HIF-1α and adaptor protein LIM and senescent cell antigen-like domains protein 1 axis promotes tubulointerstitial fibrosis by interacting with vimentin in angiotensin II-induced hypertension.

BACKGROUND AND PURPOSE: Activation of the renin-angiotensin system, as a hallmark of hypertension and chronic kidney diseases (CKD) is the key pathophysiological factor contributing to the progression of tubulointerstitial fibrosis. LIM and senescent cell antigen-like domains protein 1 (LIMS1) plays an essential role in controlling of cell behaviour through the formation of complexes with other proteins. Here, the function and regulation of LIMS1 in angiotensin II (Ang II)-induced hypertension and tubulointerstitial fibrosis was investigated. EXPERIMENTAL APPROACH: C57BL/6 mice were treated with Ang II to induce tubulointerstitial fibrosis. Hypoxia-inducible factor-1α (HIF-1α) renal tubular-specific knockout mice or LIMS1 knockdown AAV was used to investigate their effects on Ang II-induced renal interstitial fibrosis. In vitro, HIF-1α or LIMS1 was knocked down or overexpressed in HK2 cells after exposure to Ang II. KEY RESULTS: Increased expression of tubular LIMS1 was observed in human kidney with hypertensive nephropathy and in murine kidney from Ang II-induced hypertension model. Tubular-specific knockdown of LIMS1 ameliorated Ang II-induced tubulointerstitial fibrosis in mice. Furthermore, we demonstrated that LIMS1 was transcriptionally regulated by HIF-1α in tubular cells and that tubular HIF-1α knockout ameliorates LIMS1-mediated tubulointerstitial fibrosis. In addition, LIMS1 promotes Ang II-induced tubulointerstitial fibrosis by interacting with vimentin. CONCLUSION AND IMPLICATIONS: We conclude that HIF-1α transcriptionally regulated LIMS1 plays a central role in Ang II-induced tubulointerstitial fibrosis through interacting with vimentin. Our finding represents a new insight into the mechanism of Ang II-induced tubulointerstitial fibrosis and provides a novel therapeutic target for progression of CKD.

Therapeutic role of miR-26a on cardiaorenal injury in mice model of angiotensin-II induced chronic kidney disease through inhibition of LIMS1/ILK pathway.

BACKGROUND: Chronic kidney disease (CKD) is associated with common pathophysiological processes, such as inflammation and fibrosis, in both the heart and the kidney. However, the underlying molecular mechanisms that drive these processes are not yet fully understood. Therefore, this study focused on the molecular mechanism of heart and kidney injury in CKD. METHODS: We generated a microRNA (miR)-26a knockout (KO) mouse model to investigate the role of miR-26a in angiotensin (Ang)-II-induced cardiac and renal injury. We performed Ang-II modeling in wild type (WT) mice and miR-26a KO mice, with six mice in each group. In addition, Ang-II-treated AC16 cells and HK2 cells were used as in vitro models of cardiac and renal injury in the context of CKD. Histological staining, immunohistochemistry, quantitative real-time polymerase chain reaction (PCR), and Western blotting were applied to study the regulation of miR-26a on Ang-II-induced cardiac and renal injury. Immunofluorescence reporter assays were used to detect downstream genes of miR-26a, and immunoprecipitation was employed to identify the interacting protein of LIM and senescent cell antigen-like domain 1 (LIMS1). We also used an adeno-associated virus (AAV) to supplement LIMS1 and explored the specific regulatory mechanism of miR-26a on Ang-II-induced cardiac and renal injury. Dunnett's multiple comparison and t-test were used to analyze the data. RESULTS: Compared with the control mice, miR-26a expression was significantly downregulated in both the kidney and the heart after Ang-II infusion. Our study identified LIMS1 as a novel target gene of miR-26a in both heart and kidney tissues. Downregulation of miR-26a activated the LIMS1/integrin-linked kinase (ILK) signaling pathway in the heart and kidney, which represents a common molecular mechanism underlying inflammation and fibrosis in heart and kidney tissues during CKD. Furthermore, knockout of miR-26a worsened inflammation and fibrosis in the heart and kidney by inhibiting the LIMS1/ILK signaling pathway; on the contrary, supplementation with exogenous miR-26a reversed all these changes. CONCLUSIONS: Our findings suggest that miR-26a could be a promising therapeutic target for the treatment of cardiorenal injury in CKD. This is attributed to its ability to regulate the LIMS1/ILK signaling pathway, which represents a common molecular mechanism in both heart and kidney tissues.

Tubule-specific cyclin-dependent kinase 12 knockdown potentiates kidney injury through transcriptional elongation defects.

Direct tubular injury caused by several medications, especially chemotherapeutic drugs, is a common cause of AKI. Inhibition or loss of cyclin-dependent kinase 12 (CDK12) triggers a transcriptional elongation defect that results in deficiencies in DNA damage repair, producing genomic instability in a variety of cancers. Notably, 10-25% of individuals developed AKI after treatment with a CDK12 inhibitor, and the potential mechanism is not well understood. Here, we found that CDK12 was downregulated in the renal tubular epithelial cells in both patients with AKI and murine AKI models. Moreover, tubular cell-specific knockdown of CDK12 in mice enhanced cisplatin-induced AKI through promotion of genome instability, apoptosis, and proliferative inhibition, whereas CDK12 overexpression protected against AKI. Using the single molecule real-time (SMRT) platform on the kidneys of CDK12RTEC+/- mice, we found that CDK12 knockdown targeted Fgf1 and Cast through transcriptional elongation defects, thereby enhancing genome instability and apoptosis. Overall, these data demonstrated that CDK12 knockdown could potentiate the development of AKI by altering the transcriptional elongation defect of the Fgf1 and Cast genes, and more attention should be given to patients treated with CDK12 inhibitors to prevent AKI.

The potential role of differentially expressed tRNA-derived fragments in high glucose-induced podocytes.

The prevalence of diabetic kidney disease (DKD) is increasing annually. Damage to and loss of podocytes occur early in DKD. tRNA-derived fragments (tRFs), originating from tRNA precursors or mature tRNAs, are associated with various illnesses. In this study, tRFs were identified, and their roles in podocyte injury induced by high-glucose (HG) treatment were explored. High-throughput sequencing of podocytes treated with HG was performed to identify differentially expressed tRFs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. The expression levels of nephrin, podocin, and desmin were measured in podocytes after overexpression of tRF-1:24-Glu-CTC-1-M2 (tRF-1:24) and concomitant HG treatment. A total of 647 tRFs were identified, and 89 differentially expressed tRFs (|log2FC| ≥ 0.585; p ≤ .05) were identified in the HG group, of which 53 tRFs were downregulated and 36 tRFs were upregulated. The 10 tRFs with the highest differential expression were detected by real-time quantitative polymerase chain reaction (RT-qPCR), and these results were consistent with the sequencing results. GO analysis revealed that the biological process, cellular component, and molecular function terms in which the tRFs were the most enriched were cellular processes, cellular anatomical entities, and binding. KEGG pathway analysis revealed that tRFs may be involved in signaling pathways related to growth hormones, phospholipase D, the regulation of stem cell pluripotency, and T-/B-cell receptors. Overexpression of tRF-1:24, one of the most differentially expressed tRFs, attenuated podocyte injury induced by HG. Thus, tRFs might be potential biomarkers for podocyte injury in DKD.

MicroRNA-26a alleviates tubulointerstitial fibrosis in diabetic kidney disease by targeting PAR4.

Our previous study found that miR-26a alleviates aldosterone-induced tubulointerstitial fibrosis (TIF). However, the effect of miR-26a on TIF in diabetic kidney disease (DKD) remains unclear. This study clarifies the role and possible mechanism of exogenous miR-26a in controlling the progression of TIF in DKD models. Firstly, we showed that miR-26a was markedly decreased in type 2 diabetic db/db mice and mouse tubular epithelial cells (mTECs) treated with high glucose (HG, 30 mM) using RT-qPCR. We then used adeno-associated virus carrying miR-26a and adenovirus miR-26a to enhance the expression of miR-26a in vivo and in vitro. Overexpressing miR-26a alleviated the TIF in db/db mice and the extracellular matrix (ECM) deposition in HG-stimulated mTECs. These protective effects were caused by reducing expression of protease-activated receptor 4 (PAR4), which involved in multiple pro-fibrotic pathways. The rescue of PAR4 expression reversed the anti-fibrosis activity of miR-26a. We conclude that miR-26a alleviates TIF in DKD models by directly targeting PAR4, which may provide a novel molecular strategy for DKD therapy.

Vitexin attenuates chronic kidney disease by inhibiting renal tubular epithelial cell ferroptosis via NRF2 activation.

BACKGROUND: Chronic kidney disease (CKD) involves a variety of pathological processes, and ferroptosis plays a vital role in CKD progression. Targeting ferroptosis is a promising strategy for the treatment of CKD. However, inhibitors of ferroptosis have not been used in the clinical treatment of CKD. Vitexin is a natural flavonoid with many biological activities and protective effects against various diseases. However, whether vitexin can prevent the progression of CKD is not known. METHODS: In vivo, the effect of vitexin on CKD was evaluated by using mouse models of unilateral ureteral obstruction (UUO) and unilateral ischemia-reperfusion (UIR). Western blotting, Sirius red staining and transmission electron microscopy were used to analyze renal tubular injury, interstitial fibrosis, and inflammation in the kidneys of UUO and UIR mice. In vitro, CCK8 assays and lipid peroxidation assays were performed to analyze cell viability and lipid peroxidation in human renal tubular epithelial cells (HK2 cells) induced by erastin. The activation of renal fibroblasts (NRK-49 F cells) was also analyzed. Additionally, an in-silico protein-drug docking model and coimmunoprecipitation were performed to determine the direct substrate of vitexin. RESULTS: In vivo, vitexin treatment significantly ameliorated renal tubular injury, interstitial fibrosis, and inflammation in the kidneys of UUO and UIR mice. Additionally, our results showed that vitexin significantly attenuated UUO- and UIR-induced ferroptosis in renal tubular epithelial cells by upregulating glutathione peroxidase 4 (GPX4) protein levels and inhibiting lipid peroxidation in mouse kidneys. In vitro, treatment with vitexin inhibited erastin-induced ferroptosis in HK2 cells. Moreover, vitexin inhibited the expression of collagen I and α-SMA (alpha-smooth muscle actin) in NRK-49 F cells induced by the supernatant of erastin-treated HK2 cells. Mechanistically, our results suggested that vitexin could activate the NRF2/heme oxygenase-1 (HO-1) pathway by inhibiting the KEAP1- and ubiquitination-mediated degradation of NRF2, thereby increasing the expression of GPX4, and further inhibiting lipid peroxidation and ferroptosis. Additionally, knockout of NRF2 greatly inhibited the antiferroptotic effects of vitexin. CONCLUSIONS: Taken together, our results indicate that vitexin can protect against renal tubular epithelial cell ferroptosis in CKD by activating the KEAP1/NRF2/HO-1 pathway and is a promising drug to treat CKD.

tRF-003634 alleviates adriamycin-induced podocyte injury by reducing the stability of TLR4 mRNA.

Podocyte injury plays a key role in the production of proteinuria and is closely related to the progression of chronic kidney disease (CKD). Alleviating podocyte injury is beneficial to prevent the occurrence and development of CKD. tRNA-derived RNA fragments (tRFs) are associated with podocytes injury processes such as protein binding, cell adhesion, synapses, the actin cytoskeleton. Our previous data showed that tRF-003634 tightly correlated with podocyte injury, while its effect remains unclear. This study aimed to investigate the role of tRF-003634 in podocyte injury and the potential mechanisms. The expression level of tRF-003634, nephrin, podocin and tRF-003634 targeted toll-like receptor 4 (TLR4) in podocytes and kidney tissues were examined by quantitative real-time PCR (qRT-PCR), western blot and immunohistochemistry. The biochemical indices were monitored and renal pathological changes were assessed by hematoxylin and eosin PAS staining. Furthermore, potential target genes of tRF-003634 were screened using high-throughput mRNA sequencing, and then confirmed by RNA pulse-chase analysis. The results showed that tRF-003634 was downregulated in adriamycin (Adr)-induced podocyte injury. Overexpression of tRF-003634 increased the expression of nephrin and podocin in vivo and in vitro and alleviated podocyte injury. Meanwhile, overexpression of tRF-003634 alleviated proteinuria and renal pathological damage. In addition, high-throughput sequencing after overexpression of tRF-003634 showed that TLR4 might be a downstream target gene. tRF-003634 can alleviate podocyte injury by reducing the stability of TLR4 mRNA, possibly by competing with TLR4 mRNA to bind to YTH domain-containing protein 1 (YTHDC1). In conclusion, tRF-003634 was underexpressed in Adr-induced podocyte injury, and its overexpression alleviated podocyte injury in vitro and in vivo by reducing the stability of TLR4 mRNA.

Satellite cell-derived exosome-mediated delivery of microRNA-23a/27a/26a cluster ameliorates the renal tubulointerstitial fibrosis in mouse diabetic nephropathy.

Renal tubulointerstitial fibrosis (TIF) is considered as the final convergent pathway of diabetic nephropathy (DN) without effective therapies currently. MiRNAs play a key role in fibrotic diseases and become promising therapeutic targets for kidney diseases, while miRNA clusters, formed by the cluster arrangement of miRNAs on chromosomes, can regulate diverse biological functions alone or synergistically. In this study, we developed clustered miR-23a/27a/26a-loaded skeletal muscle satellite cells-derived exosomes (Exos) engineered with RVG peptide, and investigated their therapeutic efficacy in a murine model of DN. Firstly, we showed that miR-23a-3p, miR-26a-5p and miR-27a-3p were markedly decreased in serum samples of DN patients using miRNA sequencing. Meanwhile, we confirmed that miR-23a-3p, miR-26a-5p and miR-27a-3p were primarily located in proximal renal tubules and highly negatively correlated with TIF in db/db mice at 20 weeks of age. We then engineered RVG-miR-23a/27a/26a cluster loaded Exos derived from muscle satellite cells, which not only enhanced the stability of miR-23a/27a/26a cluster, but also efficiently delivered more miR-23a/27a/26a cluster homing to the injured kidney. More importantly, administration of RVG-miR-23a/27a/26a-Exos (100 µg, i.v., once a week for 8 weeks) significantly ameliorated tubular injury and TIF in db/db mice at 20 weeks of age. We revealed that miR-23a/27a/26a-Exos enhanced antifibrotic effects by repressing miRNA cluster-targeting Lpp simultaneously, as well as miR-27a-3p-targeting Zbtb20 and miR-26a-5p-targeting Klhl42, respectively. Knockdown of Lpp by injection of AAV-Lpp-RNAi effectively ameliorated the progression of TIF in DN mice. Taken together, we established a novel kidney-targeting Exo-based delivery system by manipulating the miRNA-23a/27a/26a cluster to ameliorate TIF in DN, thus providing a promising therapeutic strategy for DN.

Expression profiles and potential roles of serum tRNA­derived fragments in diabetic nephropathy.

Diabetic nephropathy (DN) is one of the most important causes of end-stage renal disease and current treatments are ineffective in preventing its progression. Transfer RNA (tRNA)-derived fragments (tRFs), which are small non-coding fragments derived from tRNA precursors or mature tRNAs, have a critical role in various human diseases. The present study aimed to investigate the expression profile and potential functions of tRFs in DN. High-throughput sequencing technology was employed to detect the differential serum levels of tRFs between DN and diabetes mellitus and to validate the reliability of the sequencing results using reverse transcription-quantitative PCR. Ultimately, six differentially expressed (DE) tRFs were identified (P<0.05; |log2fold change| ≥1), including three upregulated (tRF5-GluCTC, tRF5-AlaCGC and tRF5-ValCAC) and three downregulated tRFs (tRF5-GlyCCC, tRF3-GlyGCC and tRF3-IleAAT). Potential functions and regulatory mechanisms of these DE tRFs were further evaluated using an applied bioinformatics-based analysis. Gene ontology analysis revealed that the DE tRFs are mainly enriched in biological processes, including axon guidance, Rad51 paralog (Rad51)B-Rad51C-Rad51D-X-Ray repair cross-complementing 2 complex, nuclear factor of activated T-cells protein binding and fibroblast growth factor-activated receptor activity. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that they are associated with axon guidance, neurotrophin signaling, mTOR signaling, AMPK signaling and epidermal growth factor receptor family signaling pathways. In conclusion, the present findings indicated that tRFs were DE in DN and may be involved in the regulation of DN pathology through multiple pathways, thereby providing a new perspective for the study of DN therapeutic targets.

Mitochondrial quality control in acute kidney disease.

Acute kidney disease (AKD) involves multiple pathogenic mechanisms,  including maladaptive repair of renal cells that are rich in mitochondria. Maintenance of mitochondrial homeostasis and quality control is crucial for normal kidney function. Mitochondrial quality control serves to maintain mitochondrial function under various conditions, including mitochondrial bioenergetics, mitochondrial biogenesis, mitochondrial dynamics (fusion and fission) and mitophagy. To date, increasing evidence indicates that mitochondrial quality control is disrupted when acute kidney disease develops. This review describes the mechanisms of mitochondria quality control in acute kidney disease, aiming to provide clues to help design new clinical treatments.

Expression profiles of tRNA­derived fragments in high glucose­treated tubular epithelial cells.

Transfer RNA-derived fragments (tRFs), a novel class of small non-coding RNA produced by the cleavage of pre- and mature tRNAs, are involved in various diseases. Renal tubulointerstitial fibrosis is a common final pathway in diabetic nephropathy (DN) in which hyperglycemia-induced tubular extracellular matrix (ECM) accumulation serves a vital role. The present study aimed to detect and investigate the role of tRFs in the accumulation of tubular ECM. Differentially expressed tRFs were analysed with high-throughput sequencing in primary mouse tubular epithelial cells treated with high glucose (HG). The Gene Ontology (GO) was used to analyze the potential molecular functions of these differentially expressed tRFs, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyze the associated signaling pathways involved in these differentially expressed tRFs. tRF-1:30-Gln-CTG-4 was overexpressed using tRF-1:30-Gln-CTG-4 mimic, followed by HG treatment. A total of 554 distinct tRFs were detected and 64 differentially expressed tRFs (fold change >2; P<0.05) were identified in tubular epithelial cells following high glucose (HG) treatment, among which 27 were upregulated and 37 were downregulated. Ten selected tRFs with the greatest difference (fold change >2; P<0.05) were verified to be consistent with small RNA-sequencing data, of which tRF-1:30-Gln-CTG-4 showed the most pronounced difference in expression and was significantly decreased in response to HG. GO analysis indicated that the differentially expressed tRFs were associated with 'cellular process', 'biological regulation' and 'metabolic process'. An analysis of the KEGG database suggested that these differentially expressed tRFs were involved in 'autophagy' and signaling pathways for 'forkhead box O', 'the mammalian target of rapamycin' and 'mitogen-activated protein kinase'. Finally, the overexpression of tRF-1:30-Gln-CTG-4 ameliorated HG-induced ECM accumulation in tubular epithelial cells. Therefore, the present study demonstrated that there may be a significant association between tRFs and HG-induced ECM accumulation in tubular epithelial cells; these differentially expressed tRFs warrant further study to explore the pathogenesis of DN.

Mitochondrial Targeted Antioxidant SKQ1 Ameliorates Acute Kidney Injury by Inhibiting Ferroptosis.

Emerging evidence suggests that ferroptosis is highly correlated with the pathogenesis of acute kidney injury (AKI). Ferroptosis, an iron-dependent form of cell death, is manifested by a toxic accumulation of lipid peroxides and ultrastructural changes in mitochondria. We herein investigated the effect of Visomitin (SKQ1), a novel mitochondria-targeting antioxidant, on several AKI models in vivo and in vitro. Our results revealed that SKQ1 treatment greatly reversed renal outcomes in cisplatin, ischemia-reperfusion injury (IRI), or folic acid-induced AKI models. These effects were reflected in attenuated levels of renal injury biomarkers, histologic indices of tubular injury, and inflammatory infiltration in the SKQ1-treated groups. Transcriptomics analysis depicted ferroptosis signaling as the most pronounced pathway downregulated after SKQ1 treatment. Consequently, administration of SKQ1 significantly ameliorated lipid peroxide accumulation and inhibited ferroptosis in the kidneys of mice with AKI. In cultured human proximal tubule epithelial cells (HK2), SKQ1 treatment markedly mitigated cisplatin-induced mitochondrial reactive oxygen species (ROS) production, resulting in lower levels of lipid peroxidation and ferroptosis. In conclusion, SKQ1 treatment protected against ischemic- or nephrotoxic-induced AKI by inhibiting ferroptosis in vivo and in vitro. These results could facilitate a broader understanding of the interaction between mitochondrial antioxidants and ferroptotic defense mechanisms, providing a possible therapeutic strategy in AKI.

Double-Negative T Cells Attenuate Cisplatin-Induced Acute Kidney Injury via Upregulating IL-10/AT2R Axis.

Objective: Our previous research showed that TCR+CD4-CD8-double-negative (DN) T cells protect renal epithelial cells from cisplatin-induced acute kidney injury (AKI). Therefore, this study is aimed at investigating the mechanism underlying the effect of DN T cells against Cis-induced AKI. Methods: HK-2 cells cultured alone or with DN T cells were treated with or without Cis. After treatment, the cell viability and death were analyzed by a CCK-8 kit and flow cytometric assay with Annexin V/PI staining, respectively. The expressions of inflammatory factors in HK-2 and DN T cells were analyzed using qPCR. The expression levels of nephrotoxicity-associated biomarkers (KIM, calbindin, and TIMP-1), Bcl-2, and angiotensin AT2 receptor (AT2R) were determined by Western blot and qPCR. Results: The administration of cisplatin significantly decreased the cell viability and AT2R expression, and increased cell death, inflammatory factors, and nephrotoxicity-associated biomarkers of HK-2 cells, while these effects were partly attenuated when cocultured with DN T cells. IL-10 expression was significantly increased in DN T cells after coculture, and cisplatin treatment aggravated this elevation. IL-10 supplementation exhibited a similar effect to coculture, whereas anti-IL-10 antibody reversed the effect of coculture on cisplatin-treated HK-2 cells. Finally, PD123319, an AT2R antagonist, also reversed the effect of IL-10 and coculture on the cell viability, death, and the expression of KIM, calbindin, TIMP-1, and Bcl-2 of cisplatin-treated HK-2 cells. Conclusions: DN T cells protected HK-2 cells from cisplatin-induced injury through IL-10/AT2R axis, which may act as a potential target for the treatment of cisplatin-induced AKI.

MiR-155 deficiency protects renal tubular epithelial cells from telomeric and genomic DNA damage in cisplatin-induced acute kidney injury.

Rationale: Cisplatin nephrotoxicity is an important cause of acute kidney injury (AKI), limiting cisplatin application in cancer therapy. Growing evidence has suggested that genome instability, telomeric dysfunction, and DNA damage were involved in the tubular epithelial cells (TECs) damage in cisplatin-induced AKI (cAKI). However, the exact mechanism is largely unknown. Methods: We subjected miR-155-/- mice and wild-type controls, as well as HK-2 cells, to cAKI models. We assessed kidney function and injury with standard techniques. The cell apoptosis and DNA damage of TECs were evaluated both in vivo and in vitro. Telomeres were measured by the fluorescence in situ hybridization. Results: The expression level of miR-155 was upregulated in cAKI. Inhibition of miR-155 expression protected cisplatin-induced AKI both in vivo and in vitro. Compared with wild-type mice, miR-155-/- mice had reduced mortality, improved renal function and pathological damage after cisplatin intervention. Moreover, inhibition of miR-155 expression attenuated TECs apoptosis and DNA damage. These protective effects were caused by increasing expression of telomeric repeat binding factor 1 (TRF1) and cyclin-dependent kinase 12 (CDK12), thereby limiting the telomeric dysfunction and the genomic DNA damage in cAKI. Conclusion: We demonstrated that miR-155 deficiency could significantly attenuate pathological damage and mortality in cAKI through inhibition of TECs apoptosis, genome instability, and telomeric dysfunction, which is possibly regulated by the increasing expression of TRF1 and CDK12. This study will provide a new molecular strategy for the prevention of cAKI.

Case Report: A Novel Non-Canonical Splice Site Variant (c.1638+7T>C) in TRPM6 Cause Primary Homagnesemia With Secondary Hocalcemia.

Objective: Primary hypomagnesemia with secondary hypocalcemia (HSH) is caused by loss-of-function mutations in the TRPM6 gene encoding the epithelial magnesium channel. It is characterized by hypomagnesemia and secondary hypocalcemia associated with neurological symptoms. Here, we aimed to investigate the genetic defects of the TRPM6 gene found in a girl from China. Methods: The genomic DNA of the proband and the parents was extracted for whole-exome sequencing. Sanger sequencing was further performed to validate the candidate variants. Subsequently, the TRPM6 gene deletion was verified by quantitative PCR (qPCR) experiment. The effect of the variant on mRNA splicing was analyzed through a minigene splice assay and reverse transcription PCR (RT-PCR) in vitro. Results: The proband presented with the symptoms of generalized seizures, tetany, and muscle spasms, which were refractory to anticonvulsant treatment. Phenotypic data indicated that the patient had hypomagnesemia, poor parathyroid hormone response, and resultant hypocalcemia. The trio whole-exome sequencing identified that the proband carried compound heterozygous variants in the TRPM6 gene, a paternally derived exon 6 deletion, and a maternally derived splicing variant (c.1638+7T>C) in exon 14. The minigene splice assay confirmed that the c.1638+7T>C variant resulted in exon 14 skipping, which caused the alteration of TRPM6 mRNA splicing. Conclusion: Our results support that the compound heterozygous variants in TRPM6 are responsible for HSH in this patient. A novel pathogenic splicing variant (c.1638+7T>C) in the intron 14 disturbs the normal TRPM6 mRNA splicing, suggesting that the non-classical splice variant plays a critical role in HSH. This variant is essential for future effective genetic diagnosis.

Rapamycin attenuated podocyte apoptosis via upregulation of nestin in Ang II-induced podocyte injury.

BACKGROUND: Angiotensin II (Ang II) contributes to the progression of glomerulosclerosis, mainly by inducing podocyte injury. Convincing evidence indicates that the mTOR inhibitor rapamycin could play a fundamental role in protection against podocyte injury. Nestin, a major cytoskeletal protein, is stably expressed in podocytes and correlates with podocyte damage. The purpose of this study was to investigate the effect of rapamycin on podocyte injury induced by Ang II and to clarify the role and mechanism of nestin in the protective effect of rapamycin of podocyte injury. METHODS AND RESULTS: We established an Ang II perfusion animal model, and the effects of rapamycin treatment on podocytes were investigated in vivo. In vitro, podocytes were stimulated with Ang II and rapamycin to observe podocyte injury, and nestin-siRNA was transfected to investigate the underlying mechanisms. We observed that Ang II induced podocyte injury both in vivo and in vitro, whereas rapamycin treatment relieved Ang II-induced podocyte injury. We further found that nestin co-localized with p-mTOR in glomeruli, and the protective effect of rapamycin was reduced by nestin-siRNA in podocytes. Moreover, co-IP indicated the interaction between nestin and p-mTOR, and nestin could affect podocyte injury via the mTOR/P70S6K signaling pathway. CONCLUSION: We demonstrated that rapamycin attenuated podocyte apoptosis via upregulation of nestin expression through the mTOR/P70S6K signaling pathway in an Ang II-induced podocyte injury.

miR-199b-5p mediates adriamycin-induced podocyte apoptosis by inhibiting the expression of RGS10.

Podocyte apoptosis is a key risk factor for the progression of kidney diseases. MicroRNA (miR)-199b-5p has been shown to be involved in cell apoptosis. However, the molecular mechanisms of miR-199b-5p in podocyte apoptosis remain uncertain. Thus, the present study aimed to investigate whether miR-199b-5p participates in the regulation of podocyte apoptosis and to elucidate the involved mechanisms of this process. A podocyte apoptosis model was constructed using adriamycin (ADR) in vitro. miR-199b-5p mimic and inhibitor were transfected in podocytes to change the expression level of miR-199b-5p. RNA expression was examined by reverse transcription-quantitative PCR. Western blotting was used to measure protein expression. Apoptosis was monitored via flow cytometry and detection of apoptosis-associated proteins. The results from the present study demonstrated that miR-199b-5p was upregulated and that regulator of G-protein signaling 10 (RGS10) was downregulated in ADR-stimulated podocytes. Overexpression of miR-199b-5p could inhibit RGS10 expression and stimulate podocyte apoptosis, whereas miR-199b-5p knockdown restored the levels of RGS10 and ameliorated podocyte apoptosis in ADR-induced podocytes. Furthermore, the effects of miR-199b-5p overexpression could be significantly reversed by RGS10 overexpression. In addition, podocyte transfection of miR-199b-5p activated the AKT/mechanistic target of rapamycin (mTOR) signaling, which was blocked following RGS10 overexpression. Taken together, the present study demonstrated that miR-199b-5p upregulation could promote podocyte apoptosis by inhibiting the expression of RGS10 through the activation of AKT/mTOR signaling.

A contactless method to measure real-time finger motion using depth-based pose estimation.

BACKGROUND: Finger mobility plays a crucial role in everyday living and is a leading indicator during hand rehabilitation and assistance tasks. Depth-based hand pose estimation is a potentially low-cost solution for the clinical and home-based measurement of symptoms of limited human finger motion. OBJECTIVE: The purpose of this study was to achieve the contactless measurement of finger motion based on depth-based hand pose estimation using Azure Kinect depth cameras and transfer learning, and to evaluate the accuracy in comparison with a three-dimensional motion analysis (3DMA) system. METHODS: Thirty participants performed a series of tasks during which their hand motions were measured concurrently using the Azure Kinect and 3DMA systems. We propose a simple and effective approach to achieving real-time hand pose estimations from single depth images using ensemble convolutional neural networks trained by a transfer learning strategy. Algorithms to calculate the finger joint motion angles are presented by tracking the locations of the 24 hand joints. To demonstrate their potential, the Azure-Kinect-based 3D finger motion measurement system and algorithms are experimentally verified through comparison with a camera-based 3DMA system, which is the gold standard. RESULTS: Our results revealed that the Azure-Kinect-based hand pose estimation system produced highly correlated measurements of hand joint coordinates. Our method achieved excellent performance in terms of the fraction of good frames ( >80%) when the error thresholds were larger than approximately 2 cm, and the range of mean error distance was 0.23--1.05 cm. For joint angles, the Azure Kinect and 3DMA systems had comparable inter-trial reliability (ICC2,1 ranging from 0.89 to 0.97) and excellent concurrent validity, with Pearsons r-values >0.90 for most measurements (range: 0.88--0.97). The 95% BlandAltman limits of agreement were narrow enough for the Azure Kinect to be considered a valid tool for the measurement of all reported joint angles of the index finger and thumb in pinching. Moreover, our method runs in real time at over 45 fps. CONCLUSION: The results of this study suggest that the proposed method has the capacity to measure the performance of fine motor skills.

Borneol inhibits CD4 + T cells proliferation by down-regulating miR-26a and miR-142-3p to attenuate asthma.

BACKGROUND: Asthma is a chronic airway inflammatory disease caused by a variety of cytokines and signaling pathways closely related to immunoregulation. Corticosteroids are the most widely used drug in the asthma treatment. However, the use of corticosteroids could cause topical side effects. So, it's important to find new drugs for asthma treatment. Our study aims to explore the pharmacological effect of borneol on asthma and its underlying mechanism. METHODS: We constructed the OVA-induced asthma model to investigate the effect of borneol on asthma in mice. HE and PAS staining was used to detect the effect of borneol on pathological change of mice with asthma. Inflammatory cytokines were measured by ELISA. qRT-PCR was used to explore the effect of borneol on microRNAs expression. Cell proliferation of CD4 + T cells was detected by CCK-8 assay and flow cytometry. Western blot was used to detect pten expression and Akt activation. RESULTS: We found that borneol significantly alleviated asthma progression in mice. Borneol inhibited CD4 + T cells infiltration in vivo and proliferation in vitro by downregulating miR-26a and miR-142-3p. miR-26a and miR-142-3p promoted CD4 + T cells proliferation in vitro through targeting Pten. Overexpression of miR-26a and miR-142-3p abolished the effect of borneol in vivo. CONCLUSION: In a word, these findings suggested that borneol attenuated asthma in mice by decreasing the CD4 + T cells infiltration. The molecular mechanism of borneol was dependent on the downregulation of miR-26a and miR-142-3p to upregulate the Pten expression.

Extraction and selection of gait recognition features using skeleton point detection and improved fuzzy decision.

It is of great importance to effectively measure gait features and recognize the signature gait patterns for gait rehabilitation. In this work, we used a skeleton point detection to extract gait features and proposed an improved fuzzy decision to select the most significant features for classifying gait patterns. Thirteen gait recognition features were extracted from the obtained skeleton points data. Taking the extracted features as an input, our improved fuzzy similarity priority decision method has obtained important sequences of all features based on the relatively important scores. Then, the ranked features were delivered in different classifiers by a sequential forward selection strategy to select the optimal feature subset. There were significant differences between groups in each of the thirteen gait recognition features (p < 0.005), indicating that all extracted features are potential influence factors for classifying gait patterns. We also found that the highest classification accuracy of 100% for gait feature subsets included the stride frequency, maximum flexion angle of knee, and toe-out angle, on the all classifiers. The results suggest that the proposed approaches are very useful in searching for the optimal feature subset in present dataset.