the ER in AVP neurons of a mouse model of familial neurohypophysial diabetes insipidus. The ERACs were enclosed by membranes, an ER chaperone and marker protein of phagophores and autophagosomes were expressed around the aggregates, and lysosomes fused with the ERACs. Moreover, lysosome-related molecules were present within the ERACs, and aggregate degradation within the ERACs was dependent on autophagic-lysosomal activity. Thus, we demonstrate that protein aggregates can be degraded by autophagic-lysosomal machinery within specialized compartments of the ER

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Efficient Messenger RNA Delivery to the Kidney Using Renal Pelvis Injection in Mice

Renal dysfunction is often associated with the inflammatory cascade, leading to non-reversible nephrofibrosis. Gene therapy has the ability to treat the pathology. However, the difficulty in introducing genes into the kidney, via either viral vectors or plasmid DNA (pDNA), has hampered its extensive clinical use. Messenger RNA (mRNA) therapeutics has recently attracted attention as alternative gene therapies. mRNA allows protein production into post-mitotic cells without the need for transport to the nuclei in the target cells. However, few studies have reported the delivery of mRNA to the kidney. In this study, we attempted to deliver mRNA to the kidney based on the principle of pressure stimulation, by administering mRNA-loaded polyplex nanomicelles via a renal pelvis injection, directly into the kidney. Compared with the administration of naked plasmid DNA (pDNA) and naked mRNA, the mRNA-loaded nanomicelles diffusely induced protein expression in a greater number of cells at the tubular epithelium for some days. The plasma creatinine (Cre) and blood urea nitrogen (BUN) levels after the administration remained similar to those of the sham-operated controls, without marked changes in histological sections. The safety and efficacy of mRNA-loaded nanomicelles would make distinct contributions to the development of mRNA therapeutics for the kidney

Efficient Messenger RNA Delivery to the Kidney Using Renal Pelvis Injection in Mice

Renal dysfunction is often associated with the inflammatory cascade, leading to non-reversible nephrofibrosis. Gene therapy has the ability to treat the pathology. However, the difficulty in introducing genes into the kidney, via either viral vectors or plasmid DNA (pDNA), has hampered its extensive clinical use. Messenger RNA (mRNA) therapeutics has recently attracted attention as alternative gene therapies. mRNA allows protein production into post-mitotic cells without the need for transport to the nuclei in the target cells. However, few studies have reported the delivery of mRNA to the kidney. In this study, we attempted to deliver mRNA to the kidney based on the principle of pressure stimulation, by administering mRNA-loaded polyplex nanomicelles via a renal pelvis injection, directly into the kidney. Compared with the administration of naked plasmid DNA (pDNA) and naked mRNA, the mRNA-loaded nanomicelles diffusely induced protein expression in a greater number of cells at the tubular epithelium for some days. The plasma creatinine (Cre) and blood urea nitrogen (BUN) levels after the administration remained similar to those of the sham-operated controls, without marked changes in histological sections. The safety and efficacy of mRNA-loaded nanomicelles would make distinct contributions to the development of mRNA therapeutics for the kidney