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We developed a Fe:ZnSe chirped pulse amplifier with a KTA-based optical parametric amplifier, achieving 0.6 mJ pulses at 4-micron wavelength. It should be inherently carrier envelope phase stable and applicable to strong field science.

Acute kidney injury (AKI) causes epithelial damage followed by subsequent repair. While successful repair restores kidney function, this process is often incomplete and can lead to chronic kidney disease (CKD) in a process called failed repair. To better understand the epigenetic reprogramming driving this AKI-to-CKD transition, we generated a single-nucleus multiomic atlas for the full mouse AKI time course, consisting of ~280,000 single-nucleus transcriptomes and epigenomes. We reveal cell-specific dynamic alterations in gene regulatory landscapes reflecting, especially, activation of proinflammatory pathways. We further generated single-nucleus multiomic data from four human AKI samples including validation by genome-wide identification of nuclear factor κB binding sites. A regularized regression analysis identifies key regulators involved in both successful and failed repair cell fate, identifying the transcription factor CREB5 as a regulator of both successful and failed tubular repair that also drives proximal tubular cell proliferation after injury. Our interspecies multiomic approach provides a foundation to comprehensively understand cell states in AKI.

In the aftermath of acute kidney injury (AKI), surviving proximal tubule epithelia repopulate injured tubules to promote repair. However, a portion of cells fail to repair [termed failed-repair proximal tubule cells (FR-PTCs)] and exert ongoing proinflammatory and profibrotic effects. To better understand the molecular drivers of the FR-PTC state, we reanalyzed a mouse ischemia-reperfusion injury single-nucleus RNA-sequencing (snRNA-seq) atlas to identify Traf2 and Nck interacting kinase ( Tnik ) to be exclusively expressed in FR-PTCs but not in healthy or acutely injured proximal tubules after AKI (2 and 6 wk) in mice. We confirmed expression of Tnik protein in injured mouse and human tissues by immunofluorescence. Then, to determine the functional role of Tnik in FR-PTCs, we depleted TNIK with siRNA in two human renal proximal tubule epithelial cell lines (primary and immortalized hRPTECs) and analyzed each by bulk RNA-sequencing. Pathway analysis revealed significant upregulation of inflammatory signaling pathways, whereas pathways associated with differentiated proximal tubules such as organic acid transport were significantly downregulated. TNIK gene knockdown drove reduced cell viability and increased apoptosis, including differentially expressed poly(ADP-ribose) polymerase ( PARP ) family members, cleaved PARP-1 fragments, and increased annexin V binding to phosphatidylserine. Together, these results indicate that Tnik upregulation in FR-PTCs acts in a compensatory fashion to suppress inflammation and promote proximal tubule epithelial cell survival after injury. Modulating TNIK activity may represent a prorepair therapeutic strategy after AKI. NEW & NOTEWORTHY The molecular drivers of successful and failed repair in the proximal tubule after acute kidney injury (AKI) are incompletely understood. We identified Traf2 and Nck interacting kinase ( Tnik ) to be exclusively expressed in failed-repair proximal tubule cells after AKI. We tested the effect of si TNIK depletion in two proximal tubule cell lines followed by bulk RNA-sequencing analysis. Our results indicate that TNIK acts to suppress inflammatory signaling and apoptosis in injured renal proximal tubule epithelial cells to promote cell survival.

SARS-CoV-2 rapidly mutates and acquires resistance to neutralizing antibodies. We report an in-silico-designed antibody that restores the neutralizing activity of a neutralizing antibody. Our previously generated antibody, UT28K, exhibited broad neutralizing activity against mutant variants; however, its efficacy against Omicron BA.1 was compromised by the mutation. Using previously determined structural information, we designed a modified-UT28K (V H T28R/N57D), UT28K-RD targeting the mutation site. In vitro and in vivo experiments demonstrated the efficacy of UT28K-RD in neutralizing Omicron BA.1. Although the experimentally determined structure partially differed from the predicted model, our study serves as a successful case of antibody design, wherein the predicted amino acid substitution enhanced the recognition of the previously elusive Omicron BA.1. We anticipate that numerous similar cases will be reported, showcasing the potential of this approach for improving protein-protein interactions. Our findings will contribute to the development of novel therapeutic strategies for highly mutable viruses, such as SARS-CoV-2., Competing Interests: Declaration of interests T.O., H.K., H.N., and H.T. have patent applications pending UT28K (JP2021-056423). T.O., H.T., Y.I., and T.H. have patent applications pending UT28K-RD (JP2022-149604)., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Introduction: A reduced salt intake is a vital lifestyle modification in the management of hypertension. Initiatives aimed at decreasing the intake of salt are based on the preference by humans for a salt taste. Salt intake behavior appears to be affected by the balance between attraction to a low salt taste and aversion to a high salt taste. However, aversion to a high salt taste has not yet been quantitively investigated in both healthy individuals and patients with chronic kidney disease (CKD)., Methods: Assessments of gustatory and aversion thresholds for salt, bitter, sour, and sweet tastes were performed using a stimulant-impregnated test strip in healthy subjects and patients with CKD., Results: In a pilot taste test of 125 healthy subjects, the number of participants with an aversive reaction increased at higher salt concentrations. The threshold for normal taste perception was arbitrarily defined as 10% NaCl, with 47.2% of healthy subjects displaying an aversive reaction. In taste tests performed by 70 patients with CKD, 10% were unable to recognize a salt taste, even at the highest concentration (20% NaCl), suggesting a significant impairment in taste perception in patients with CKD. Only 15.7% of patients with CKD exhibited a normal aversion to NaCl, whereas 78.6% showed the complete loss of aversion to salt., Conclusion: The present results confirmed the anticipated aversive response to a high salt taste in humans and demonstrated its impairment in patients with CKD, implying that patients with CKD have reduced resistance to a high salt intake., (© 2024 International Society of Nephrology. Published by Elsevier Inc.)

The immense public health burden of diabetic kidney disease (DKD) has led to an increase in research on the pathophysiology of advanced DKD. The present study focused on the significance of proinflammatory vascular cell adhesion molecule 1 (VCAM1)+ tubules in DKD progression. A retrospective cohort study of DKD patients showed that the percentage of VCAM1+ tubules in kidney samples was correlated with poor renal outcomes. We established an advanced DKD model by partial resection of the kidneys of db/db mice and demonstrated that it closely resembled the human advanced DKD phenotype, with tissue hypoxia, tubular DNA damage, tissue inflammation, and high tubular VCAM1 expression. Luseogliflozin ameliorated tissue hypoxia and proinflammatory responses, including VCAM1+ expression, in tubules. These findings suggest the potential of tubular VCAM1 as a histological marker for poor DKD outcomes. SGLT2 inhibitors may attenuate tissue hypoxia and subsequent tissue inflammation in advanced DKD, thereby ameliorating tubular injury., Competing Interests: T. Kusaba reports receiving commercial research support from Taisho Pharmaceutical and speaker’s bureau honoraria from AstraZeneca., (© 2024 The Author(s).)

Introduction: Patients with polymerase epsilon (POLE) mutation (POLEmut) subtype, MMR-deficient (MMR-d) subtype as classified by The Cancer Genome Atlas (TCGA), and a high tumor mutation burden (TMB-high) potentially benefit from immunotherapy. However, characteristics of the cytological morphology within these populations remain unknown., Methods: DNA extracted from formalin-fixed paraffin-embedded tissues was subjected to next-generation sequencing analysis. Genomic mutations related to gynecological cancers, TMB, and microsatellite instability were analyzed and were placed in four TCGA classification types. The following morphological cytological investigations were conducted on endometrial cancer using a liquid-based preparation method, prior to the commencement of initial treatment: (i) cytological backgrounds; (ii) differences between each count of neutrophils and lymphocytes as described below., Results: Insignificant differences in the cytological background patterns of TCGA groups and TMB status were found. Although there was no significant difference in neutrophil count (p = 0.955) in the TCGA groups, POLEmut and MMR-d had significantly higher lymphocyte counts than no specific molecular profile (NSMP) (p = 0.019 and 0.037, respectively); furthermore, p53mut also tended to be significant (p = 0.064). Lymphocyte counts in TMB-high were also significantly greater than TMB-low (p = 0.002). POLEmut showed a positive correlation between TMB levels and lymphocyte counts. For predicting patients with POLEmut plus MMR-d, lymphocyte counts demonstrated a superior diagnostic accuracy of area under the curve (AUC) (0.70, 95% CI: 0.57-0.84), with a cutoff value of 26 high-power field., Conclusion: Lymphocyte count using liquid-based cytology for patients with endometrial cancer may predict POLEmut plus MMR-d of TCGA groups and TMB-high in those who can benefit from immunotherapy., (© 2024 S. Karger AG, Basel.)

Background/aim: Among the four genomic subtypes of endometrial cancer, distinguishing between the DNA polymerase epsilon mutation (POLEmut) and no specific molecular profile (NSMP) subtypes requires genomic profiling owing to the lack of surrogate immunohistochemical markers. We have previously found that, histologically, the POLEmut-subtype exhibits surface epithelial slackening (SES). Therefore, to improve subtype identification, we aimed to extract cytological features corresponding to SES in POLEmut-subtype cervical cytology specimens., Materials and Methods: We analyzed 104 endometrial cancer cervical cytology specimens, with integrative diagnosis confirmation via histology, immunohistochemistry, and genomic profiling. Cytological features were evaluated for the presence of atypical glandular cells, atypical cell appearance in single cells and clusters, and cytological SES and the presence of tumor-infiltrating inflammatory cells in clusters., Results: Based on cervical cytology, the POLEmut- and p53mut-subtypes exhibited more frequent atypical cells in smaller clusters, giant tumor cells, and cytological SES patterns than the NSMP-subtype. Tumor-infiltrating lymphocytes were frequent in the POLEmut- and mismatch repair-deficient subtypes., Conclusion: Histologically-detected SES as well as other endometrial cancer features may be preserved in the atypical cell clusters observed in cervical cytology specimens. Cytological detection of SES and of smaller clusters of atypical cells and inflammatory cells with moderate atypia are suggestive of POLEmut-subtype. Integrative diagnosis including genomic profiling remains critical for diagnostic confirmation., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

SpCas9 and AsCas12a are widely utilized as genome-editing tools in human cells. However, their relatively large size poses a limitation for delivery by cargo-size-limited adeno-associated virus (AAV) vectors. The type V-F Cas12f from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been harnessed as a compact genome-editing tool. Here, we developed an approach, combining deep mutational scanning and structure-informed design, to successfully generate two AsCas12f activity-enhanced (enAsCas12f) variants. Remarkably, the enAsCas12f variants exhibited genome-editing activities in human cells comparable with those of SpCas9 and AsCas12a. The cryoelectron microscopy (cryo-EM) structures revealed that the mutations stabilize the dimer formation and reinforce interactions with nucleic acids to enhance their DNA cleavage activities. Moreover, enAsCas12f packaged with partner genes in an all-in-one AAV vector exhibited efficient knock-in/knock-out activities and transcriptional activation in mice. Taken together, enAsCas12f variants could offer a minimal genome-editing platform for in vivo gene therapy., Competing Interests: Declaration of interests T.H., S.N.O., R.N., Y.K., S.M., T.O., A.H., and O.N. have filed a patent application related to this work., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

SARS-CoV-2 Omicron subvariants have evolved to evade receptor-binding site (RBS) antibodies that exist in diverse individuals as public antibody clones. We rationally selected RBS antibodies resilient to mutations in emerging Omicron subvariants. Y489 was identified as a site of virus vulnerability and a common footprint of broadly neutralizing antibodies against the subvariants. Multiple Y489-binding antibodies were encoded by public clonotypes and additionally recognized F486, potentially accounting for the emergence of Omicron subvariants harboring the F486V mutation. However, a subclass of antibodies broadly neutralized BA.4/BA.5 variants via hydrophobic binding sites of rare clonotypes along with high mutation-resilience under escape mutation screening. A computationally designed antibody based on one of the Y489-binding antibodies, NIV-10/FD03, was able to bind XBB with any 486 mutation and neutralized XBB.1.5. The structural basis for the mutation-resilience of this Y489-binding antibody group may provide important insights into the design of therapeutics resistant to viral escape., (© 2023. The Author(s).)

Nonalcoholic steatohepatitis (NASH) is a progressive disorder with aberrant lipid accumulation and subsequent inflammatory and profibrotic response. Therapeutic efforts at lipid reduction via increasing cytoplasmic lipolysis unfortunately worsens hepatitis due to toxicity of liberated fatty acid. An alternative approach could be lipid reduction through autophagic disposal, i.e., lipophagy. We engineered a synthetic adaptor protein to induce lipophagy, combining a lipid droplet-targeting signal with optimized LC3-interacting domain. Activating hepatocyte lipophagy in vivo strongly mitigated both steatosis and hepatitis in a diet-induced mouse NASH model. Mechanistically, activated lipophagy promoted the excretion of lipid from hepatocytes, thereby suppressing harmful intracellular accumulation of nonesterified fatty acid. A high-content compound screen identified alpelisib and digoxin, clinically-approved compounds, as effective activators of lipophagy. Administration of alpelisib or digoxin in vivo strongly inhibited the transition to steatohepatitis. These data thus identify lipophagy as a promising therapeutic approach to prevent NASH progression., (© 2023. The Author(s).)

Streptozotocin (STZ), an anti-cancer drug that is primarily used to treat neuroendocrine tumors (NETs) in clinical settings, is incorporated into pancreatic β-cells or proximal tubular epithelial cells through the glucose transporter, GLUT2. However, its cytotoxic effects on kidney cells have been underestimated and the underlying mechanisms remain unclear. We herein demonstrated that DNA damage and subsequent p53 signaling were responsible for the development of STZ-induced tubular epithelial injury. We detected tubular epithelial DNA damage in NET patients treated with STZ. Unbiased transcriptomics of STZ-treated tubular epithelial cells in vitro showed the activation of the p53 signaling pathway. STZ induced DNA damage and activated p53 signaling in vivo in a dose-dependent manner, resulting in reduced membrane transporters. The pharmacological inhibition of p53 and sodium-glucose transporter 2 (SGLT2) mitigated STZ-induced epithelial injury. However, the cytotoxic effects of STZ on pancreatic β-cells were preserved in SGLT2 inhibitor-treated mice. The present results demonstrate the proximal tubular-specific cytotoxicity of STZ and the underlying mechanisms in vivo. Since the cytotoxic effects of STZ against β-cells were not impaired by dapagliflozin, pretreatment with an SGLT2 inhibitor has potential as a preventative remedy for kidney injury in NET patients treated with STZ., (© 2023. The Author(s).)

Pulmonary arterial hypertension (PAH) is a fatal disease characterized by a progressive increase in pulmonary artery pressure caused by pathological pulmonary artery remodeling. Here, we demonstrate that endothelial cell (EC) senescence plays a negative role in pulmonary hypertension via juxtacrine interaction with smooth muscle cells (SMCs). By using EC-specific progeroid mice, we discovered that EC progeria deteriorated vascular remodeling in the lungs, and exacerbated pulmonary hypertension in mice. Mechanistically, senescent ECs overexpressed Notch ligands, which resulted in increased Notch signaling and activated proliferation and migration capacities in neighboring SMCs. Pharmacological inhibition of Notch signaling reduced the effects of senescent ECs on SMCs functions in vitro , and improved the worsened pulmonary hypertension in EC-specific progeroid mice in vivo . Our findings show that EC senescence is a critical disease-modifying factor in PAH and that EC-mediated Notch signaling is a pharmacotherapeutic target for the treatment of PAH, particularly in the elderly., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

Significance Statement: Understanding the mechanisms underlying adaptive and maladaptive renal repair after AKI and their long-term consequences is critical to kidney health. The authors used lineage tracing of cycling cells and single-nucleus multiomics (profiling transcriptome and chromatin accessibility) after AKI. They demonstrated that AKI triggers a cell-cycle response in most epithelial and nonepithelial kidney cell types. They also showed that maladaptive proinflammatory proximal tubule cells (PTCs) persist until 6 months post-AKI, although they decreased in abundance over time, in part, through cell death. Single-nucleus multiomics of lineage-traced cells revealed regulatory features of adaptive and maladaptive repair. These included activation of cell state-specific transcription factors and cis-regulatory elements, and effects in PTCs even after adaptive repair, weeks after the injury event., Background: AKI triggers a proliferative response as part of an intrinsic cellular repair program, which can lead to adaptive renal repair, restoring kidney structure and function, or maladaptive repair with the persistence of injured proximal tubule cells (PTCs) and an altered kidney structure. However, the cellular and molecular understanding of these repair programs is limited., Methods: To examine chromatin and transcriptional responses in the same cell upon ischemia-reperfusion injury (IRI), we combined genetic fate mapping of cycling ( Ki67+ ) cells labeled early after IRI with single-nucleus multiomics-profiling transcriptome and chromatin accessibility in the same nucleus-and generated a dataset of 83,315 nuclei., Results: AKI triggered a broad cell cycle response preceded by cell type-specific and global transcriptional changes in the nephron, the collecting and vascular systems, and stromal and immune cell types. We observed a heterogeneous population of maladaptive PTCs throughout proximal tubule segments 6 months post-AKI, with a marked loss of maladaptive cells from 4 weeks to 6 months. Gene expression and chromatin accessibility profiling in the same nuclei highlighted differences between adaptive and maladaptive PTCs in the activity of cis-regulatory elements and transcription factors, accompanied by corresponding changes in target gene expression. Adaptive repair was associated with reduced expression of genes encoding transmembrane transport proteins essential to kidney function., Conclusions: Analysis of genome organization and gene activity with single-cell resolution using lineage tracing and single-nucleus multiomics offers new insight into the regulation of renal injury repair. Weeks to months after mild-to-moderate IRI, maladaptive PTCs persist with an aberrant epigenetic landscape, and PTCs exhibit an altered transcriptional profile even following adaptive repair., (Copyright © 2023 by the American Society of Nephrology.)

The major concern with COVID-19 therapeutic monoclonal antibodies is the loss of efficacy against continuously emerging variants of SARS-CoV-2. To predict antibody efficacy against future Omicron subvariants, we conducted deep mutational scanning (DMS) encompassing all single mutations of the receptor-binding domain of the BA.2 strain utilizing an inverted infection assay with an ACE2-harboring virus and library spike-expressing cells. In the case of bebtelovimab, which preserves neutralization activity against BA.2 and BA.5, a broad range of amino acid substitutions at K444, V445, and G446, and some substitutions at P499 and T500, were indicated to achieve the antibody escape. Among subvariants with current rises in case numbers, BA2.75 with G446S partially evaded neutralization by bebtelovimab, while complete evasion was observed in XBB with V445P and BQ.1 with K444T. This is consistent with the DMS results against BA.2, highlighting the potential of DMS as a predictive tool for antibody escape.

Based on recent clinical trials using sodium-glucose co-transporter 2 inhibitor (SGLT2i) demonstrating the significant improvement of outcomes of diabetic kidney disease (DKD), the paradigm shift from "glomerulocentric" to "tubule centric" pathophysiology in DKD progression has been highlighted. Several responsible mechanisms for renoprotective effects by SGLT2i have been proposed recently, but the changes in proximal tubule-specific gene expression by SGLT2i in diabetic mice have not been elucidated. We report the analysis of the proximal tubular-specific pathway, demonstrating the downregulation of oxidative phosphorylation in dapagliflozin-treated db/db mice, a type 2 diabetic model. After 8-week treatment of dapagliflozin for db/db mice having a proximal tubule-specific tdTomato reporter, tdTomato-positive cells were isolated by FACS. Pathway analysis of RNA sequencing of isolated tubular epithelia revealed that oxidative phosphorylation was downregulated in dapagliflozin-treated mice. However, depletion of renal tissue ATP content in db/db mice was ameliorated by dapagliflozin administration. Pimonidazole staining demonstrated renal cortical tissue hypoxia in db/db mice, which was improved by dapagliflozin administration. This study suggests that dapagliflozin can ameliorate the excessive oxygen and ATP consumption, and subsequent tissue hypoxia in the diabetic kidney, which may explain, in part, the responsible mechanisms of the renoprotective effects of dapagliflozin., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s).)

The Omicron (B.1.1.529) SARS-CoV-2 variant contains an unusually high number of mutations in the spike protein, raising concerns of escape from vaccines, convalescent serum, and therapeutic drugs. Here, we analyzed the degree to which Omicron pseudo-virus evades neutralization by serum or therapeutic antibodies. Serum samples obtained 3 months after two doses of BNT162b2 vaccination exhibited 18-fold lower neutralization titers against Omicron than parental virus. Convalescent serum samples from individuals infected with the Alpha and Delta variants allowed similar frequencies of Omicron breakthrough infections. Domain-wise analysis using chimeric spike proteins revealed that this efficient evasion was primarily achieved by mutations clustered in the receptor binding domain but that multiple mutations in the N-terminal domain contributed as well. Omicron escaped a therapeutic cocktail of imdevimab and casirivimab, whereas sotrovimab, which targets a conserved region to avoid viral mutation, remains effective. Angiotensin-converting enzyme 2 (ACE2) decoys are another virus-neutralizing drug modality that are free, at least in theory, from complete escape. Deep mutational analysis demonstrated that an engineered ACE2 molecule prevented escape for each single-residue mutation in the receptor binding domain, similar to immunized serum. Engineered ACE2 neutralized Omicron comparably to the Wuhan strain and also showed a therapeutic effect against Omicron infection in hamsters and human ACE2 transgenic mice. Similar to previous SARS-CoV-2 variants, some sarbecoviruses showed high sensitivity against engineered ACE2, confirming the therapeutic value against diverse variants, including those that are yet to emerge.

Heart failure is frequently accompanied by kidney failure and co-incidence of these organ failures worsens the mortality in patients with heart failure. Recent clinical observations revealed that increased kidney venous pressure, rather than decreased cardiac output, causes the deterioration of kidney function in patients with heart failure. However, the underlying pathophysiology is unknown. Here, we found that decreased blood flow velocity in peritubular capillaries by kidney congestion and upregulation of endothelial nuclear factor-κB (NF-κB) signaling synergistically exacerbate kidney injury. We generated a novel mouse model with unilateral kidney congestion by constriction of the inferior vena cava between kidney veins. Intravital imaging highlighted the notable dilatation of peritubular capillaries and decreased kidney blood flow velocity in the congestive kidney. Damage after ischemia reperfusion injury was exacerbated in the congestive kidney and accumulation of polymorphonuclear leukocytes within peritubular capillaries was noted at the acute phase after injury. Similar results were obtained in vitro, in which polymorphonuclear leukocytes adhesion on activated endothelial cells was decreased in flow velocity-dependent manner but cancelled by inhibition of NF-κB signaling. Pharmacological inhibition of NF-κB for the mice subjected by both kidney congestion and ischemia reperfusion injury ameliorated the accumulation of polymorphonuclear leukocytes and subsequent exacerbation of kidney injury. Thus, our study demonstrates the importance of decreased blood flow velocity accompanying activated NF-κB signaling in aggravation of kidney injury. Hence, inhibition of NF-κB signaling may be a therapeutic candidate for the vicious cycle between heart and kidney failure with increased kidney venous pressure., (Copyright © 2021 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Kidney hypertrophy is a common clinical feature in patients with diabetes and is associated with poor renal outcomes. Initial cell proliferation followed by cellular hypertrophy are considered the responsible mechanisms for diabetic kidney hypertrophy. However, whether similar responses against hyperglycemia continue in the chronic phase in diabetes is unclear. We performed lineage tracing analysis of proximal tubular epithelia using novel type 2 diabetic mice with a tamoxifen-inducible proximal tubule-specific fluorescent reporter. Clonal analysis of proximal tubular epithelia demonstrated that the labeled epithelia proliferated in type 2 diabetic mice. Based on the histological analysis and protein/DNA ratio of sorted labeled tubular epithelia, there was no evidence of cellular hypertrophy in type 2 diabetic mice. Lineage tracing and histological analyses of streptozocin-induced type 1 diabetes also revealed that cellular proliferation occurs in the chronic phase of type 1 diabetes induction. According to our study, epithelial proliferation accompanied by SGLT2 upregulation, rather than cellular hypertrophy, predominantly occurs in the hypertrophic kidney in both type 1 and type 2 diabetes. An increased number of SGLT2+ tubular epithelia may be an adaptive response against hyperglycemia, and linked to the hyper-reabsorption of sodium and glucose observed in type 2 diabetes patients., (© 2022. The Author(s).)

Cisplatin is a commonly used anticancer drug, but nephrotoxicity is a dose-limiting adverse effect. Recent experimental and clinical observations have demonstrated that multiple injections of cisplatin induce the transition to chronic kidney disease; however, the underlying mechanisms remain unclear. We found that multiple injections of higher doses of cisplatin in a shorter interval affected the severity of kidney injury, causing kidney fibrosis to develop at a later time point. An additional injection of cisplatin during the recovery period after a prior injury, when proximal tubule epithelia are actively proliferating, induced substantial tubular injury by inducing more severe DNA damage than that induced by a single injection. Lineage tracing analysis of proximal tubular epithelia demonstrated that the tubular epithelia that underwent multiple rounds of cell division after multiple injections of cisplatin existed at the chronic phase, and these populations often expressed vcam1 + , suggesting the induction of proinflammatory failed-repair tubular epithelia. Our study revealed that as cisplatin exerts cytotoxic effects on actively proliferating cells, additional cisplatin injections before the completion of tubular repair exacerbates kidney injury through cumulative DNA damage. Appropriate both the setting of dosage and dosing intervals, with careful monitoring, are essential to prevent nephrotoxicity of repeated cisplatin treatment in cancer patients., (© 2021. The Author(s).)

Background: Genomic examination of cytology specimens is often performed on cell blocks or conventional smears rather than on liquid-based cytology (LBC) specimens. Since LBC specimens preserve high-quality DNA, cancer genome profiling using next-generation sequencing (NGS) is also attainable from residual LBC specimens. One of the advantages of using LBC specimens for NGS is that it allows direct extraction of DNA from residual specimens, avoiding a sacrifice of smear slides and minimizing genomic profiling processing time., Methods: Endometrial LBC specimens were subjected to NGS analysis to validate the practicality of rapid cancer genomic profiling in a pathology laboratory. The extracted DNA was subjected to NGS using a customized cancer gene panel comprising 56 genes and 17 microsatellite regions. The workflow strategy was defined, and the processing time estimated for specimen sampling, cell counting, NGS run, and genome profiling., Results: NGS analysis of most LBC specimens revealed somatic mutations, tumor mutation burden, and microsatellite instability, which were almost identical to those obtained from formalin-fixed paraffin-embedded tissues. The processing time for direct NGS analysis and cancer genomic profiling of the residual LBC specimens was approximately 5 days., Conclusion: The residual LBC specimens collected using endometrial cytology were verified to carry a high tumor fraction for NGS analysis and could serve as an alternate source for rapid molecular classification and diagnosis of endometrial cancers, as a routine process in a pathology laboratory., (© 2021 Wiley Periodicals LLC.)

SARS-CoV-2 has mutated during the global pandemic leading to viral adaptation to medications and vaccinations. Here we describe an engineered human virus receptor, ACE2, by mutagenesis and screening for binding to the receptor binding domain (RBD). Three cycles of random mutagenesis and cell sorting achieved sub-nanomolar affinity to RBD. Our structural data show that the enhanced affinity comes from better hydrophobic packing and hydrogen-bonding geometry at the interface. Additional disulfide mutations caused the fixing of a closed ACE2 conformation to avoid off-target effects of protease activity, and also improved structural stability. Our engineered ACE2 neutralized SARS-CoV-2 at a 100-fold lower concentration than wild type; we also report that no escape mutants emerged in the co-incubation after 15 passages. Therapeutic administration of engineered ACE2 protected hamsters from SARS-CoV-2 infection, decreased lung virus titers and pathology. Our results provide evidence of a therapeutic potential of engineered ACE2.

Tubular atrophy is a common pathological feature of kidney fibrosis. Although fibroblasts play a predominant role in tissue fibrosis, the role of repairing tubular epithelia in tubular atrophy is unclear. We demonstrated the essential role of focal adhesion kinase (FAK)-mediated intratubular epithelial-mesenchymal transition (EMT) in the pathogenesis of tubular atrophy after severe ischemia-reperfusion injury (IRI). Actively proliferating tubular epithelia undergoing intratubular EMT were noted in the acute phase of severe IRI, resulting in tubular atrophy in the chronic phase, reflecting failed tubular repair. Furthermore, FAK was phosphorylated in the tubular epithelia in the acute phase of severe IRI, and its inhibition ameliorated both tubular atrophy and interstitial fibrosis in the chronic phase after injury. In vivo clonal analysis of single-labeled proximal tubular epithelial cells after IRI using proximal tubule reporter mice revealed substantial clonal expansion after IRI, reflecting active epithelial proliferation during repair. The majority of these proliferating epithelia were located in atrophic and nonfunctional tubules, and FAK inhibition was sufficient to prevent tubular atrophy. In vitro, transforming growth factor-β induced FAK phosphorylation and an EMT phenotype, which was also prevented by FAK inhibition. In an in vitro tubular epithelia gel contraction assay, transforming growth factor-β treatment accelerated gel contraction, which was suppressed by FAK inhibition. In conclusion, injury-induced intratubular EMT is closely related to tubular atrophy in a FAK-dependent manner.

After acute kidney injury (AKI), patients either recover or alternatively develop fibrosis and chronic kidney disease. Interactions between injured epithelia, stroma, and inflammatory cells determine whether kidneys repair or undergo fibrosis, but the molecular events that drive these processes are poorly understood. Here, we use single nucleus RNA sequencing of a mouse model of AKI to characterize cell states during repair from acute injury. We identify a distinct proinflammatory and profibrotic proximal tubule cell state that fails to repair. Deconvolution of bulk RNA-seq datasets indicates that this failed-repair proximal tubule cell (FR-PTC) state can be detected in other models of kidney injury, increasing during aging in rat kidney and over time in human kidney allografts. We also describe dynamic intercellular communication networks and discern transcriptional pathways driving successful vs. failed repair. Our study provides a detailed description of cellular responses after injury and suggests that the FR-PTC state may represent a therapeutic target to improve repair., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The DNA damage response after kidney injury induces cell cycle arrest in renal tubular epithelial cells, resulting in the secretion of pro-fibrotic cytokines, thereby promoting interstitial fibrosis in a paracrine manner. Phosphorylation of ataxia-telangiectasia mutated (ATM) is the initial step in the DNA damage response and subsequent cell cycle arrest; however, the effects of ATM inhibition on the injured kidney have not been explored. Pharmacological ATM inhibition by KU55933 in cisplatin-treated mice did not ameliorate, but instead exacerbated cisplatin-induced DNA damage and tubular injury, thereby increasing mortality. Analysis of isolated tubular epithelia by FACS from bigenic SLC34a1-CreERt2; R26tdTomato proximal tubular-specific reporter mice revealed that KU55933 upregulated p53 and subsequent pro-apoptotic signaling in tubular epithelia of cisplatin-treated mice, leading to marked mitochondrial injury and apoptosis. In addition, KU55933 attenuated several DNA repair processes after cisplatin treatment, including single-strand DNA repair and Fanconi anemia pathways, suggesting that DNA repair after dual treatment of cisplatin and KU55933 was not sufficient to prevent the cisplatin-induced tubular injury. Our study suggested that ATM inhibition does not increase DNA repair after cisplatin-induced DNA damage and exacerbates tubular injury through the upregulation of p53-dependent pro-apoptotic signaling. Acute kidney injury must be carefully monitored when ATM inhibitors become available in clinical practice in the future.

Diabetic nephropathy is characterized by damage to both the glomerulus and tubulointerstitium, but relatively little is known about accompanying cell-specific changes in gene expression. We performed unbiased single-nucleus RNA sequencing (snRNA-seq) on cryopreserved human diabetic kidney samples to generate 23,980 single-nucleus transcriptomes from 3 control and 3 early diabetic nephropathy samples. All major cell types of the kidney were represented in the final dataset. Side-by-side comparison demonstrated cell-type-specific changes in gene expression that are important for ion transport, angiogenesis, and immune cell activation. In particular, we show that the diabetic thick ascending limb, late distal convoluted tubule, and principal cells all adopt a gene expression signature consistent with increased potassium secretion, including alterations in Na + /K + -ATPase, WNK1 , mineralocorticoid receptor, and NEDD4L expression, as well as decreased paracellular calcium and magnesium reabsorption. We also identify strong angiogenic signatures in glomerular cell types, proximal convoluted tubule, distal convoluted tubule, and principal cells. Taken together, these results suggest that increased potassium secretion and angiogenic signaling represent early kidney responses in human diabetic nephropathy., Competing Interests: Conflict of interest statement: In separate work, B.D.H. consults for and receives grant support from Janssen to study mouse diabetic nephropathy. B.D.H. also consults for and holds equity in Chinook Therapeutics. Neither relationship is related to the contents of this manuscript., (Copyright © 2019 the Author(s). Published by PNAS.)

High-throughput single-cell assays increasingly require special consideration in experimental design, sample multiplexing, batch effect removal, and data interpretation. Here, we describe a lentiviral barcode-based multiplexing approach, CellTag Indexing, which uses predefined genetic barcodes that are heritable, enabling cell populations to be tagged, pooled, and tracked over time in the same experimental replicate. We demonstrate the utility of CellTag Indexing by sequencing transcriptomes using a variety of cell types, including long-term tracking of cell engraftment and differentiation in vivo. Together, this presents CellTag Indexing as a broadly applicable genetic multiplexing tool that is complementary with existing single-cell technologies.

Background: A challenge for single-cell genomic studies in kidney and other solid tissues is generating a high-quality single-cell suspension that contains rare or difficult-to-dissociate cell types and is free of both RNA degradation and artifactual transcriptional stress responses., Methods: We compared single-cell RNA sequencing (scRNA-seq) using the DropSeq platform with single-nucleus RNA sequencing (snRNA-seq) using sNuc-DropSeq, DroNc-seq, and 10X Chromium platforms on adult mouse kidney. We validated snRNA-seq on fibrotic kidney from mice 14 days after unilateral ureteral obstruction (UUO) surgery., Results: A total of 11,391 transcriptomes were generated in the comparison phase. We identified ten clusters in the scRNA-seq dataset, but glomerular cell types were absent, and one cluster consisted primarily of artifactual dissociation - induced stress response genes. By contrast, snRNA-seq from all three platforms captured a diversity of kidney cell types that were not represented in the scRNA-seq dataset, including glomerular podocytes, mesangial cells, and endothelial cells. No stress response genes were detected. Our snRNA-seq protocol yielded 20-fold more podocytes compared with published scRNA-seq datasets (2.4% versus 0.12%, respectively). Unexpectedly, single-cell and single-nucleus platforms had equivalent gene detection sensitivity. For validation, analysis of frozen day 14 UUO kidney revealed rare juxtaglomerular cells, novel activated proximal tubule and fibroblast cell states, and previously unidentified tubulointerstitial signaling pathways., Conclusions: snRNA-seq achieves comparable gene detection to scRNA-seq in adult kidney, and it also has substantial advantages, including reduced dissociation bias, compatibility with frozen samples, elimination of dissociation-induced transcriptional stress responses, and successful performance on inflamed fibrotic kidney., (Copyright © 2019 by the American Society of Nephrology.)

Injured tubular epithelium exhibits cellular plasticity in that it can dedifferentiate, reenter the cell cycle, and subsequently either redifferentiate or adopt a chronically injured phenotype. Although some nephrogenic genes are reexpressed during injury and repair, developmental pathways are only partially recapitulated and the process is more accurately viewed as an entirely new program intrinsic to the regenerative response to injury. Recent advances in our understanding of the molecular circuitry underpinning epithelial plasticity have come from bulk, cell-specific, and single-cell transcriptomic analyses. These results have begun to define the signaling pathways and gene regulatory networks governing the epithelial injury response. In this review, we highlight recent transcriptomic analyses in kidney injury, repair and fibrosis, and outline the ways that these studies are improving our understanding of kidney regeneration., (© 2019 S. Karger AG, Basel.)

Kidney organoids derived from human pluripotent stem cells have great utility for investigating organogenesis and disease mechanisms and, potentially, as a replacement tissue source, but how closely organoids derived from current protocols replicate adult human kidney is undefined. We compared two directed differentiation protocols by single-cell transcriptomics of 83,130 cells from 65 organoids with single-cell transcriptomes of fetal and adult kidney cells. Both protocols generate a diverse range of kidney cells with differing ratios, but organoid-derived cell types are immature, and 10%-20% of cells are non-renal. Reconstructing lineage relationships by pseudotemporal ordering identified ligands, receptors, and transcription factor networks associated with fate decisions. Brain-derived neurotrophic factor (BDNF) and its cognate receptor NTRK2 were expressed in the neuronal lineage during organoid differentiation. Inhibiting this pathway improved organoid formation by reducing neurons by 90% without affecting kidney differentiation, highlighting the power of single-cell technologies to characterize and improve organoid differentiation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background Single-cell genomics techniques are revolutionizing our ability to characterize complex tissues. By contrast, the techniques used to analyze renal biopsy specimens have changed little over several decades. We tested the hypothesis that single-cell RNA-sequencing can comprehensively describe cell types and states in a human kidney biopsy specimen. Methods We generated 8746 single-cell transcriptomes from a healthy adult kidney and a single kidney transplant biopsy core by single-cell RNA-sequencing. Unsupervised clustering analysis of the biopsy specimen was performed to identify 16 distinct cell types, including all of the major immune cell types and most native kidney cell types, in this biopsy specimen, for which the histologic read was mixed rejection. Results Monocytes formed two subclusters representing a nonclassical CD16+ group and a classic CD16- group expressing dendritic cell maturation markers. The presence of both monocyte cell subtypes was validated by staining of independent transplant biopsy specimens. Comparison of healthy kidney epithelial transcriptomes with biopsy specimen counterparts identified novel segment-specific proinflammatory responses in rejection. Endothelial cells formed three distinct subclusters: resting cells and two activated endothelial cell groups. One activated endothelial cell group expressed Fc receptor pathway activation and Ig internalization genes, consistent with the pathologic diagnosis of antibody-mediated rejection. We mapped previously defined genes that associate with rejection outcomes to single cell types and generated a searchable online gene expression database. Conclusions We present the first step toward incorporation of single-cell transcriptomics into kidney biopsy specimen interpretation, describe a heterogeneous immune response in mixed rejection, and provide a searchable resource for the scientific community., (Copyright © 2018 by the American Society of Nephrology.)

Clinical and experimental studies have shown that sodium glucose co-transporter 2 inhibitors (SGLT2i) contribute to the prevention of diabetic kidney disease progression. In order to clarify its pharmacological effects on the molecular mechanisms underlying the development of diabetic kidney disease, we administered different doses of the SGLT2i, ipragliflozin, to type 2 diabetic mice. A high-dose ipragliflozin treatment for 8 weeks lowered blood glucose levels and reduced urinary albumin excretion. High- and low-dose ipragliflozin both inhibited renal and glomerular hypertrophy, and reduced NADPH oxidase 4 expression and subsequent oxidative stress. Analysis of glomerular phenotypes using glomeruli isolation demonstrated that ipragliflozin preserved podocyte integrity and reduced oxidative stress. Regarding renal tissue hypoxia, a short-term ipragliflozin treatment improved oxygen tension in the kidney cortex, in which SGLT2 is predominantly expressed. We then administered ipragliflozin to type 1 diabetic mice and found that high- and low-dose ipragliflozin both reduced urinary albumin excretion. In conclusion, we confirmed dose-dependent differences in the effects of ipragliflozin on early diabetic nephropathy in vivo. Even low-dose ipragliflozin reduced renal cortical hypoxia and abnormal hemodynamics in early diabetic nephropathy. In addition to these effects, high-dose ipragliflozin exerted renoprotective effects by reducing oxidative stress in tubular epithelia and glomerular podocytes.

Purpose To evaluate the accuracy of the remote-controlled robotic computed tomography (CT)-guided needle insertion in phantom and animal experiments. Materials and Methods In a phantom experiment, 18 robotic and manual insertions each were performed with 19-gauge needles by using CT fluoroscopic guidance for the evaluation of the equivalence of accuracy of insertion between the two groups with a 1.0-mm margin. Needle insertion time, CT fluoroscopy time, and radiation exposure were compared by using the Student t test. The animal experiments were approved by the institutional animal care and use committee. In the animal experiment, five robotic insertions each were attempted toward targets in the liver, kidneys, lungs, and hip muscle of three swine by using 19-gauge or 17-gauge needles and by using conventional CT guidance. The feasibility, safety, and accuracy of robotic insertion were evaluated. Results The mean accuracies of robotic and manual insertion in phantoms were 1.6 and 1.4 mm, respectively. The 95% confidence interval of the mean difference was -0.3 to 0.6 mm. There were no significant differences in needle insertion time, CT fluoroscopy time, or radiation exposure to the phantom between the two methods. Effective dose to the physician during robotic insertion was always 0 μSv, while that during manual insertion was 5.7 μSv on average (P < .001). Robotic insertion was feasible in the animals, with an overall mean accuracy of 3.2 mm and three minor procedure-related complications. Conclusion Robotic insertion exhibited equivalent accuracy as manual insertion in phantoms, without radiation exposure to the physician. It was also found to be accurate in an in vivo procedure in animals. © RSNA, 2017 Online supplemental material is available for this article.

Objective: The custom-homebuilding company, Cosmic Garden Co. Ltd., located in Okayama City, Japan was established in 1997 and uses specific natural ore powder (SNOP) in wall materials and surveys customers in order to improve allergic symptoms., Methods: To investigate the biological effects of SNOP, patients with a pollen allergy were recruited to stay in a room surrounded by cloth containing SNOP (CCSNOP), and their symptoms and various biological parameters were compared with those of individuals staying in a room surrounded by control non-woven cloth (NWC). Each stay lasted 60 min. Before and immediately after the stay, a questionnaire regarding allergic symptoms, as well as POMS (Profile of Mood Status) and blood sampling, was performed. Post-stay minus pre-stay values were calculated and compared between CCSNOP and NWC groups., Results: Results indicated that some symptoms, such as nasal obstruction and lacrimation, improved, and POMS evaluation showed that patients were calmer following a stay in CCSNOP. Relative eosinophils, non-specific Ig E, epidermal growth factor, monocyte chemotactic protein-1, and tumor necrosis factor-α increased following a stay in CCSNOP., Conclusion: This ore powder improved allergic symptoms, and long-term monitoring involving 1 to 2 months may be necessary to fully explore the biological and physical effects of SNOP on allergic patients., (Copyright © 2016 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

Reconstitution of total nephron segments after resection in the adult kidney has not been achieved; however, whether the neonatal kidney can maintain the capacity for neo-nephrogenesis after resection is unknown. We performed partial resection of the kidney in neonatal rats on postnatal days 1 (P1x kidney) and 4 (P4x kidney) and examined morphological changes and relevant factors. The P1x kidney bulged into the newly formed cortex from the wound edge, while nephrogenesis failure was prominent in the P4x kidney. Twenty-eight days post-resection, the glomerular number, cortex area, and collecting duct were preserved in the P1x kidney, whereas these parameters were markedly decreased in the P4x kidney. During normal development, Six2 expression and Six2+ nephron progenitor cells in the cap mesenchyme both rapidly disappear after birth. However, time course analysis for the P1x kidney showed that Six2 expression and Six2+ cells were well preserved in the tissue surrounding the resected area even 2 days after resection. In conclusion, our results indicate that kidneys in early neonate rats retain the capability for neo-nephrogenesis after resection; however, this ability is lost soon after birth, which may be attributed to a declining amount of Six2+ cells.

Sonoporation can deliver agents to target local organs by systemic administration, while decreasing the associated risk of adverse effects. Sonoporation has been used for a variety of materials and in a variety of organs. Herein, we demonstrated that local sonoporation to the kidney can offer highly efficient transfer of oligonucleotides, which were systemically administrated to the tubular epithelium with high specificity. Ultrasonic wave irradiation to the kidney collapsed the microbubbles and transiently affected the glomerular filtration barrier and increased glomerular permeability. Oligonucleotides were passed through the barrier all at once and were absorbed throughout the tubular epithelium. Tumor necrosis factor alpha (TNFα), which plays a central role in renal ischemia-reperfusion injury, was targeted using small interfering RNA (siRNA) with renal sonoporation in a murine model. The reduction of TNFα expression after single gene transfer significantly inhibited the expression of kidney injury markers, suggesting that systemic administration of siRNA under temporary and local sonoporation could be applicable in the clinical setting of ischemic acute kidney injury.

Whole organ tissue engineering for various organs, including the heart, lung, liver, and kidney, has demonstrated promising results for end-stage organ failure. However, the sodium dodecyl sulfate (SDS)-based protocol for standard decellularization has drawbacks such as clot formation in vascularized transplantation and poor cell engraftment in recellularization procedures. Preservation of the surface milieu of extracellular matrices (ECMs) might be crucial for organ generation based on decellularization/recellularization engineering. We examined a novel detergent, sodium lauryl ether sulfate (SLES), to determine whether it could overcome the drawbacks associated with SDS using rat heart and kidney. Both organs were perfused in an antegrade fashion with either SLES or SDS. Although immunohistochemistry for collagen I, IV, laminin, and fibronectin showed similar preservation in both detergents, morphological analysis using scanning electron microscopy and an assay of glycosaminoglycan content on ECMs showed that SLES-treated tissues had better-preserved ECMs than SDS-treated tissues. Mesenteric transplantation revealed SLES did not induce significant inflammation, as opposed to SDS. Platelet adhesion to decellularized tissues was significantly reduced with SLES. Overall, SLES could replace older detergents such as SDS in the decellularization process for generation of transplantable recellularized organs., (© 2015 Wiley Periodicals, Inc.)

A 66-year-old man undergoing peritoneal dialysis (PD) was admitted to our hospital for treatment of PD-related peritonitis. Culture of the PD fluid revealed the presence of Citrobacter freundii, and therapy with ceftazidime was started intraperitoneally. The cell count in PD fluid slowly decreased over time during the first 2 weeks of treatment, but increased again on the 14th hospital day. A second culture of the PD fluid revealed the presence of Enterococcus species. A switch in antibiotic therapy to vancomycin did not improve the cell count in the PD fluid. A third culture of the PD fluid revealed the presence of Stenotrophomonas maltophilia. The PD was discontinued and the catheter removed on the 28th hospital day. Examination of the catheter revealed that the inner tip was coated with a fibrous sheet of cells, suggesting biofilm formation. Following catheter removal, the patient was administered intravenous ciprofloxacin, and the inflammatory reaction started to disappear immediately and had completely disappeared after 1 week of treatment. Microscopic analysis of the fibrous structure on the catheter revealed multiple layers of various inflammatory cells. Immunostaining revealed the presence of CD44-positive polynuclear cells, indicating neutrophils, facing the catheter lumen. CD68-positive cells, indicating macrophages, were observed in the following layer, and keratin-positive cells, indicating peritoneal mesothelial cells, were present at the bottom of the structure. Based on the immediate improvement of PD-related peritonitis after catheter removal, we presumed that this biofilm contributed to the intractability of the patient's peritonitis. Morphological analysis of catheter revealed that both the mesothelial cells and the various inflammatory cells may have contributed to biofilm development.

A 70-year-old woman was admitted to our hospital for repair of vascular access for maintenance hemodialysis. She had been undergoing the maintenance hemodialysis for 20 years, however, her underlying renal disease had not been identified. The laboratory data on admission revealed marked thrombocytopenia with giant platelets and a Dohle body-like cytoplasmic inclusion body in granulocytes. The same hematological abnormalities were also detected in the peripheral blood smear of her daughter. We suspected hereditary macrothrombocytopenia and performed gene analysis of the MYH9 gene that encodes the nonmuscle myosin heavy chain-II A (NMMHC- II A). Mutational analysis showed the heterozygous mutation, c. 1841 G>A, in exon 38 of the MYH9 gene (E1841 K). We further examined intracellular NMMHC- II A localization in granulocytes by immunofluorescent analysis. The results revealed that one or two NMMHC- II A-positive granules were observed in neutrophils, whereas these granules were not detected in the granulocytes of normal healthy volunteers. From these analyses, we diagnosed her disease as MYH9 disorder, especially as a May-Hegglin abnormality. Thrombocytopenia is sometimes observed in maintenance hemodialysis patients. To avoid inappropriate medical intervention for the thrombocytopenia, MYH9 disorders should be differentiated.

Extensive simultaneous multisegment laminectomy (ESML) for treatment of ossification of the posterior longitudinal ligament (OPLL) in the cervical region is a technique which cuts the laminae on the median line and on the pedicles with an air drill, and then, the right and left halves of laminae are lifted simultaneously. This technique provides a protection to the spinal cord that swells instantaneously after decompression. A total of 155 cases were followed up for more than 1 year. According to the Japanese Orthopedic Association's evaluation criteria, 127 cases (81.9%) showed some improvement; specifically, 57 cases (36.8%) were rated excellent; 28 (18.1%), good; 42 (27.1%), fair; 11 (7.1%), unchanged; and 17 (11.0%) had poor results.