Enhancing nasopharyngeal carcinoma cell radiosensitivity by suppressing AKT/mTOR via CENP-N knockdown.

OBJECTIVE: Investigating the impact of centromere protein N (CENP-N) on radiosensitivity of nasopharyngeal carcinoma (NPC) cells. METHODS: Using immunohistochemistry and immunofluorescence to detect CENP-N expression in tissues from 35 patients with radiosensitive or radioresistant NPC. Assessing the effect of combined CENP-N knockdown and radiotherapy on various cellular processes by CCK-8, colony formation, flow cytometry, and Western blotting. Establishing a NPC xenograft model. When the tumor volume reached 100 mm3, a irradiation dose of 6 Gy was given, and the effects of the combined treatment were evaluated in vivo using immunofluorescence and Western blotting techniques. RESULTS: The level of CENP-N was significantly reduced in radiosensitive tissues of NPC (p < 0.05). Knockdown of CENP-N enhanced NPC radiosensitivity, resulting in sensitizing enhancement ratios (SER) of 1.44 (5-8 F) and 1.16 (CNE-2Z). The combined treatment showed significantly higher levels of proliferation suppression, apoptosis, and G2/M phase arrest (p < 0.01) compared to either CENP-N knockdown alone or radiotherapy alone. The combined treatment group showed the highest increase in Bax and γH2AX protein levels, whereas the protein Cyclin D1 exhibited the greatest decrease (p < 0.01). However, the above changes were reversed after treatment with AKT activator SC79. In vivo, the mean volume and weight of tumors in the radiotherapy group were 182 ± 54 mm3 and 0.16 ± 0.03 g. The mean tumor volume and weight in the combined treatment group were 84 ± 42 mm3 and 0.04 ± 0.01 g. CONCLUSION: Knockdown of CENP-N can enhance NPC radiosensitivity by inhibiting AKT/mTOR.

Allergen-induced CD11c + dendritic cell pyroptosis aggravates allergic rhinitis.

BACKGROUND: Pyroptosis is crucial for controlling various immune cells. However, the role of allergen-induced CD11c + dendritic cell (DC) pyroptosis in allergic rhinitis (AR) remains unclear. METHODS: Mice were grouped into the control group, AR group and necrosulfonamide-treated AR group (AR + NSA group). The allergic symptom scores, OVA-sIgE titres, serum IL-1ß/IL-18 levels, histopathological characteristics and T-helper cell-related cytokines were evaluated. CD11c/GSDMD-N-positive cells were examined by immunofluorescence analysis. Murine CD11c + bone marrow-derived DCs (BMDCs) were induced in vitro, stimulated with OVA/HDM, treated with necrosulfonamide (NSA), and further cocultured with lymphocytes to assess BMDC function. An adoptive transfer murine model was used to study the role of BMDC pyroptosis in allergic rhinitis. RESULTS: Inhibiting GSDMD-N-mediated pyroptosis markedly protected against Th1/Th2/Th17 imbalance and alleviated inflammatory responses in the AR model. GSDMD-N was mainly coexpressed with CD11c (a DC marker) in AR mice. In vitro, OVA/HDM stimulation increased pyroptotic morphological abnormalities and increased the expression of pyroptosis-related proteins in a dose-dependent manner; moreover, inhibiting pyroptosis significantly decreased pyroptotic morphology and NLRP3, C-Caspase1 and GSDMD-N expression. In addition, OVA-induced BMDC pyroptosis affected CD4 + T-cell differentiation and related cytokine levels, leading to Th1/Th2/Th17 cell imbalance. However, the Th1/Th2/Th17 cell immune imbalance was significantly reversed by NSA. Adoptive transfer of OVA-loaded BMDCs promoted allergic inflammation, while the administration of NSA to OVA-loaded BMDCs significantly reduced AR inflammation. CONCLUSION: Allergen-induced dendritic cell pyroptosis promotes the development of allergic rhinitis through GSDMD-N-mediated pyroptosis, which provides a clue to allergic disease interventions. Video Abstract.

Clinicopathological features of neuronal intranuclear inclusion disease diagnosed by skin biopsy.

STUDY OBJECTIVES: Neuronal intranuclear inclusion disease (NIID) is a rare progressive neurodegenerative disorder, with complex and diverse of clinical manifestations characterized by eosinophilic hyaline inclusions in neurons and somatic cells. Due to the improvement in diagnostic methods, NIID is being increasingly diagnosed. METHODS: Herein, we reported three NIID cases, which were diagnosed by skin biopsy and FMR1 gene, after DWI showed the characteristic corticomedullary junction hyperintensity. Then we reviewed all the published cases of NIID in PubMed, which were diagnosed by the same method. RESULTS: We discussed 15 NIID cases, including three cases diagnosed by us. The average age was 63.4 ± 14.0 years. The average time from onset of symptom to diagnosis was 5.4 ± 7.9 years. Nine cases had dementia or cognitive impairment. Three cases presented with encephalitis. Three cases showed bladder dysfunction and two cases only presented with dizziness and headache. Two cases showed acute neurological deficit mimicking stroke. All cases were diagnosed by skin biopsy, after DWI showed abnormal corticomedullary junction hyperintensity. Ten cases showed inclusions in sweat gland cells, and seven cases in adipocytes, sweat gland cells, and fibroblasts. EMG was performed in five cases, four of whom had abnormal results, showing simultaneous involvement of motor and sensory nerves. CONCLUSIONS: The results indicated that inclusions were more easily detected in sweat gland cells in skin biopsy. The early stage of NIID could only characterized by autonomic nerve function involvement. Combined autonomic nerve dysfunction might be another relatively common manifestation in NIID.

Urinary small extracellular vesicles derived CCL21 mRNA as biomarker linked with pathogenesis for diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) is a leading cause of renal failure, whereas the effective and early diagnostic biomarkers are still lacking. METHODS: Fourteen cytokines and chemokines mRNA were detected in urinary extracellular vesicles (EVs) from the screening cohort including 4 healthy controls (HC), 4 diabetes mellitus (DM) and 4 biopsy-proven DN patients, and was validated in another 16 HC and 15 DM and 28 DN patients. Correlation analysis was performed between the candidate biomarkers and clinic parameters as well as kidney histological changes. The findings were also confirmed in DN rat model with single injection of STZ. RESULTS: The number of small EVs secreted in urine was increased in DN patients compared to DM patients and healthy controls, with expression of AQP1 (a marker of proximal tubules) and AQP2 (a marker of distal/collecting tubules). Small EVs derived CCL21 mRNA increased significantly in DN patients and correlated with level of proteinuria and eGFR. Interestingly, elevated CCL21 mRNA from urine small EVs was observed in DN patients with normal renal function and could discriminate early DN patients from DM more efficiently compared to eGFR and proteinuria. CCL21 also showed an accurate diagnostic ability in distinguishing incipient from overt DN. Histologically, CCL21 mRNA expression increased progressively with the deterioration of tubulointerstitial inflammation and showed the highest level in nodular sclerosis group (class III) in DN patients. Remarkable infiltration of CD3 positive T cells including both CD4 and CD8 positive T cell population were observed in DN patients with high-CCL21 expression. Besides, accumulation of CD3 positive T cells correlated with level of urinary small EVs derived CCL21 and co-localized with CCL21 in the tubulointerstitium in DN patients. Finally, the correlation of CCL21 expression in renal cortex and urinary small EVs was confirmed in STZ-induced DN rat model. CONCLUSIONS: Urinary small EVs derived CCL21 mRNA may serve as early biomarker for identifying DN linked with pathogenesis. CCL21 mRNA mediated T cell infiltration may constitute the key mechanism of chronic inflammation in DN.

Calcium-sensing receptor activation attenuates collagen expression in renal proximal tubular epithelial cells.

316: F1006-F1015, 2019. First published March 6, 2019; doi: 10.1152/ajprenal.00413.2018 .-Experimental studies have shown that pharmacological activation of calcium-sensing receptor (CaSR) attenuates renal fibrosis in some animal models beyond modification of bone and mineral homeostasis; however, its underlying mechanisms remain largely unknown. Since excessive collagen deposition is the key feature of fibrosis, the present study aimed to examine whether CaSR was involved in the regulation of collagen expression in rats with adenine diet-induced renal fibrosis and in profibrotic transforming growth factor (TGF)-ß1-treated renal proximal tubular epithelial cells (PTECs). The results showed that the CaSR agonist cinacalcet significantly attenuated renal collagen accumulation and tubular injury in adenine diet-fed rats. Additionally, the in vitro experiment showed that profibrotic TGF-ß1 significantly increased the expression of collagen and decreased CaSR expression at the mRNA and protein levels in a concentration- and time-dependent manner. Furthermore, the CaSR CRISPR activation plasmid and cinacalcet partially abrogated the upregulation of collagen induced by TGF-ß1 treatment. Blockade of CaSR by the CRISPR/Cas9 KO plasmid or the pharmacological antagonist Calhex231 further enhanced TGF-ß1-induced collagen expression. Mechanistic experiments found that Smad2 phosphorylation and Snail expression were markedly increased in PTECs treated with TGF-ß1, whereas the CaSR CRISPR activation plasmid and cinacalcet substantially suppressed this induction. In summary, this study provides evidence for a direct renal tubular epithelial protective effect of CaSR activation in renal fibrosis, possibly through suppression of collagen expression in PTECs.

Urinary Exosomes and Exosomal CCL2 mRNA as Biomarkers of Active Histologic Injury in IgA Nephropathy.

IgA nephropathy (IgAN) features variable renal pathology and a heterogeneous clinical course. Our aim was to search noninvasive biomarkers from urinary exosomes for IgAN patients; membrane nephropathy and minimal change disease were included as other glomerulopathy controls. Transmission electron microscopy and nanoparticle tracking analysis confirmed the size and morphology characteristic of urinary exosomes. Exosome markers (Alix and CD63) as well as renal cell markers [aquaporin 2 (AQP2) and nephrin] were detected, which indicate the renal origin of urinary exosomes. Exosome excretion was increased markedly in IgAN patients compared with controls and correlated with levels of proteinuria and tubular injury. More important, urinary exosome excretion correlated with greater histologic activity (mesangial hypercellularity, crescents, and endocapillary hypercellularity). Profiling of the inflammation-related mRNA revealed that exosomal chemokine (C-C motif) ligand 2 (CCL2) was up-regulated in IgAN patients. In a validation study, CCL2 was exclusively highly expressed in IgAN patients compared with healthy controls as well as minimal change disease and membrane nephropathy patients. Also, a correlation between exosomal CCL2 and estimated glomerular filtration rate levels was found in IgAN. Exosomal CCL2 was correlated with tubulointerstitial inflammation and C3 deposition. High CCL2 levels at the time of renal biopsy were associated with subsequent deterioration in renal function. Thus, urinary exosomes and exosomal CCL2 mRNA are promising biomarkers reflecting active renal histologic injury and renal function deterioration in IgAN.

Morphology and Evaluation of Renal Fibrosis.

With continuing damage, both the indigenous cells of the cortex and medulla, and inflammatory cells are involved in the formation and development of renal fibrosis. Furthermore, interactions among the glomerular, tubular, and interstitial cells contribute to the process by excessive synthesis and decreased degradation of extracellular matrix. The morphology of kidney is different from pathological stages of diseases and changes with various causes. At the end stage of the disease, the kidneys are symmetrically contracted with diffuse granules. Most glomeruli show diffuse fibrosis and hyaline degeneration, and intervening tubules become atrophied. Renal interstitium shows obvious hyperplasia of fibrous tissues with marked infiltration of lymphocytes, mononuclear cells, and plasma cells. The renal arterioles are wall thickening frequently because of hyaline degeneration. Morphologic analysis based on Masson staining of the kidney tissues has been regarded as the golden standard to evaluate the visual fibrosis. However, the present studies have found that the evaluation system has poor repeatability. Several computer-aided image analysis techniques have been used to assess interstitial fibrosis. It is possible that the evaluation of renal fibrosis is carried out by the artificial intelligence renal biopsy pathological diagnosis system in the near future.

Exosomal CCL2 from Tubular Epithelial Cells Is Critical for Albumin-Induced Tubulointerstitial Inflammation.

Albuminuria is a key instigator of tubulointerstitial inflammation associated with CKD, but the mechanism through which filtered albumin propagates renal injury remains unclear. In this study, we explored the role in this process of exosome mRNA released from tubular epithelial cells (TECs). Compared with control mice, acute and chronic kidney injury models had more exosomes containing inflammatory cytokine mRNA, particularly the chemokine CCL2, in kidneys and urine. In vitro stimulation of TECs with BSA recapitulated this finding. Notably, the internalization of purified TEC exosomes by cultured macrophages increased if TECs were exposed to BSA. Macrophage internalization of exosomes from BSA-treated TECs led to an enhanced inflammatory response and macrophage migration, but CCL2 silencing in TECs prevented these effects. Using a GFP-CCL2 fusion mRNA construct, we observed direct transfer of CCL2 mRNA from TEC exosomes to macrophages. Mice subjected to tail vein injection of purified BSA-treated TEC exosomes developed tubular injury with renal inflammatory cell infiltration. However, injection of exosomes from BSA-treated CCL2-deficient TECs induced less severe kidney inflammation. Finally, in patients with IgA nephropathy, the increase of proteinuria correlated with augmented urinary excretion of exosomes with exaggerated expression of CCL2 mRNA. Moreover, the level of CCL2 mRNA in urinary exosomes correlated closely with levels of renal interstitial macrophage infiltration in these patients. Our studies demonstrate that the increasing release of exosomes that transfer CCL2 mRNA from TECs to macrophages constitutes a critical mechanism of albumin-induced tubulointerstitial inflammation.

A rat model of SHPT with bone abnormalities in CKD induced by adenine and a high phosphorus diet.

The study of parathyroid hyperplasia with bone disease as a critical manifestation of chronic kidney disease-mineral and bone disorders (CKD-MBDs) is challenging due to the lack of a suitable research model. Here, we established a rat model with secondary hyperparathyroidism (SHPT) and bone disease induced by adenine and a high phosphorous diet and analyzed the skeletal characteristics. We performed blood analysis, emission computed tomography (ECT), dual energy X-ray absorptiometry (DEXA), micro-computed tomography (micro-CT), bone histomorphometry, and bone mechanical tests. The CKD rats with SHPT induced by adenine and a high phosphorus diet showed severe abnormalities in calcium and phosphorus metabolism and exhibited parathyroid hyperplasia. The bone mineral density (BMD) of femurs and lumbar vertebrae was significantly lower in the CKD rats than in the control (CTL) rats. The cortical and trabecular bone parameters of femurs showed significant bone loss. In addition, we found decreases in ultimate force, work to failure, stiffness, and elastic modulus in the CKD rats. In conclusion, our findings demonstrated that the CKD rats with SHPT induced by adenine and a high phosphorus diet may serve as a useful model for skeletal analysis in CKD with SHPT.

Urinary vimentin mRNA as a potential novel biomarker of renal fibrosis.

Renal fibrosis is a histological outcome of chronic kidney disease (CKD) progression. However, the noninvasive detection of renal fibrosis remains a challenge. Here we constructed a renal fibrosis target mRNA array and used it to detect urinary mRNAs of CKD patients for investigating potential noninvasive biomarkers of renal fibrosis. We collected urine samples from 39 biopsy-proven CKD patients and 11 healthy controls in the training set. Urinary mRNA profiles of 86 genes showed a total of 21 mRNAs that were differentially expressed between CKD patients and controls (P < 0.05), and vimentin (VIM) mRNA demonstrated the highest change fold of 9.99 in CKD vs. controls with robust correlations with decline of renal function and severity of tubulointerstitial fibrosis. Additionally, VIM mRNA further differentiated patients with moderate-to-severe fibrosis from none-to-mild fibrosis group with an area of the curve of 0.796 (P = 0.008). A verification of VIM mRNA in the urine of an additional 96 patients and 20 controls showed that VIM is not only well correlated with renal function parameters but also correlated with proteinuria and renal fibrosis scores. Multiple logistic regression and receiver-operating characteristics analysis further showed that urine VIM mRNA is the best predictive parameter of renal fibrosis compared with estimated glomerular filtration rate, serum creatinine, and blood urea nitrogen. In addition, there is no improved predictive performance for the composite biomarkers to predict renal fibrosis severity compared with a single gene of VIM. Overall, urinary VIM mRNA might serve as a novel independent noninvasive biomarker to monitor the progression of kidney fibrosis.

[Effect of Cordyceps sinensis powder on renal oxidative stress and mitochondria functions in 5/6 nephrectomized rats].

OBJECTIVE: To observe the effect of Cordyceps sinensis (CS) powder on renal oxidative stress and mitochondria functions in 5/6 nephrectomized rats, and to primarily explore its possible mechanisms. METHODS: Totally 30 male Sprague-Dawley rats were divided into the sham-operation group, the model group, and the treatment group by random digit table, 10 in each group. A chronic kidney disease (CKD) rat model was prepared by one step 5/6 nephrectomy. Rats in the treatment group were intragastrically administered with CS powder solution at the daily dose of 2 g/kg, once per day. Equal volume of double distilled water was intragastrically administered to rats in the sham-operation group and the model group. All medication lasted for 12 weeks. The general condition of rats, their body weight, blood pressure, 24 h proteinuria, urinary N-acetyl-ß-D-glucosaminidase (NAG), serum creatinine (SCr) , and blood urea nitrogen (BUN) were assessed before surgery, at week 2, 4, 6, 8, 10, and 10 after surgery. Pathological changes of renal tissues were observed under light microscope. Morphological changes of mitochondria in renal tubular epithelial cells were observed under transmission electron microscope. Activities of antioxidant enzymes including reduced glutathione (GSH), manganese superoxide dismutase (MnSOD), and malondialdehyde (MDA) in fresh renal tissue homogenate were detected. Mitochondria of renal tissues were extracted to detect levels of mitochondrial membrane potential and changes of reactive oxygen species (ROS). And expressions of cytochrome-C (Cyto-C) and prohibitin in both mitochondria and cytoplasm of the renal cortex were also measured by Western blot. RESULTS: (1) Compared with the sham-operation group, body weight was significantly decreased at week 2 (P <0. 01), but blood pressure increased at week 4 (P <0. 05) in the model group. Compared with the model group, body weight was significantly increased at week 12 (P <0. 01), but blood pressure decreased at week 8 (P < 0. 01) in the treatment group. (2) Compared with the sham-operation group, 24 h proteinuria, urinary NAG, blood SCr and BUN significantly increased in the model group (all P <0. 01). Compared with the model group, blood and urinary biochemical indices all significantly decreased in the treatment group (all P <0. 01). (3) Results of pathological renal scoring: Glomerular sclerosis index, scoring for tubulointerstitial fibrosis, degree of tubulointerstitial inflammatory infiltration were all obviously higher in the model group than in the sham-operation group (all P <0. 01). All the aforesaid indices were more obviously improved in the treatment group than in the model group (all P <0. 01). (4) Compared with the sham-operation group, activities of MnSOD and GSH-Px were significantly reduced, but MDA contents obviously increased in the renal cortex of the model group (all P <0. 01). Compared with the model group, activities of MnSOD and GSH-Px obviously increased (P <0. 05, P <0. 01), but MDA contents obviously decreased in the renal cortex of the treatment group (P <0. 01). (5) Compared with the sham-operation group, the mitochondrial membrane potential significantly decreased, but ROS levels significantly increased in the model group (all P <0.01). Compared with the model group, mitochondrial transmembrane potential increased in the treatment group, thereby inhibiting the tendency of increased production of ROS (both P < 0. 01). (6) Results of Western blot showed that, compared with the sham-operation group, expression levels of mitochondrial Cyto-C and Prohibitin were significantly reduced in the renal cortex (P <0. 01), but significantly elevated in the cytoplasm of the model group (P <0. 01). Compared with the model group, each index was obviously improved in the treatment group with statistical difference (P <0. 05, P <0. 01). CONCLUSION: CS powder had renal protection, and its mechanism might partially depend on in- hibition of oxidative stress and protection for mitochondria.

Renal cell carcinoma in an adult-onset ESRD patient with nephronophthisis harboring NPHP3 deletion: A case report.

Background: Nephronophthisis (NPHP) is a rare autosomal recessive inherited tubulointerstitial nephropathy, the most prevalent genetic cause of end-stage renal disease (ESRD) in children. Convincing evidence indicated that the overall prevalence of NPHP in adult-onset ESRD is very likely to be an underestimation. Therefore, understanding the genetic background and clinicopathologic features of adult-onset NPHP is warranted. Case presentation: we reported one intriguing case with concurrent NPHP3 c.2694-2_2694-1delAG (splicing) variant and c.1082C > G (p.S361C) variant. A 48-year-old male was admitted to our hospital, complained about renal dysfunction for 10 years, and found right renal space-occupying lesion for 1 week. One of the most interesting clinical features is adult-onset ESRD, which differs from previous cases. Another discovery of this study is that the NPHP harboring NPHP3 deletion may be associated with clear cell renal cell carcinoma. Conclusion: In conclusion, we report two mutations in the NPHP3 gene that cause NPHP with adult-onset ESRD and renal clear cell carcinoma in a Chinese family, enriching the clinical features of NPHP.

MicroRNA-29c in urinary exosome/microvesicle as a biomarker of renal fibrosis.

Micro (mi)RNAs are frequently dysregulated in the development of renal fibrosis. Exosomes are small membrane vesicles that could be isolated from urine secreted from all nephron segments. Here we sought to observe for the first time whether miRNA in urine exosome could serve as a potential biomarker of renal fibrosis. Urine samples were collected from 32 chronic kidney disease (CKD) patients who underwent kidney biopsy and 7 controls. Exosome was isolated and confirmed by immunogold staining of exosome marker. Members of miR-29, miR-200, and RNU6B as endogenous control were detected by RT quantitative PCR. Electronic microscopy verified a typical shape of exosome with average size of 65.1 nm and labeled it with anti-CD9 and anti-aquaporin 2 antibody. Members of miR-29 and miR-200 are readily measured with reduced levels compared with controls (P < 0.05) and can robustly distinguish CKD from controls [area under the curve (AUC) varied from 0.902 to 1 by receiver operating characteristics analysis]. miR-29c correlated with both estimated glomerular filtration rate (r = 0.362; P < 0.05) and degree of tubulointerstitial fibrosis (r = -0.359; P < 0.05) for CKD patients. Moreover, miRNA in exosome was decreased in mild fibrosis group compared with moderated to severe group. miR-29a and miR-29c could predict degree of tubulointerstitial fibrosis with AUC of 0.883 and 0.738 (P < 0.05). The sensitivity and specificity for distinguishing mild from moderate to severe fibrosis were 93.8 and 81.3% with the use of miR-29a and 68.8 and 81.3% for miR-29c. Overall, miR-29c in urinary exosome correlates with both renal function and degree of histological fibrosis, suggesting it as a novel, noninvasive marker for renal fibrosis.

Pirfenidone inhibits macrophage infiltration in 5/6 nephrectomized rats.

Tubulointerstitial macrophage infiltration is a hallmark of chronic kidney disease involved in the progression of renal fibrosis. Pirfenidone is a newly identified antifibrotic drug, the potential mechanism of which remains unclear. The aim of this study was to investigate the effects of pirfenidone on M1/M2 macrophage infiltration in nephrectomized rats. Nephrectomized rats were treated with pirfenidone by gavage for 12 wk. Twenty-four hour urinary protein, N-acetyl-ß-D-glycosaminidase (NAG) activity, systolic blood pressure, and C-reactive protein were determined. Paraffin-embedded sections were stained for CD68, CCR7, and CD163 macrophages. Monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1α (MIP-1α), as well as M1 and M2 macrophages secretory markers, were evaluated by real-time RT-PCR and Western blotting analysis. Pirfenidone significantly improved the elevated proteinuria and NAG activity from week 2 onward after surgery. Pirfenidone attenuated interstitial fibrosis and decreased expression of fibrotic markers including transforming growth factor-ß(1), connective tissue growth factor, α-smooth muscle actin, fibronectin, and fibroblast-specific protein-1. Pirfenidone significantly decreased the infiltrating macrophages. The number of M1 and M2 macrophages was significantly lower after pirfenidone treatment. MCP-1 and MIP-1α were increased in nephrectomized rats at mRNA and protein levels. Pirfenidone treatment significantly inhibited their expression. The TNF-α, IL-6, and nitric oxide synthases-2 expressed by M1 macrophages were increased in nephrectomized rats, and pirfenidone significantly attenuated their expression. Pirfenidone treatment also significantly decreased arginase-1, dectin-1, CD206, and CD86 expressed by M2 macrophages. Thus pirfenidone inhibits M1 and M2 macrophage infiltration in 5/6 nephrectomized rats, which suggests its efficacy in the early and late periods of renal fibrosis.

Inflammatory stress exacerbates the progression of cardiac fibrosis in high-fat-fed apolipoprotein E knockout mice via endothelial-mesenchymal transition.

Background Chronic inflammation plays a crucial role in the progression of cardiac fibrosis. This study investigated whether inflammation exacerbated the progression of cardiac fibrosis in high-fat-fed apolipoprotein E knockout (ApoE KO) mice via endothelial-mesenchymal transition (EndMT). Methods Twenty-four male ApoE KO mice were divided into normal chow diet (Control), high-fat diet (HFD), or high-fat diet plus 10% casein injection (inflamed) groups for 8 weeks. The body weight of ApoE KO mice was measured at each week. The lipid profile and serum amyloid A (SAA) levels were examined using clinical biochemistry and enzyme-linked immunosorbent assays, respectively. Cardiac lipid and collagen accumulation was visualised with haematoxylin-eosin (HE) and Masson's trichrome staining. EndMT-related molecule expression was examined by immunohistochemistry and Western blotting. Results SAA levels were increased in the inflamed group compared with the HFD and control groups, suggesting that inflammation was successfully induced. There were no differences in body weight among three groups at each week. Interestingly, inflammation significantly reduced serum total cholesterol, triglyceride, and low-density lipoprotein (LDL) levels compared with the HFD mice. However, both foam cell formation in cardiac blood vessels and cardiac collagen deposition were increased in the inflamed group, as demonstrated by HE and Masson trichrome staining. Furthermore, inflammation reduced protein expression of CD31 and increased protein expression of alpha-smooth muscle actin (α-SMA) and collagen I, which contribute to cardiac EndMT. Conclusions Inflammatory stress exacerbates the progression of cardiac fibrosis in high-fat-fed ApoE KO mice via EndMT, suggesting that hyperlipidaemia and inflammation act synergistically to redistribute plasma lipids to cardiac tissues and accelerate the progression of cardiac fibrosis.

Activation of renin-angiotensin system is involved in dyslipidemia-mediated renal injuries in apolipoprotein E knockout mice and HK-2 cells.

BACKGROUND: Dyslipidemia and activation of renin-angiotensin system (RAS) contribute to the progression of chronic kidney disease (CKD). This study investigated possible synergistic effects of intrarenal RAS activation with hyperlipidemia in renal injuries. METHODS: Apolipoprotein knockout mice were fed with normal chow diet (control) or high fat diet (HF group) for eight weeks. Human proximal tubular epithelial cell line (HK-2) was treated without (control) or with cholesterol (30 µg/ml) plus 25-hydroxycholesterol (1 µg/ml) (lipid group) for 24 hours. The plasma lipid profile and RAS components were determined by clinical biochemistry assay and radiommunoassay, respectively. Collagen deposition in kidneys was evaluated by Masson-staining. The gene and protein expressions of molecules involved in RAS components and biomarkers of epithelial mesenchymal transition (EMT) were examined by real-time PCR, immunochemical staining, and Western blot. RESULTS: The mice fed with high-fat diet showed significant hyperlipidemia with collagen deposition in renal tubular interstitium compared to controls. The plasma levels of renin, angiotensin I, and angiotensin II were no difference in two groups. However, the kidneys of HF group showed up-regulated RAS components, which were positively associated with increased plasma levels of triglyceride, total cholesterol, and LDL. These effects were further confirmed by in vitro studies. Lipid loading induced HK-2 cells underwent EMT, which was closely associated with the increased expressions of intracellular RAS components. CONCLUSIONS: Local RAS activation was involved in hyperlipidemia-mediated renal injuries, suggesting that there are synergistic effects resulting from RAS activation with hyperlipidemia that accelerates the progression of CKD.

FTY720 attenuates tubulointerstitial inflammation and fibrosis in subtotally nephrectomized rats.

Tubulointerstitial fibrosis is a common pathway that leads to kidney failure, and persistent tubulointerstitial inflammation is a key event in the development of tubulointerstitial fibrosis. The new immunosuppressive drug FTY720 modifies lymphocyte migration into injured tissues by sequestering lymphocytes within secondary lymphoid organs. However, its therapeutic effect on tubulointerstitial inflammation and fibrosis had not been well understood. This study was designed to explore the effect of FTY720 on tubulointerstitial inflammation and fibrosis in subtotally nephrectomized (SNX) rats. In total, 24 male Sprague-Dawley rats were used. Seven days after 5/6 nephrectomy, rats were randomized to FTY720 (1 mg/kg/d) and placebo-treated groups. Sham-operated rats served as controls. FTY720 significantly attenuated the rise in proteinuria, serum creatinine, urea nitrogen and N-acetyl-ß-D-glucosaminidase activity in SNX rats, and reduced the count of peripheral white blood cells and lymphocytes in SNX rats. Morphological analysis revealed that there was severe tubulointerstitial inflammation and fibrosis in SNX group and much more tubulointerstitial infiltrating inflammatory cells with high expression of CD3, CD4, CD8, CD20, CD68, CD163 and CCR-7 in SNX group, as compared with the controls, but the lesions were attenuated significantly by treatment with FTY720. Furthermore, the expressions of proinflammatory molecules (IL-6, TNF-α and MCP-1), profibrotic molecule (TGF-ß1) and production of extracellular matrix proteins such as fibronectin and types I and III collagens were upregulated in SNX rats. FTY720 administration significantly reduced these abnormalities. In summary, FTY720 exerts therapeutic effects on tubulointerstitial fibrosis in SNX rats by inhibiting the tubulointerstitial inflammatory response.

[Inflammation accelerates lipid dysregulation mediated cardiac fibrosis through enhancing myocardial endothelial-to-mesenchymal transition].

OBJECTIVE: Dyslipidemia and chronic inflammation are risk factors of cardiac fibrosis. This study was aimed to investigate their possible synergetic effects and underlying mechanisms on progression of cardiac fibrosis in apolipoprotein E knockout (ApoE -/-) mice. METHODS: Twenty-four ApoE-/- mice were divided into normal chow diet (control), high fat diet (HFD group), and HFD plus subcutaneously injection of 10% casein (inflammation group) for 8 weeks. Lipid profile and serum amyloid A (SAA) were examined by clinical biochemical assays and Enzyme-Linked Immunosorbent Assay, respectively. Hematoxylin-eosin staining (HE) and Masson staining were used to evaluate the myocardial accumulation of lipid and collagen. Collagen I protein expression was detected by immunohistochemical staining. Endothelial-to-mesenchymal transition related protein expressions were determined by Western blot. RESULTS: Serum SAA level was significantly higher in inflammation group [(127.42 ± 26.99) ng/ml] than in control [(15.40 ± 7.62) ng/ml] and HFD [(8.17 ± 0.72) ng/ml] group (all P < 0.01).However serum levels of triglyceride, total cholesterol, and low density lipoprotein (LDL) cholesterol were significantly higher in HFD group than in inflammation and control groups[TG (7.53 ± 2.05) mmol/L vs. (3.43 ± 0.79) mmol/L; TC (27.80 ± 3.99) mmol/L vs. (14.94 ± 1.92) mmol/L;LDL-C (11.56 ± 2.56) mmol/L vs. (9.46 ± 1.31) mmol/L, all P < 0.05) . Foam cell formation in cardiac vessels, myocardial collagen deposit, protein expressions of collagen I, CD31, and alpha-smooth muscle actin (α-SMA) were all significantly higher in inflammation group than in HFD group (all P < 0.05) suggesting that inflammation contributes to the phenotype endothelial-to-mesenchymal transition in heart. CONCLUSION: Inflammation exacerbates dyslipidemia mediated cardiac fibrosis in ApoE-/- mice partly through enhancing myocardial endothelial-to-mesenchymal transition.

Long-term consequences of regulatory T-cell-specific knockout of Notch2 in immune homeostasis.

AIMS: To investigate the long-term alterations in immune function and spontaneous inflammation in mice following specific knockout of Notch2 (Notch2KO) in Treg cells. MAIN METHODS: A Treg cell-specific Notch2 knockout mouse model was constructed, and the mice were named Notch2KO mice. The pathological changes and inflammatory cell infiltration in the lungs, skin, and liver of the mice at 2, 6, 9, and 12 months of age were evaluated by HE staining. The expression of Th1/Th2/Th17/Treg transcription factors was detected by Western blotting. The proportion of CD4 + T-cell subsets was determined by flow cytometry. The levels of Th1/Th2/Th17/Treg cytokines were measured by enzyme-linked immunosorbent assays (ELISAs). KEY FINDINGS: The expression level of Notch2 in Treg cells from the Notch2KO mice was significantly decreased compared with that in Treg cells from the control mice (P < 0.05). HE staining showed that compared with the control mice, the Notch2KO mice displayed spontaneous inflammation and had a large amount of inflammatory cell infiltration in the lungs and skin (P < 0.05). The number of Treg cells, the expression level of Foxp3, and the level of IL-10 were reduced in the Notch2KO mice compared with the control mice (P < 0.05), and these metrics further decreased with increasing age (P < 0.05). In contrast, the number of Th1/Th2 cells, the expression level of T-bet/GATA3, and the levels of Th1 cytokines (IFN-γ)/Th2 cytokines (IL-4, IL-5, and IL-13) were significantly increased in the Notch2KO mice (P < 0.05), and these metrics further increased with increasing age (P < 0.05). There was no significant change in the number of Th17 cells, the expression of RORγt, or the level of IL-17. Further analysis showed that the balance of Th1/Th2 and Treg/Th17 cells in the Notch2KO mice was shifted, and the ratio showed a downward trend over time (P < 0.05). SIGNIFICANCE: The number and function of Treg cells can be severely inhibited by a specific knockout of Notch2 in Treg cells, leading to immune disorders that gradually worsen over time.

Specific knockout of Notch2 in Treg cells significantly inhibits the growth and proliferation of head and neck squamous cell carcinoma in mice.

OBJECTIVE: To investigate the effect of Notch2 gene knockout in Treg cells on head and neck squamous cell carcinoma (HNSCC) in mice. METHODS: A mouse model of HNSCC was constructed. Flow cytometry and immunofluorescence were used to examine the numbers of related immune cells and programmed cell death in tumor cells in the spleen and tumor microenvironment of mice. Western blotting was used to measure the expression of related proteins in tumor tissues. RESULTS: The tumor volume of regulatory T (Treg) cell-specific Notch2-knockout mice (experimental group) was significantly smaller than that of control mice (control group) (P < 0.05). Compared with those in the control group, the number of Treg cells and the expression of Ki67 in Treg cells in the spleen and tumor tissue were significantly decreased in the experimental group, while the numbers of CD45+ hematopoietic cells, CD4+ T cells, CD8+ T cells, T helper 1 (Th1) cells, CD11b+ cells (macrophages), and CD11b+CD11c+ cells (dendritic cells) and the expression of Ki67 in CD4+ T cells and CD8+ T cells were significantly increased (P < 0.05). There was no significant difference in the number of Th2 cells between the two groups (P > 0.05). Immunofluorescence analysis showed that the numbers of CD4+ T cells and CD8+ T cells in the tumor tissue in the experimental group were significantly higher than those in the control group (P < 0.05). Compared with that in the control group, programmed cell death in the experimental group was significantly increased (P < 0.05). Moreover, the expression levels of NLRP3, Caspase-1 and GSDMD in the tumor tissues of the experimental group were higher than those in the control group (P < 0.01), while the expression levels of BCL2, Bax, ATG5, LC3 and p62 were not significantly different (P > 0.05). CONCLUSIONS: Specific knockout of the Notch2 gene in Treg cells significantly decreases the function of Treg cells, inhibits the growth of HNSCC and improves the immune microenvironment in mice, thus effectively treating HNSCC.