Identification and Expression Analysis of R2R3-MYB Transcription Factors Associated with Flavonoid Biosynthesis in Panax quinquefolius.

Panax quinquefolius L. is an important medicinal plant, and flavonoids are among its main secondary metabolites. The R2R3-MYB transcription factor plays an irreplaceable role in plant growth, development, and secondary metabolism. In our study, we identified 159 R2R3-MYBs and analyzed their physical and chemical properties in P. quinquefolius. The protein length of 159 PqMYBs varied from 107 to 1050 amino acids. The molecular weight ranged from 12.21 to 116.44 kDa. The isoelectric point was between 4.57 and 10.34. We constructed a phylogenetic tree of P. quinquefolius and Arabidopsis thaliana R2R3-MYB family members, and PqMYB members were divided into 33 subgroups. Transcriptome data analysis showed that the expression patterns of PqMYBs in root, leaf, and flower were significantly different. Following the MeJA treatment of seedlings, five candidate PqMYB genes demonstrated a response. A correlation analysis of PqMYBs and candidate flavonoid pathway genes showed that PqMYB2, PqMYB46, and PqMYB72 had correlation coefficients that were higher than 0.8 with PqCHS, PqANS4, and PqCCoAMT10, respectively. Furthermore, a transient expression assay confirmed that the three PqMYBs were localized in the nucleus. We speculated that these three PqMYBs were related to flavonoid biosynthesis in P. quinquefolius. These results provided a theoretical basis and a new perspective for further understanding the R2R3-MYB gene family and the biosynthesis mechanism of secondary metabolites in P. quinquefolius.

Prevention of age-related truncation of γ-glutamylcysteine ligase catalytic subunit (GCLC) delays cataract formation.

A sharp drop in lenticular glutathione (GSH) plays a pivotal role in age-related cataract (ARC) formation. Despite recognizing GSH's importance in lens defense for decades, its decline with age remains puzzling. Our recent study revealed an age-related truncation affecting the essential GSH biosynthesis enzyme, the γ-glutamylcysteine ligase catalytic subunit (GCLC), at aspartate residue 499. Intriguingly, these truncated GCLC fragments compete with full-length GCLC in forming a heterocomplex with the modifier subunit (GCLM) but exhibit markedly reduced enzymatic activity. Crucially, using an aspartate-to-glutamate mutation knock-in (D499E-KI) mouse model that blocks GCLC truncation, we observed a notable delay in ARC formation compared to WT mice: Nearly 50% of D499E-KI mice remained cataract-free versus ~20% of the WT mice at their age of 20 months. Our findings concerning age-related GCLC truncation might be the key to understanding the profound reduction in lens GSH with age. By halting GCLC truncation, we can rejuvenate lens GSH levels and considerably postpone cataract onset.

The prognostic value of LAYN in HPV-related head and neck squamous cell carcinoma and its influence on immune cell infiltration.

BACKGROUND: HPV-positive head and neck squamous cell carcinoma (HNSCC) exhibits different characteristics from HPV-negative tumors in terms of tumor development, clinical features, treatment response, and prognosis. Layilin (LAYN), which contains homology with C-type lectins, plays a critical role in tumorigenesis and cancer progression. However, the prognostic value of LAYN and the relationship between LAYN and immune infiltration levels in HPV-related HNSCC patients still require a comprehensive understanding. Herein, we aimed to assess the prognostic value of LAYN and to investigate its underlying immunological function in HPV-related HNSCC. METHODS: Through various bioinformatics methods, we analyzed the data from The Cancer Genome Atlas (TCGA), Tumor Immune Estimation Resource (TIMER) and Gene Expression Profiling Interactive Analysis (GEPIA) databases to explore the potential underlying oncogenic impression of LAYN, including the relevance of LAYN to survival outcomes, clinicopathological factors, immune cell infiltration, and immune marker sets in HPV-related HNSCC. The expression levels of LAYN and HPV were also verified in HNSCC patient tissues. RESULTS: LAYN was differentially expressed in a variety of tumors. The expression of LAYN in HNSCC was significantly higher than that in adjacent normal tissues (P < 0.0001), and high expression of LAYN was correlated with poor overall survival (OS) in HNSCC patients (Hazard Ratio (HR) = 1.3, P = 0.035). Moreover, LAYN expression level in HPV-positive HNSCC patients was significantly lower than that in HPV-negative patients, with HPV-positive HNSCC patients displaying a trend of favorable prognosis. In addition, the relationship between LAYN expression and immune infiltration levels in HPV-positive HNSCC group was less tightly correlated than that in HPV-negative HNSCC group, and there was a strong relationship between LAYN expression and markers of M2 macrophage (P < 0.001) and exhausted T cells (P < 0.05) in HPV-negative HNSCC. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis suggested that LAYN potentially influenced tumor progression through HPV infection and other cancer-related pathways. CONCLUSIONS: LAYN might contribute to tumorigenesis via its positive correlation with immune checkpoint molecules and tumor-associated macrophages (TAMs). Our study might provide a novel prognostic biomarker and latent therapeutic target for the treatment of HPV-related HNSCC.

Autophagy activates EGR1 via MAPK/ERK to induce FGF2 in renal tubular cells for fibroblast activation and fibrosis during maladaptive kidney repair.

Macroautophagy/autophagy contributes to maladaptive kidney repair by inducing pro-fibrotic factors such as FGF2 (fibroblast growth factor 2), but the underlying mechanism remains elusive. Here, we show that EGR1 (early growth response 1) was induced in injured proximal tubules after ischemic acute kidney injury (AKI) and this induction was suppressed by autophagy deficiency in inducible, renal tubule-specific atg7 (autophagy related 7) knockout (iRT-atg7 KO) mice. In cultured proximal tubular cells, TGFB1 (transforming growth factor beta 1) induced EGR1 and this induction was also autophagy dependent. Egr1 knockdown in tubular cells reduced FGF2 expression during TGFB1 treatment, leading to less FGF2 secretion and decreased paracrine effects on fibroblasts. ChIP assay detected an increased binding of EGR1 to the Fgf2 gene promoter in TGFB1-treated tubular cells. Both Fgf2 and Egr1 transcription was inhibited by FGF2 neutralizing antibody, suggesting a positive feedback for EGR1-mediated FGF2 autoregulation. This feedback was confirmed using fgf2-deficient tubular cells and fgf2-deficient mice. Upstream of EGR1, autophagy deficiency in mice suppressed MAPK/ERK (mitogen-activated protein kinase) activation in post-ischemic renal tubules. This inhibition correlated with SQSTM1/p62 (sequestosome 1) aggregation and its sequestration of MAPK/ERK. SQSTM1/p62 interacted with MAPK/ERK and blocked its activation during TGFB1 treatment in autophagy-deficient tubular cells. Inhibition of MAPK/ERK suppressed EGR1 and FGF2 expression in maladaptive tubules, leading to the amelioration of renal fibrosis and improvement of renal function. These results suggest that autophagy activates MAPK/ERK in renal tubular cells, which induces EGR1 to transactivate FGF2. FGF2 is then secreted into the interstitium to stimulate fibroblasts for fibrogenesis.Abbreviation: 3-MA: 3-methyladenine; ACTA2/α-SMA: actin alpha 2, smooth muscle, aorta; ACTB/ß-actin: actin, beta; AKI: acute kidney injury; aa: amino acid; ATG/Atg: autophagy related; BUN: blood urea nitrogen; ChIP: chromatin immunoprecipitation; CKD: chronic kidney disease; CM: conditioned medium; COL1A1: collagen, type I, alpha 1; COL4A1: collagen, type IV, alpha 1; CQ: chloroquine; DBA: dolichos biflorus agglutinin; EGR1: early growth response 1; ELK1: ELK1, member of ETS oncogene family; FGF2: fibroblast growth factor 2; FN1: fibronectin 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HAVCR1/KIM-1: hepatitis A virus cellular receptor 1; IP: immunoprecipitation; LIR: LC3-interacting region; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAP2K/MEK: mitogen-activated protein kinase kinase; MAPK: mitogen-activated protein kinase; NFKB: nuclear factor kappa B; PB1: Phox and Bem1; PFT: pifithrin α; PPIB/cyclophilin B: peptidylprolyl isomerase B; RT-qPCR: real time-quantitative PCR; SQSTM1/p62: sequestosome 1; TGFB1/TGF-ß1: transforming growth factor beta 1; VIM: vimentin.

Multimodality CT imaging contributes to improving the diagnostic accuracy of solitary pulmonary nodules: a multi-institutional and prospective study.

BACKGROUND: Solitary pulmonary nodules (SPNs) are one of the most common chest computed tomography (CT) abnormalities clinically. We aimed to investigate the value of non-contrast enhanced CT (NECT), contrast enhanced CT (CECT), CT perfusion imaging (CTPI), and dual- energy CT (DECT) used for differentiating benign and malignant SPNs with a multi-institutional and prospective study. PATIENTS AND METHODS: Patients with 285 SPNs were scanned with NECT, CECT, CTPI and DECT. Differences between the benign and malignant SPNs on NECT, CECT, CTPI, and DECT used separately (NECT combined with CECT, DECT, and CTPI were methods of A, B, and C) or in combination (Method A + B, A + C, B + C, and A + B + C) were compared by receiver operating characteristic curve analysis. RESULTS: Multimodality CT imaging showed higher performances (sensitivities of 92.81% to 97.60%, specificities of 74.58% to 88.14%, and accuracies of 86.32% to 93.68%) than those of single modality CT imaging (sensitivities of 83.23% to 85.63%, specificities of 63.56% to 67.80%, and accuracies of 75.09% to 78.25%, all p < 0.05). CONCLUSIONS: SPNs evaluated with multimodality CT imaging contributes to improving the diagnostic accuracy of benign and malignant SPNs. NECT helps to locate and evaluate the morphological characteristics of SPNs. CECT helps to evaluate the vascularity of SPNs. CTPI using parameter of permeability surface and DECT using parameter of normalized iodine concentration at the venous phase both are helpful for improving the diagnostic performance.

Blocking ribosomal protein S6 phosphorylation inhibits podocyte hypertrophy and focal segmental glomerulosclerosis.

Ribosomal protein S6 (rpS6) phosphorylation mediates the hypertrophic growth of kidney proximal tubule cells. However, the role of rpS6 phosphorylation in podocyte hypertrophy and podocyte loss during the pathogenesis of focal segmental glomerulosclerosis (FSGS) remains undefined. Here, we examined rpS6 phosphorylation levels in kidney biopsy specimens from patients with FSGS and in podocytes from mouse kidneys with Adriamycin-induced FSGS. Using genetic and pharmacologic approaches in the mouse model of FSGS, we investigated the role of rpS6 phosphorylation in podocyte hypertrophy and loss during development and progression of FSGS. Phosphorylated rpS6 was found to be markedly increased in the podocytes of patients with FSGS and Adriamycin-induced FSGS mice. Genetic deletion of the Tuberous sclerosis 1 gene in kidney glomerular podocytes activated mammalian target of rapamycin complex 1 signaling to rpS6 phosphorylation, resulting in podocyte hypertrophy and pathologic features similar to those of patients with FSGS including podocyte loss, leading to segmental glomerulosclerosis. Since protein phosphatase 1 is known to negatively regulate rpS6 phosphorylation, treatment with an inhibitor increased phospho-rpS6 levels, promoted podocyte hypertrophy and exacerbated formation of FSGS lesions. Importantly, blocking rpS6 phosphorylation (either by generating congenic rpS6 knock-in mice expressing non-phosphorylatable rpS6 or by inhibiting ribosomal protein S6 kinase 1-mediated rpS6 phosphorylation with an inhibitor) significantly blunted podocyte hypertrophy, inhibited podocyte loss, and attenuated formation of FSGS lesions. Thus, our study provides genetic and pharmacologic evidence indicating that specifically targeting rpS6 phosphorylation can attenuate the development of FSGS lesions by inhibiting podocyte hypertrophy and associated podocyte depletion.

Mitotic Activation Around Wound Edges and Epithelialization Repair in UVB-Induced Capsular Cataracts.

Purpose: Ultraviolet B (UVB) has been well documented to induce capsular cataracts; however, the mechanism of the lens epithelial cell-mediated repair process after UVB irradiation is not fully understood. The purpose of this study was to better understand lens epithelial cell repair after UVB-induced epithelium damage. Method: C57BL/6J mice were irradiated by various doses of UVB. Lens morphology and lens capsule opacity were monitored by slit lamp, darkfield microscopy, and phase-contrast microscopy. Lens epithelial cell mitotic activation and cell apoptosis were measured by immunohistochemistry. Lens epithelial ultrastructure was analyzed by transmission electron microscopy. Results: UVB irradiation above a dose of 2.87 kJ/m2 triggered lens epithelial cell apoptosis and subcapsular cataract formation, with a ring-shaped structure composed of multilayered epithelial cell clusters manifesting a dense ring-shaped capsular cataract. The epithelial cells immediately outside the edge of the ring-shaped aggregates transitioned to mitotically active cells and performed wound healing through the epithelialization process. However, repairs ceased when lens epithelial cells made direct contact, and scar-like tissue in the center of the anterior capsule remained even by 6 months after UVB irradiation. Conclusions: Our present study demonstrates that normally quiescent lens epithelial cells can be reactivated for epithelialization repair in response to UV-induced damage.

Predictive value of plasma volume status for contrast-induced nephropathy in patients with heart failure undergoing PCI.

AIMS: Contrast-induced nephropathy remains a common complication of coronary procedure and increases poor outcomes, especially in patients with heart failure. Plasma volume expansion relates to worsening prognosis of heart failure. We hypothesized that calculated plasma volume status (PVS) might provide predictive utility for contrast-induced nephropathy in patients with heart failure undergoing elective percutaneous coronary intervention (PCI). METHODS AND RESULTS: We enrolled 441 patients with heart failure undergoing elective PCI from 2012 to 2018. Pre-procedural estimated PVS by the Duarte's formula (Duarte-ePVS) and Kaplan-Hakim formula (KH-ePVS) were calculated for all patients. CIN was defined as an absolute serum creatinine (SCr) increase ≥0.5 mg/dL or a relative increase ≥25% compared with the baseline value within 48 h of contrast medium exposure. We assessed the association between PVS and CIN in patients with heart failure undergoing elective PCI. In 441 patients, 28 (6.3%) patients developed CIN. The median Duarte-ePVS was 4.44 (3.87, 5.13) and the median KH-ePVS was -0.03 (-0.09, 0.05). The best cutoff values for Duarte-ePVS and KH-ePVS to predict CIN were 4.64 (with 78.6% sensitivity and 61.7% specificity) and 0.04 (with 64.5% sensitivity and 75.5% specificity), respectively. After adjusting for potential confounding variables, KH-ePVS > 0.04 [odds ratio (OR) 2.685, 95% confidence interval (CI) 1.012-7.123, P = 0.047] remained significantly associated with CIN whereas Duarte-ePVS was not. CONCLUSIONS: Pre-procedural KH-ePVS is an independent risk factor for CIN in patients with heart failure undergoing elective PCI. The best cutoff point of KH-ePVS for predicting CIN was 0.04.

Metformin effectively treats Tsc1 deletion-caused kidney pathology by upregulating AMPK phosphorylation.

Tuberous sclerosis complex (TSC) is characterized by hamartomatous lesions in multiple organs, with most patients developing polycystic kidney disease and leading to a decline of renal function. TSC is caused by loss-of-function mutations in either Tsc1 or Tsc2 gene, but currently, there is no effective treatment for aberrant kidney growth in TSC patients. By generating a renal proximal tubule-specific Tsc1 gene-knockout (Tsc1 ptKO) mouse model, we observed that Tsc1 ptKO mice developed aberrantly enlarged kidneys primarily due to hypertrophy and proliferation of proximal tubule cells, along with some cystogenesis, interstitial inflammation, and fibrosis. Mechanistic studies revealed inhibition of AMP-activated protein kinase (AMPK) phosphorylation at Thr-172 and activation of Akt phosphorylation at Ser-473 and Thr-308. We therefore treated Tsc1 ptKO mice with the AMPK activator, metformin, by daily intraperitoneal injection. Our results indicated that metformin increased the AMPK phosphorylation, but decreased the Akt phosphorylation. These signaling modulations resulted in inhibition of proliferation and induction of apoptosis in the renal proximal tubule cells of Tsc1 ptKO mice. Importantly, metformin treatment effectively prevented aberrant kidney enlargement and cyst growth, inhibited inflammatory response, attenuated interstitial fibrosis, and protected renal function. The effects of metformin were further confirmed by in vitro experiments. In conclusion, this study indicates a potential therapeutic effect of metformin on Tsc1 deletion-induced kidney pathology, although currently metformin is primarily prescribed to treat patients with type 2 diabetes.

Fibrinogen-to-Albumin Ratio Predicts Contrast-Induced Nephropathy in Patients after Emergency Percutaneous Coronary Intervention.

BACKGROUND: The aim of the present study was to investigate the association between fibrinogen-to-albumin ratio (FAR) with contrast-induced nephropathy (CIN) in patients undergoing emergency percutaneous coronary intervention (PCI). METHODS: 565 patients with emergency PCI were consecutively enrolled. The primary outcome was CIN defined as either a 25% increase in baseline serum creatinine levels or a 0.5 mg/dL (44 µmol/L) increase in absolute serum creatinine levels within 72 h after the contrast medium exposure. Logistic regression analysis was applied to analyze whether FAR was an independent risk factor for CIN. RESULTS: Overall, 29 (5.1%) patients developed CIN. Compared with the patients without CIN, the patients developing CIN had lower albumin (39.79 ± 3.95 vs. 37.14 ± 5.21, P=0.012) and higher fibrinogen levels (3.51 ± 0.94 vs. 4.14 ± 0.96, P < 0.001). In the multivariate logistic analysis, FAR was an independent predictor of CIN (OR = 3.97; 95% CI, 1.61-9.80; P=0.003) along with perihypotension, age >75 years, and LVEF <45%, and 0.106 was the optimal cutoff value of preprocedural FAR to predict CIN. CONCLUSION: Preprocedural levels of FAR were associated with CIN in patients after emergency PCI.

The TNF-derived TIP peptide activates the epithelial sodium channel and ameliorates experimental nephrotoxic serum nephritis.

In mice, the initial stage of nephrotoxic serum-induced nephritis (NTN) mimics antibody-mediated human glomerulonephritis. Local immune deposits generate tumor necrosis factor (TNF), which activates pro-inflammatory pathways in glomerular endothelial cells (GECs) and podocytes. Because TNF receptors mediate antibacterial defense, existing anti-TNF therapies can promote infection; however, we have previously demonstrated that different functional domains of TNF may have opposing effects. The TIP peptide mimics the lectin-like domain of TNF, and has been shown to blunt inflammation in acute lung injury without impairing TNF receptor-mediated antibacterial activity. We evaluated the impact of TIP peptide in NTN. Intraperitoneal administration of TIP peptide reduced inflammation, proteinuria, and blood urea nitrogen. The protective effect was blocked by the cyclooxygenase inhibitor indomethacin, indicating involvement of prostaglandins. Targeted glomerular delivery of TIP peptide improved pathology in moderate NTN and reduced mortality in severe NTN, indicating a local protective effect. We show that TIP peptide activates the epithelial sodium channel(ENaC), which is expressed by GEC, upon binding to the channel's α subunit. In vitro, TNF treatment of GEC activated pro-inflammatory pathways and decreased the generation of prostaglandin E2 and nitric oxide, which promote recovery from NTN. TIP peptide counteracted these effects. Despite the capacity of TIP peptide to activate ENaC, it did not increase mean arterial blood pressure in mice. In the later autologous phase of NTN, TIP peptide blunted the infiltration of Th17 cells. By countering the deleterious effects of TNF through direct actions in GEC, TIP peptide could provide a novel strategy to treat glomerular inflammation.

GP73, a novel TGF-ß target gene, provides selective regulation on Smad and non-Smad signaling pathways.

Increased GP73 expression in hepatocytes from patients with acute hepatitis, through disease progression to cirrhosis and chronic liver disease suggests that progressive tissue remodeling and fibrogenesis are driving forces for GP73 upregulation. Nevertheless, details about regulation of GP73 expression and its biological functions remain elusive and await further characterization. In this study, we demonstrate that GP73 is a direct target of TGF-ß1 transcriptional regulation. Its induced expression inhibits TGF-ß-Smad mediated growth suppression. On the other hand, elevated GP73 results in upregulation of ERK/Akt signaling induced by TGF-ß1. Mechanistically, upregulation of lipid raft and caveolae-1 induced by GP73 overexpression mediates its regulatory effect on TGF-ß1 signaling. Notably, lipid raft expression is elevated in HCC tumors and tissues with higher GP73 expression yield more intensive Flotillin staining. Our results establish the linkage between GP73 and TGF-ß signaling, indicating that GP73 may promote HCC tumorigenesis by selectively regulating TGF-ß signaling through lipid raft modulation.

Diurnal rhythm of follicle-stimulating hormone is associated with nonalcoholic fatty liver disease in a Chinese elderly population.

OBJECTIVE: Previous studies have found that impairment of the circadian clock appears to contribute to the development of nonalcoholic fatty liver disease (NAFLD) and the circulating follicle-stimulating hormone (FSH) level showed a diurnal cycle. A recent study reported that a lower FSH level was associated with NAFLD. However, the effects of the diurnal rhythm of FSH on NAFLD have not been reported. The aim of this study was to evaluate whether the diurnal rhythm of FSH was associated with NAFLD in an elderly population. STUDY DESIGN: We performed a cross-sectional study among 71 elderly patients between August 2015 and November 2015 at Fujian Provincial Hospital. Anthropometrics and tests for laboratory were performed for each patient. FSH was determined by radioimmunoassay. The FSH receptor (FSHR) expression was identified in liver and ovary tissue by immunohistochemical staining. NAFLD was diagnosed by sonographic features. RESULTS: Of the 71 patients, 33 (42.9%) had NAFLD on their ultrasound. There were no significant differences between subjects with NAFLD and those without NAFLD in terms of age, sex, body mass index, waist-to-hip ratio, fasting plasma glucose, postload plasma glucose, liver enzyme, triglycerides, total cholesterol, high-density lipoprotein-cholesterol and low-density lipoprotein-cholesterol. Both the serum FSH levels of 8AM and 0AM showed no differences between the groups. The proportion of the 'normal' diurnal rhythm of FSH was higher among the patients with NAFLD (78.1% vs. 52.6%, P = .027). After adjusting for all potential confounders, the fully adjusted odds ratios (OR) of diurnal rhythm of FSH for NAFLD was 3.86 (95%CI: 1.01, 14.81, P = .049). Immunohistochemical staining showed that the FSHR protein was detected in human ovarian and hepatic tissues. CONCLUSIONS: These results suggest that the 'normal' diurnal rhythm of FSH was independently associated with NAFLD in an elderly population. This study provides a novel insight into the diurnal rhythm of FSH in the pathogenesis of NAFLD.

GP73 regulates Hepatic Steatosis by enhancing SCAP-SREBPs interaction.

Elevated Golgi phosphoprotein 2 (GP73, also known as GOLPH2 or GOLM1) expression in serum and liver, which can be induced by viral infection and cytokine treatments, is intimately connected with liver disease, including acute hepatitis, cirrhosis and hepatocellular carcinoma (HCC). However, its pathogenic roles in hepatic diseases have never been clarified in detail. Here, we showed that the upregulated GP73 is indispensable for SREBPs activation and lipogenesis. Notably, GP73 overexpression enhanced SCAP-SREBPs binding and its Golgi trafficking even under cholesterol sufficiency. Consistent with these functional findings, GP73 blockage could alleviate tunicamycin-induced liver steatosis by reducing SREBPs activation. A significant positive correlation of GP73 with genes in lipid metabolism pathway was also identified in liver cancer based on data from The Cancer Genome Atlas (TCGA) dataset. Our findings revealed previously unrecognized role of GP73 in lipid metabolism.

DNA methylation protects against cisplatin-induced kidney injury by regulating specific genes, including interferon regulatory factor 8.

DNA methylation is an epigenetic mechanism that regulates gene transcription without changing primary nucleotide sequences. In mammals, DNA methylation involves the covalent addition of a methyl group to the 5-carbon position of cytosine by DNA methyltransferases (DNMTs). The change of DNA methylation and its pathological role in acute kidney injury (AKI) remain largely unknown. Here, we analyzed genome-wide DNA methylation during cisplatin-induced AKI by reduced representation bisulfite sequencing. This technique identified 215 differentially methylated regions between the kidneys of control and cisplatin-treated animals. While most of the differentially methylated regions were in the intergenic, intronic, and coding DNA sequences, some were located in the promoter or promoter-regulatory regions of 15 protein-coding genes. To determine the pathological role of DNA methylation, we initially examined the effects of the DNA methylation inhibitor 5-aza-2'-deoxycytidine and showed it increased cisplatin-induced apoptosis in a rat kidney proximal tubular cell line. We further established a kidney proximal tubule-specific DNMT1 (PT-DNMT1) knockout mouse model, which showed more severe AKI during cisplatin treatment than wild-type mice. Finally, interferon regulatory factor 8 (Irf8), a pro-apoptotic factor, was identified as a hypomethylated gene in cisplatin-induced AKI, and this hypomethylation was associated with a marked induction of Irf8. In the rat kidney proximal tubular cells, the knockdown of Irf8 suppressed cisplatin-induced apoptosis, supporting a pro-death role of Irf8 in renal tubular cells. Thus, DNA methylation plays a protective role in cisplatin-induced AKI by regulating specific genes, such as Irf8.

Autophagy is activated to protect against podocyte injury in adriamycin-induced nephropathy.

Podocytes are highly differentiated epithelial cells wrapping glomerular capillaries to form the filtration barrier in kidneys. As such, podocyte injury or dysfunction is a critical pathogenic event in glomerular disease. Autophagy plays an important role in the maintenance of the homeostasis and function of podocytes. However, it is less clear whether and how autophagy contributes to podocyte injury in glomerular disease. Here, we have examined the role of autophagy in adriamycin-induced nephropathy, a classic model of glomerular disease. We show that autophagy was induced by adriamycin in cultured podocytes in vitro and in podocytes in mice. In cultured podocytes, activation of autophagy with rapamycin led to the suppression of adriamycin-induced apoptosis, whereas inhibition of autophagy with chloroquine enhanced podocyte apoptosis during adriamycin treatment. To determine the role of autophagy in vivo, we established an inducible podocyte-specific autophagy-related gene 7 knockout mouse model (Podo-Atg7-KO). Compared with wild-type littermates, Podo-Atg7-KO mice showed higher levels of podocyte injury, glomerulopathy, and proteinuria during adriamycin treatment. Together, these observations support an important role of autophagy in protecting podocytes under the pathological conditions of glomerular disease, suggesting the therapeutic potential of autophagy induction.

Exosome production and its regulation of EGFR during wound healing in renal tubular cells.

Kidney repair following injury involves the reconstitution of a structurally and functionally intact tubular epithelium. Growth factors and their receptors, such as EGFR, are important in the repair of renal tubules. Exosomes are cell-produced small (~100 nm in diameter) vesicles that contain and transfer proteins, lipids, RNAs, and DNAs between cells. In this study, we examined the relationship between exosome production and EGFR activation and the potential role of exosome in wound healing. EGFR activation occurred shortly after scratch wounding in renal tubular cells. Wound repair after scratching was significantly promoted by EGF and suppressed by EGFR inhibitor gefitinib. Interestingly, scratch wounding induced a significant increase of exosome production. The exosome production was decreased by EGF and increased by gefitinib, suggesting a suppressive role of EGFR signaling in exosome production. Conversely, inhibition of exosome release by GW4869 and manumycin A markedly increased EGFR activation and promoted wound healing. Moreover, exosomes derived from scratch-wounding cells could inhibit wound healing. Collectively, the results indicate that wound healing in renal tubular cells is associated with EGFR activation and exosome production. Although EGFR activation promotes wound healing, released exosomes may antagonize EGFR activation and wound healing.

Protein Kinase Cδ Suppresses Autophagy to Induce Kidney Cell Apoptosis in Cisplatin Nephrotoxicity.

Nephrotoxicity is a major adverse effect in cisplatin chemotherapy, and renoprotective approaches are unavailable. Recent work unveiled a critical role of protein kinase Cδ (PKCδ) in cisplatin nephrotoxicity and further demonstrated that inhibition of PKCδ not only protects kidneys but enhances the chemotherapeutic effect of cisplatin in tumors; however, the underlying mechanisms remain elusive. Here, we show that cisplatin induced rapid activation of autophagy in cultured kidney tubular cells and in the kidneys of injected mice. Cisplatin also induced the phosphorylation of mammalian target of rapamycin (mTOR), p70S6 kinase downstream of mTOR, and serine/threonine-protein kinase ULK1, a component of the autophagy initiating complex. In vitro, pharmacologic inhibition of mTOR, directly or through inhibition of AKT, enhanced autophagy after cisplatin treatment. Notably, in both cells and kidneys, blockade of PKCδ suppressed the cisplatin-induced phosphorylation of AKT, mTOR, p70S6 kinase, and ULK1 resulting in upregulation of autophagy. Furthermore, constitutively active and inactive forms of PKCδ respectively enhanced and suppressed cisplatin-induced apoptosis in cultured cells. In mechanistic studies, we showed coimmunoprecipitation of PKCδ and AKT from lysates of cisplatin-treated cells and direct phosphorylation of AKT at serine-473 by PKCδin vitro Finally, administration of the PKCδ inhibitor rottlerin with cisplatin protected against cisplatin nephrotoxicity in wild-type mice, but not in renal autophagy-deficient mice. Together, these results reveal a pathway consisting of PKCδ, AKT, mTOR, and ULK1 that inhibits autophagy in cisplatin nephrotoxicity. PKCδ mediates cisplatin nephrotoxicity at least in part by suppressing autophagy, and accordingly, PKCδ inhibition protects kidneys by upregulating autophagy.

Blocking rpS6 Phosphorylation Exacerbates Tsc1 Deletion-Induced Kidney Growth.

The molecular mechanisms underlying renal growth and renal growth-induced nephron damage remain poorly understood. Here, we report that in murine models, deletion of the tuberous sclerosis complex protein 1 (Tsc1) in renal proximal tubules induced strikingly enlarged kidneys, with minimal cystogenesis and occasional microscopic tumorigenesis. Signaling studies revealed hyperphosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and increased phosphorylation of ribosomal protein S6 (rpS6) in activated renal tubules. Notably, knockin of a nonphosphorylatable rpS6 in these Tsc1-mutant mice exacerbated cystogenesis and caused drastic nephron damage and renal fibrosis, leading to kidney failure and a premature death rate of 67% by 9 weeks of age. In contrast, Tsc1 single-mutant mice were all alive and had far fewer renal cysts at this age. Mechanistic studies revealed persistent activation of mammalian target of rapamycin complex 1 (mTORC1) signaling causing hyperphosphorylation and consequent accumulation of 4E-BP1, along with greater cell proliferation, in the renal tubules of Tsc1 and rpS6 double-mutant mice. Furthermore, pharmacologic treatment of Tsc1 single-mutant mice with rapamycin reduced hyperphosphorylation and accumulation of 4E-BP1 but also inhibited phosphorylation of rpS6. Rapamycin also exacerbated cystic and fibrotic lesions and impaired kidney function in these mice, consequently leading to a premature death rate of 40% within 2 weeks of treatment, despite destroying tumors and decreasing kidney size. These findings indicate that Tsc1 prevents aberrant renal growth and tumorigenesis by inhibiting mTORC1 signaling, whereas phosphorylated rpS6 suppresses cystogenesis and fibrosis in Tsc1-deleted kidneys.

Phosphatidylinositol 3-kinase signaling determines kidney size.

Kidney size adaptively increases as mammals grow and in response to the loss of 1 kidney. It is not clear how kidneys size themselves or if the processes that adapt kidney mass to lean body mass also mediate renal hypertrophy following unilateral nephrectomy (UNX). Here, we demonstrated that mice harboring a proximal tubule-specific deletion of Pten (Pten(ptKO)) have greatly enlarged kidneys as the result of persistent activation of the class I PI3K/mTORC2/AKT pathway and an increase of the antiproliferative signals p21(Cip1/WAF) and p27(Kip1). Administration of rapamycin to Pten(ptKO) mice diminished hypertrophy. Proximal tubule-specific deletion of Egfr in Pten(ptKO) mice also attenuated class I PI3K/mTORC2/AKT signaling and reduced the size of enlarged kidneys. In Pten(ptKO) mice, UNX further increased mTORC1 activation and hypertrophy in the remaining kidney; however, mTORC2-dependent AKT phosphorylation did not increase further in the remaining kidney of Pten(ptKO) mice, nor was it induced in the remaining kidney of WT mice. After UNX, renal blood flow and amino acid delivery to the remaining kidney rose abruptly, followed by increased amino acid content and activation of a class III PI3K/mTORC1/S6K1 pathway. Thus, our findings demonstrate context-dependent roles for EGFR-modulated class I PI3K/mTORC2/AKT signaling in the normal adaptation of kidney size and PTEN-independent, nutrient-dependent class III PI3K/mTORC1/S6K1 signaling in the compensatory enlargement of the remaining kidney following UNX.