Single-cell multi-omics in the medicinal plant Catharanthus roseus.

Advances in omics technologies now permit the generation of highly contiguous genome assemblies, detection of transcripts and metabolites at the level of single cells and high-resolution determination of gene regulatory features. Here, using a complementary, multi-omics approach, we interrogated the monoterpene indole alkaloid (MIA) biosynthetic pathway in Catharanthus roseus, a source of leading anticancer drugs. We identified clusters of genes involved in MIA biosynthesis on the eight C. roseus chromosomes and extensive gene duplication of MIA pathway genes. Clustering was not limited to the linear genome, and through chromatin interaction data, MIA pathway genes were present within the same topologically associated domain, permitting the identification of a secologanin transporter. Single-cell RNA-sequencing revealed sequential cell-type-specific partitioning of the leaf MIA biosynthetic pathway that, when coupled with a single-cell metabolomics approach, permitted the identification of a reductase that yields the bis-indole alkaloid anhydrovinblastine. We also revealed cell-type-specific expression in the root MIA pathway.

Integration of cell differentiation and initiation of monoterpenoid indole alkaloid metabolism in seed germination of Catharanthus roseus.

In Catharanthus roseus, monoterpenoid indole alkaloids (MIAs) are produced through the cooperation of four cell types, with final products accumulating in specialized cells known as idioblasts and laticifers. To explore the relationship between cellular differentiation and cell type-specific MIA metabolism, we analyzed the expression of MIA biosynthesis in germinating seeds. Embryos from immature and mature seeds were observed via stereomicroscopy, fluorescence microscopy, and electron microscopy. Time-series MIA and iridoid quantification, along with transcriptome analysis, were conducted to determine the initiation of MIA biosynthesis. In addition, the localization of MIAs was examined using alkaloid staining and imaging mass spectrometry (IMS). Laticifers were present in embryos before seed maturation. MIA biosynthesis commenced 12 h after germination. MIAs accumulated in laticifers of embryos following seed germination, and MIA metabolism is induced after germination in a tissue-specific manner. These findings suggest that cellular morphological differentiation precedes metabolic differentiation. Considering the well-known toxicity and defense role of MIAs in matured plants, MIAs may be an important defense strategy already in the delicate developmental phase of seed germination, and biosynthesis and accumulation of MIAs may require the tissue and cellular differentiation.

In vivo characterization of key iridoid biosynthesis pathway genes in catnip (Nepeta cataria).

MAIN CONCLUSION: Using virus-induced gene silencing, we demonstrated that the enzymes GES, ISY, and MLPL are responsible for nepetalactone biosynthesis in Nepeta cataria. Nepetalactone is the main iridoid that is found in the Nepeta genus and is well-known for its psychoactive effect on house cats. Moreover, there is a burgeoning interest into the effect of nepetalactone on insects. Although the enzymes for nepetalactone biosynthesis have been biochemically assayed in vitro, validation of the role that these enzymes have in planta has not been demonstrated. Virus-induced gene silencing (VIGS) is a silencing method that relies on transient transformation and is an approach that has been particularly successful when applied to a variety of non-model plants. Here, we use a recently designed visual-marker dependent VIGS system to demonstrate that the nepetalactone biosynthetic enzymes GES, ISY, and MLPL impact nepetalactone biosynthesis in Nepeta cataria.

Improved virus-induced gene silencing allows discovery of a serpentine synthase gene in Catharanthus roseus.

Specialized metabolites are chemically complex small molecules with a myriad of biological functions. To investigate plant-specialized metabolite biosynthesis more effectively, we developed an improved method for virus-induced gene silencing (VIGS). We designed a plasmid that incorporates fragments of both the target gene and knockdown marker gene (phytoene desaturase, PDS), which identifies tissues that have been successfully silenced in planta. To demonstrate the utility of this method, we used the terpenoid indole alkaloid (TIA) pathway in Madagascar periwinkle (Catharanthus roseus) as a model system. Catharanthus roseus is a medicinal plant well known for producing many bioactive compounds, such as vinblastine and vincristine. Our VIGS method enabled the discovery of a previously unknown biosynthetic enzyme, serpentine synthase (SS). This enzyme is a cytochrome P450 (CYP) that produces the ß-carboline alkaloids serpentine and alstonine, compounds with strong blue autofluorescence and potential pharmacological activity. The discovery of this enzyme highlights the complexity of TIA biosynthesis and demonstrates the utility of this improved VIGS method for discovering unidentified metabolic enzymes in plants.

Differential regulation of fluorescent alkaloid metabolism between idioblast and lacticifer cells during leaf development in Catharanthus roseus seedlings.

Bioactive specialized (secondary) metabolites are indispensable for plant development or adjustment to their surrounding environment. In many plants, these specialized metabolites are accumulated in specifically differentiated cells. Catharanthus roseus is a well-known medicinal plant known for producing many kinds of monoterpenoid indole alkaloids (MIAs). C. roseus has two types of specifically differentiated cells accumulating MIAs, so-called idioblast cells and laticifer cells. In this study, we compared each of the cells as they changed during seedling growth, and found that the fluorescent metabolites accumulated in these cells were differentially regulated. Analysis of fluorescent compounds revealed that the fluorescence observed in these cells was emitted from the compound serpentine. Further, we found that the serpentine content of leaves increased as leaves grew. Our findings suggest that idioblast cells and laticifer cells have different biological roles in MIA biosynthesis and its regulation.

The formation of perinucleolar bodies is important for normal leaf development and requires the zinc-finger DNA-binding motif in Arabidopsis ASYMMETRIC LEAVES2.

In Arabidopsis, the ASYMMETRIC LEAVES2 (AS2) protein plays a key role in the formation of flat symmetric leaves via direct repression of the abaxial gene ETT/ARF3. AS2 encodes a plant-specific nuclear protein that contains the AS2/LOB domain, which includes a zinc-finger (ZF) motif that is conserved in the AS2/LOB family. We have shown that AS2 binds to the coding DNA of ETT/ARF3, which requires the ZF motif. AS2 is co-localized with AS1 in perinucleolar bodies (AS2 bodies). To identify the amino acid signals in AS2 required for formation of AS2 bodies and function(s) in leaf formation, we constructed recombinant DNAs that encoded mutant AS2 proteins fused to yellow fluorescent protein. We examined the subcellular localization of these proteins in cells of cotyledons and leaf primordia of transgenic plants and cultured cells. The amino acid signals essential for formation of AS2 bodies were located within and adjacent to the ZF motif. Mutant AS2 that failed to form AS2 bodies also failed to rescue the as2-1 mutation. Our results suggest the importance of the formation of AS2 bodies and the nature of interactions of AS2 with its target DNA and nucleolar factors including NUCLEOLIN1. The partial overlap of AS2 bodies with perinucleolar chromocenters with condensed ribosomal RNA genes implies a correlation between AS2 bodies and the chromatin state. Patterns of AS2 bodies in cells during interphase and mitosis in leaf primordia were distinct from those in cultured cells, suggesting that the formation and distribution of AS2 bodies are developmentally modulated in plants.

The complexity of intercellular localisation of alkaloids revealed by single-cell metabolomics.

Catharanthus roseus is a medicinal plant well known for producing bioactive compounds such as vinblastine and vincristine, which are classified as terpenoid indole alkaloids (TIAs). Although the leaves of this plant are the main source of these antitumour drugs, much remains unknown on how TIAs are biosynthesised from a central precursor, strictosidine, to various TIAs in planta. Here, we have succeeded in showing, for the first time in leaf tissue of C. roseus, cell-specific TIAs localisation and accumulation with 10 µm spatial resolution Imaging mass spectrometry (Imaging MS) and live single-cell mass spectrometry (single-cell MS). These metabolomic studies revealed that most TIA precursors (iridoids) are localised in the epidermal cells, but major TIAs including serpentine and vindoline are localised instead in idioblast cells. Interestingly, the central TIA intermediate strictosidine also accumulates in both epidermal and idioblast cells of C. roseus. Moreover, we also found that vindoline accumulation increases in laticifer cells as the leaf expands. These discoveries highlight the complexity of intercellular localisation in plant specialised metabolism.

Cell-specific localization of alkaloids in Catharanthus roseus stem tissue measured with Imaging MS and Single-cell MS.

Catharanthus roseus (L.) G. Don is a medicinal plant well known for producing antitumor drugs such as vinblastine and vincristine, which are classified as terpenoid indole alkaloids (TIAs). The TIA metabolic pathway in C. roseus has been extensively studied. However, the localization of TIA intermediates at the cellular level has not been demonstrated directly. In the present study, the metabolic pathway of TIA in C. roseus was studied with two forefront metabolomic techniques, that is, Imaging mass spectrometry (MS) and live Single-cell MS, to elucidate cell-specific TIA localization in the stem tissue. Imaging MS indicated that most TIAs localize in the idioblast and laticifer cells, which emit blue fluorescence under UV excitation. Single-cell MS was applied to four different kinds of cells [idioblast (specialized parenchyma cell), laticifer, parenchyma, and epidermal cells] in the stem longitudinal section. Principal component analysis of Imaging MS and Single-cell MS spectra of these cells showed that similar alkaloids accumulate in both idioblast cell and laticifer cell. From MS/MS analysis of Single-cell MS spectra, catharanthine, ajmalicine, and strictosidine were found in both cell types in C. roseus stem tissue, where serpentine was also accumulated. Based on these data, we discuss the significance of TIA synthesis and accumulation in the idioblast and laticifer cells of C. roseus stem tissue.

Correction to: Space-time analysis of gravitropism in etiolated Arabidopsis hypocotyls using bioluminescence imaging of the IAA19 promoter fusion with a destabilized luciferase reporter.

The article Space-time analysis of gravitropism in etiolated Arabidopsis hypocotyls using bioluminescence imaging of the IAA19 promoter fusion with a destabilized luciferase reporter, written by Kotaro T. Yamamoto, Masaaki K. Watahiki, Jun Matsuzaki, Soichirou Satoh and Hisayo Shimizu, was originally published electronically on the publisher's internet portal (currently SpringerLink) on 10 April 2017 without open access.

The neuroprotective effect of latanoprost acts via klotho-mediated suppression of calpain activation after optic nerve transection.

Latanoprost was first developed for use in glaucoma therapy as an ocular hypotensive agent targeting the prostaglandin F2α (FP) receptor. Subsequently, latanoprost showed a neuroprotective effect, an additional pharmacological action. However, although it is well-known that latanoprost exerts an ocular hypotensive effect via the FP receptor, it is not known whether this is also true of its neuroprotective effect. Klotho was firstly identified as the gene linked to the suppression of aging phenotype: the defect of klotho gene in mice results aging phenotype such as hypokinesis, arteriosclerosis, and short lifespan. After that, the function of klotho was also reported to maintain calcium homeostasis and to exert a neuroprotective effect in various models of neurodegenerative disease. However, the function of klotho in eyes including retina is still poorly understood. Here, we show that klotho is a key factor underlying the neuroprotective effect of latanoprost during post-axotomy retinal ganglion cell (RGC) degeneration. Importantly, a quantitative RT-PCR gene expression analysis of klotho in sorted rat retinal cells revealed that the highest expression level of klotho in the retina was in the RGCs. Latanoprost acid, the biologically active form of latanoprost, inhibits post-traumatic calpain activation and concomitantly facilitates the expression and shedding of klotho in axotomized RGCs. This expression profile is a good match with the localization, not of the FP receptor, but of organic anion transporting polypeptide 2B1, known as a prostaglandin transporter, in the ocular tissue. Furthermore, an organic anion transporting polypeptide 2B1 inhibitor suppressed latanoprost acid-mediated klotho shedding ex vivo, whereas an FP receptor antagonist did not. The klotho fragments shed from the RGCs reduced the intracellular level of reactive oxygen species, and a specific klotho inhibitor accelerated and increased RGC death after axotomy. We conclude that the shed klotho fragments might contribute to the attenuation of axonal injury-induced calpain activation and oxidative stress, thereby protecting RGCs from post-traumatic neuronal degeneration.

Space-time analysis of gravitropism in etiolated Arabidopsis hypocotyls using bioluminescence imaging of the IAA19 promoter fusion with a destabilized luciferase reporter.

Imaging analysis was carried out during the gravitropic response of etiolated Arabidopsis hypocotyls, using an IAA19 promoter fusion of destabilized luciferase as a probe. From the bright-field images we obtained the local deflection angle to the vertical, A, local curvature, C, and the partial derivative of C with respect to time, [Formula: see text]. These were determined every 19.9 µm along the curvilinear length of the hypocotyl, at ~10 min intervals over a period of ~6 h after turning hypocotyls through 90° to the horizontal. Similarly from the luminescence images we measured the luminescence intensity of the convex and concave flanks of the hypocotyl as well as along the median of the hypocotyl, to determine differential expression of auxin-inducible IAA19. Comparison of these parameters as a function of time and curvilinear length shows that the gravitropic response is composed of three successive elements: the first and second curving responses and a decurving response (autostraightening). The maximum of the first curving response occurs when A is 76° along the entire length of the hypocotyl, suggesting that A is the sole determinant in this response; in contrast, the decurving response is a function of both A and C, as predicted by the newly-proposed graviproprioception model (Bastien et al., Proc Natl Acad Sci USA 110:755-760, 2013). Further, differential expression of IAA19, with higher expression in the convex flank, is observed at A = 44°, and follows the Sachs' sine law. This also suggests that IAA19 is not involved in the first curving response. In summary, the gravitropic response of Arabidopsis hypocotyls consists of multiple elements that are each determined by separate principles.

Bleeding sites in elderly trauma patients who required massive transfusion: a comparison with younger patients.

INTRODUCTION: Among elderly patients with severe trauma, the sites of massive hemorrhage and their clinical characteristics are not well understood. Therefore, we investigated the sites of massive hemorrhage in patients with severe trauma, and compared the results for younger and elderly patients. METHODS: A cohort of severe trauma patients (Injury Severity Score ≥16) admitted from March 2007 to December 2014 was reviewed retrospectively. The inclusion criterion was massive bleeding, which was defined as bleeding that required the transfusion of ≥10 red cell concentrate units within 24 hours of admission, or as cases of early death that occurred despite continuous blood transfusion and before the patient could receive ≥10 red cell concentrate units within the first 24 hours after their admission. RESULTS: Eighty-four patients met our inclusion criterion. The younger group (<65 years old) included 40 patients (48%), whereas the older group (≥65 years old) included 44 patients (52%). The percentage of nondiagnosable cases at the primary survey (massive bleeding due to multisite damage caused by a bone fracture or contusion, retroperitoneal hematoma without a pelvic ring fracture and with stable pelvic ring fracture) was 14% in the younger group and 40% in the older group (odds ratio, 3.92; 95% confidence interval, 1.37-11.27, P = .017). CONCLUSIONS: Even if no abnormalities are observed at the primary survey of elderly patients with severe trauma, physicians should consider the possibility of massive bleeding.

Reduced phototropism in pks mutants may be due to altered auxin-regulated gene expression or reduced lateral auxin transport.

Phototropism allows plants to orient their photosynthetic organs towards the light. In Arabidopsis, phototropins 1 and 2 sense directional blue light such that phot1 triggers phototropism in response to low fluence rates, while both phot1 and phot2 mediate this response under higher light conditions. Phototropism results from asymmetric growth in the hypocotyl elongation zone that depends on an auxin gradient across the embryonic stem. How phototropin activation leads to this growth response is still poorly understood. Members of the phytochrome kinase substrate (PKS) family may act early in this pathway, because PKS1, PKS2 and PKS4 are needed for a normal phototropic response and they associate with phot1 in vivo. Here we show that PKS proteins are needed both for phot1- and phot2-mediated phototropism. The phototropic response is conditioned by the developmental asymmetry of dicotyledonous seedlings, such that there is a faster growth reorientation when cotyledons face away from the light compared with seedlings whose cotyledons face the light. The molecular basis for this developmental effect on phototropism is unknown; here we show that PKS proteins play a role at the interface between development and phototropism. Moreover, we present evidence for a role of PKS genes in hypocotyl gravi-reorientation that is independent of photoreceptors. pks mutants have normal levels of auxin and normal polar auxin transport, however they show altered expression patterns of auxin marker genes. This situation suggests that PKS proteins are involved in auxin signaling and/or lateral auxin redistribution.

Application of clotrimazole via a novel controlled release device provides potent retinal protection.

Age-related macular degeneration is the leading cause of legal blindness among older individuals. Therefore, the development of new therapeutic agents and optimum drug delivery systems for its treatment are crucial. In this study, we investigate whether clotrimazole (CLT) is capable of protecting retinal cells against oxidative-induced injury and the possible inhibitory effect of a sustained CLT-release device against light-induced retinal damage in rats. In vitro results indicated pretreatment of immortalized retinal pigment epithelium cells (RPE-J cells) with 10-50 µM CLT before exposure to oxygen/glucose deprivation conditions for 48 h decreased the extent of cell death, attenuated the percentage of reactive oxygen species-positive cells, and decreased the levels of cleaved caspase-3. The device consists of a separately fabricated reservoir, a CLT formulation, and a controlled release cover, which are made of poly(ethyleneglycol) dimethacrylate (PEGDM) and tri(ethyleneglycol) dimethacrylate (TEGDM). The release rate of CLT was successfully tuned by changing the ratio of PEGDM/TEGDM in the cover. In vivo results showed that use of a CLT-loaded device lessened the reduction of electroretinographic amplitudes after light exposure. These findings indicate that the application of a polymeric CLT-loaded device may be a promising method for the treatment of some retinal disorders.

The role of calpain in an in vivo model of oxidative stress-induced retinal ganglion cell damage.

PURPOSE: In this study, we set out to establish an in vivo animal model of oxidative stress in the retinal ganglion cells (RGCs) and determine whether there is a link between oxidative stress in the RGCs and the activation of calpain, a major part of the apoptotic pathway. MATERIALS AND METHODS: Oxidative stress was induced in the RGCs of C57BL/6 mice by the intravitreal administration of 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH, 30mM, 2µl). Control eyes were injected with 2µl of vehicle. Surviving Fluorogold (FG)-labeled RGCs were then counted in retinal flat mounts. Double staining with CellROX and Annexin V was performed to investigate the co-localization of free radical generation and apoptosis. An immunoblot assay was used both to indirectly evaluate calpain activation in the AAPH-treated eyes by confirming α-fodrin cleavage, and also to evaluate the effect of SNJ-1945 (a specific calpain inhibitor: 4% w/v, 100mg/kg, intraperitoneal administration) in these eyes. RESULTS: Intravitreal administration of AAPH led to a significant decrease in FG-labeled RGCs 7days after treatment (control: 3806.7±575.2RGCs/mm(2), AAPH: 3156.1±371.2RGCs/mm(2), P<0.01). CellROX and Annexin V signals were co-localized in the FG-labeled RGCs 24h after AAPH injection. An immunoblot assay revealed a cleaved α-fodrin band that increased significantly 24h after AAPH administration. Intraperitoneally administered SNJ-1945 prevented the cleavage of α-fodrin and had a neuroprotective effect against AAPH-induced RGC death (AAPH: 3354.0±226.9RGCs/mm(2), AAPH+SNJ-1945: 3717.1±614.6RGCs/mm(2), P<0.01). CONCLUSION: AAPH administration was an effective model of oxidative stress in the RGCs, showing that oxidative stress directly activated the calpain pathway and induced RGC death. Furthermore, inhibition of the calpain pathway protected the RGCs after AAPH administration.

Critical neuroprotective roles of heme oxygenase-1 induction against axonal injury-induced retinal ganglion cell death.

Although axonal damage induces significant retinal ganglion cell (RGC) death, small numbers of RGCs are able to survive up to 7 days after optic nerve crush (NC) injury. To develop new treatments, we set out to identify patterns of change in the gene expression of axonal damage-resistant RGCs. To compensate for the low density of RGCs in the retina, we performed retrograde labeling of these cells with 4Di-10ASP in adult mice and 7 days after NC purified the RGCs with fluorescence-activated cell sorting. Gene expression in the cells was determined with a microarray, and the expression of Ho-1 was determined with quantitative PCR (qPCR). Changes in protein expression were assessed with immunohistochemistry and immunoblotting. Additionally, the density of Fluoro-gold-labeled RGCs was counted in retinas from mice pretreated with CoPP, a potent HO-1 inducer. The microarray and qPCR analyses showed increased expression of Ho-1 in the post-NC RGCs. Immunohistochemistry also showed that HO-1-positive cells were present in the ganglion cell layer (GCL), and cell counting showed that the proportion of HO-1-positive cells in the GCL rose significantly after NC. Seven days after NC, the number of RGCs in the CoPP-treated mice was significantly higher than in the control mice. Combined pretreatment with SnPP, an HO-1 inhibitor, suppressed the neuroprotective effect of CoPP. These results reflect changes in HO-1 activity to RGCs that are a key part of RGC survival. Upregulation of HO-1 signaling may therefore be a novel therapeutic strategy for glaucoma.

Light-dependent gravitropism and negative phototropism of inflorescence stems in a dominant Aux/IAA mutant of Arabidopsis thaliana, axr2.

Gravitropism and phototropism of the primary inflorescence stems were examined in a dominant Aux/IAA mutant of Arabidopsis, axr2/iaa7, which did not display either tropism in hypocotyls. axr2-1 stems completely lacked gravitropism in the dark but slowly regained it in light condition. Though wild-type stems showed positive phototropism, axr2 stems displayed negative phototropism with essentially the same light fluence-response curve as the wild type (WT). Application of 1-naphthaleneacetic acid-containing lanolin to the stem tips enhanced the positive phototropism of WT, and reduced the negative phototropism of axr2. Decapitation of stems caused a small negative phototropism in WT, but did not affect the negative phototropism of axr2. p-glycoprotein 1 (pgp1) pgp19 double mutants showed no phototropism, while decapitated double mutants exhibited negative phototropism. Expression of auxin-responsive IAA14/SLR, IAA19/MSG2 and SAUR50 genes was reduced in axr2 and pgp1 pgp19 stems relative to that of WT. These suggest that the phototropic response of stem is proportional to the auxin supply from the shoot apex, and that negative phototropism may be a basal response to unilateral blue-light irradiation when the levels of auxin or auxin signaling are reduced to the minimal level in the primary stems. In contrast, all of these treatments reduced or did not affect gravitropism in wild-type or axr2 stems. Tropic responses of the transgenic lines that expressed axr2-1 protein by the endodermis-specific promoter suggest that AXR2-dependent auxin response in the endodermis plays a more crucial role in gravitropism than in phototropism in stems but no significant roles in either tropism in hypocotyls.

Feedback control of automatic navigation for cyborg cockroach without external motion capture system.

Due to their size and locomotion ability, cockroaches are favorable as hybrid robot platforms in search and rescue (SAR) missions. However, cockroaches most likely approach the corner area and stay for an uncertain time. This natural behavior will hinder the utilization of cyborg cockroaches in SAR missions under rubble, unstructured, and unknown areas. Therefore, we proposed onboard automatic obstacle avoidance and human detection that can run on the wireless backpack stimulator without an external motion capture system. A low-power and small-size Time of Flight (ToF) sensor was selected as a distance measurement sensor, while a low-resolution thermopile array sensor was applied for human presence detection. The implemented feedback control based on IMU and ToF sensors has successfully navigated the cyborg cockroach to avoid obstacles and escape from the sharp corners in the laboratory unstructured area without stopping or being trapped. It could also recognize the human presence when the human was in front of it in real-time. Due to its performance, the random forest classifier was implemented as an embedded human detection system. It could achieve the highest accuracy at a distance of around 25 cm (92.5%) and the lowest accuracy at about 100 cm (70%).

Clinical profile of patients with diabetic ketoacidosis and hyperglycemic hyperosmolar syndrome in Japan: a multicenter retrospective cohort study.

INTRODUCTION: Diabetic ketoacidosis (DKA) and hyperglycemic hyperosmolar syndrome (HHS) are life-threatening complications of diabetes mellitus. Their clinical profiles have not been fully investigated. METHODS: A multicenter retrospective cohort study was conducted in 21 acute care hospitals in Japan. Patients included were adults aged 18 or older who had been hospitalized from January 1, 2012, to December 31, 2016 due to DKA or HHS. The data were extracted from patient medical records. A four-group comparison (mild DKA, moderate DKA, severe DKA, and HHS) was performed to evaluate outcomes. RESULTS: A total of 771 patients including 545 patients with DKA and 226 patients with HHS were identified during the study period. The major precipitating factors of disease episodes were poor medication compliance, infectious diseases, and excessive drinking of sugar-sweetened beverages. The median hospital stay was 16 days [IQR 10-26 days]. The intensive care unit (ICU) admission rate was 44.4% (mean) and the rate at each hospital ranged from 0 to 100%. The in-hospital mortality rate was 2.8% in patients with DKA and 7.1% in the HHS group. No significant difference in mortality was seen among the three DKA groups. CONCLUSIONS: The mortality rate of patients with DKA in Japan is similar to other studies, while that of HHS was lower. The ICU admission rate varied among institutions. There was no significant association between the severity of DKA and mortality in the study population. TRIAL REGISTRATION: This study is registered in the UMIN clinical Trial Registration System (UMIN000025393, Registered 23th December 2016).

High versus low chloride load in adult hyperglycemic emergencies with acute kidney injury: a multicenter retrospective cohort study.

Hyperglycemic emergencies frequently lead to acute kidney injury (AKI) and require treatment with large amount of intravenous fluids. However, the effects of chloride loading on this population have not yet been investigated. We conducted a multicenter, retrospective, cohort study in 21 acute-care hospitals in Japan. The study included hospitalized adult patients with diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic syndrome (HHS) who had AKI upon arrival. The patients were classified into high and low chloride groups based on the amount of chloride administered within the first 48 h of their arrival. The primary outcome was recovery from AKI; secondary outcome was major adverse kidney events within 30 days (MAKE30), including mortality and prolonged renal failure. A total of 390 patients with AKI, including 268 (69%) with DKA and 122 (31%) with HHS, were included in the study. Using the criteria of Kidney Disease Improving Global Outcomes, the severity of AKI in the patients was Stage 1 (n = 159, 41%), Stage 2 (n = 121, 31%), and Stage 3 (n = 110, 28%). The analysis showed no significant difference between the two groups in recovery from AKI (adjusted hazard ratio, 0.96; 95% CI 0.72-1.28; P = 0.78) and in MAKE30 (adjusted odds ratio, 0.91; 95% CI 0.45-1.76; P = 0.80). Chloride loading with fluid administration had no significant impact on recovery from AKI in patients with hyperglycemic emergencies.Trial Registration This study was registered in the UMIN clinical trial registration system (UMIN000025393, registered December 23, 2016).