Alpha-kinase1 promotes tubular injury and interstitial inflammation in diabetic nephropathy by canonical pyroptosis pathway

BACKGROUND: Alpha-kinase 1 (ALPK1) is a master regulator in inflammation and has been proved to promote renal fibrosis by promoting the production of IL-1ß in diabetic nephropathy (DN) mice. Pyroptosis is involved in high glucose (HG)-induced tubular cells injury, characterized by activation of Gasdermin D (GSDMD) and the release of IL-1ß and IL-18, resulting in inflammatory injury in DN. It is reasonable to assume that ALPK1 is involved in pyroptosis-related tubular injury in DN. However, the mechanism remains poorly defined. METHODS: Immunohistochemistry (IHC) staining was performed to detect the expression of pyroptosis- and fibrosis-related proteins in renal sections of DN patients and DN mice. DN models were induced through injection of streptozotocin combined with a high-fat diet. Protein levels of ALPK1, NF-κB, Caspase-1, GSDMD, IL-1ß, IL-18 and α-SMA were detected by Western blot. HK-2 cells treated with high-glucose (HG) served as an in vitro model. ALPK1 small interfering RNA (siRNA) was transfected into HK-2 cells to down-regulate ALPK1. The pyroptosis rates were determined by flow cytometry. The concentrations of IL-1ß and IL-18 were evaluated by ELISA kits. Immunofluorescence staining was used to observe translocation of NF-κB and GSDMD. RESULTS: The heat map of differentially expressed genes showed that ALPK1, Caspase-1 and GSDMD were upregulated in the DN group. The expression levels of ALPK1, Caspase-1, GSDMD and CD68 were increased in renal biopsy tissues of DN patients by IHC. ALPK1expression and CD68+ macrophages were positively correlated with tubular injury in DN patients. Western blot analysis showed increased expressions of ALPK1, phospho-NF-κB P65, GSDMD-NT, and IL-1ß in renal tissues of DN mice and HK-2 cells, accompanied with increased renal fibrosis-related proteins (FN, α-SMA) and macrophages infiltration in interstitial areas. Inhibition of ALPK1 attenuated HG-induced upregulation expressions of NF-κB, pyroptosis-related proteins Caspase-1, GSDMD-NT, IL-1ß, IL-18, α-SMA, and pyroptosis level in HK-2 cells. Also, the intensity and nuclear translocation of NF-κB and membranous translocation of GSDMD were ameliorated in HG-treated HK-2 cells after treatment with ALPK1 siRNA. CONCLUSIONS: Our data suggest that ALPK1/NF-κB pathway initiated canonical caspase-1-GSDMD pyroptosis pathway, resulting in tubular injury and interstitial inflammation of DN.

Pathological findings in COVID-19: A conventional autopsy-based study from India

Background & objectives: Autopsy study has been considered the gold standard method for studying the effects of any disease on the body. Since COVID-19 is a novel disease, autopsy is crucial to understand its pathophysiology. This study was conducted to analyze the microscopic and macroscopic findings of various organs in COVID-19 and to associate those findings with clinical observations and laboratory findings. Methods: Conventional invasive autopsies were performed on 33 patients with COVID-19 from September 7, 2020 to December 23, 2020. All the organs were removed by routine dissection techniques and preserved in 10 per cent formalin. The tissues were processed and stained according to standard practices using haematoxylin-eosin (H & E) and periodic acid-schiff (PAS) stain. Results: The study included 28 males and 5 females with a median age of 61 yr (range 30-90 yr). Massive pulmonary oedema and thrombi in the lungs were the characteristic features macroscopically. On microscopic examination, diffuse alveolar damage in the exudative/proliferative phase was found in 29 (87.88%) cases. Among the other notable microscopic findings were bronchopneumonia and lung abscesses due to secondary bacterial infection (n=17, 51.52%), acute tubular injury (n=21, 63.64%) and thrombi in the lungs, heart, and kidneys. Interpretation & conclusions: COVID-19 primarily affected the respiratory and the renal systems in the vast majority of severely affected patients in our study. We also found signs of hypercoagulability, as evidenced by widespread thrombi in multiple organs, along with a raised d-dimer level and a hyperinflammatory state manifested by elevated inflammatory markers. Our autopsy findings and altered laboratory investigations support

Upregulation of Megalin, Cubilin, NGAL mRNA expression in kidney may represent tubular injury and apoptosis in chronic condition of rat diabetic model

@#Introduction: Diabetes mellitus (DM) leads to microvascular injury development and produces diabetes nephropathy (DN) with proteinuria, tubular injury, apoptosis and autophagy with upregulation of Bax, BASP and mTORC-1. Megalin, Cubilin and Neutrophil Gelatinase Associated Lipocalin (NGAL) play role in acute pathological condition of kidney injury, however its expression in chronic and slowly progressive kidney injury such as DN has not been elucidated yet. This study focuses upregulation of Megalin, Cubilin and NGAL in association with tubular injury and apoptosis in DN condition. Materials and methods: Diabetic condition was performed with intraperitoneal injection of Streptozotocin 60 mg/kg body weight (BW) in Sprague Dawley rats (2 months old, n=24), and were kept for 1, 2, and 4 months (DM1, DM2, and DM4, respectively). Control group was injected with NaCl 0.9%. Serum glucose level and proteinuria score were assessed, furthermore tubular injury score was quantified based on Periodic-Acid Schiff (PAS) staining. Reverse Transcriptase-PCR (RT-PCR) was carried out for NGAL, Megalin, Cubilin, m-TOR, Bax, and BASP-1 mRNA expression. Data were analyzed using SPSS 22 software. Results: DM led to kidney injury in this model with significant higher glucose level, proteinuria and tubular injury, especially in DM4 group which represented chronic phase of DN and CKD. These findings associated with upregulation of Megalin,Cubilin and NGAL mRNA expression in DM groups, especially in DM4 group. DM4 group also revealed higher expression of Bax, BASP and mTOR mRNA expression which demonstrated apoptosis. Conclusion: Megalin, Cubilin and NGAL upregulation may represent tubular injury and apoptosis as progression of DN.

Imbalance between Endothelin-1 and eNOS Expression Associates with Tubular Injury in Mice with 5/6 Subtotal Nephrectomy

@#Introduction: Chronic Kidney Diseases (CKD) leads to kidney fibrosis which characterized by tubular injury and atrophy with interstitial fibrosis. Endothelin-1 (ET-1) and endothelial Nitrite Oxide Synthase (eNOS) are known to play role in CKD and kidney fibrosis, although their correlation with tubulo-interstitial injury have not been understood clearly. Methods: 5/6 Subtotal Nephrectomy (SN) was performed in male Swiss Background mice to induce CKD. Sham operation (SO, n=5) procedure was performed on mice as control. The mice were sacrificed in day 7 (1N, n=5) and day 28 (4N, n=5) after operation. We measured creatinine serum to assess renal function. Tubular injury score was quantified based on Periodic-Acid Schiff (PAS) staining. Prepro-ET-1 and eNOS were quantified using RT-PCR. Results: SN_1N and SN_4N groups had significant higher of serum creatinine and tubular injury from SO group. Densitometry analysis of RT-PCR revealed up-regulation of prepro-ET-1 mRNA expression in SN_1N and SN_4N (p<0.05 vs SO). Meanwhile, we found a significant increase of eNOS expression in SN_1N, and then it reduced significantly in SN_4N. We found significant parallel correlation between ET-1 and tubular injury expression (r: 0.768;p<0,05), meanwhile there were insignificant inverse correlation between eNOS and tubular injury (r: -0.354;p>0.05). Conclusion: eNOS might play role as a counterbalance in the up regulation of ET-1 in acute condition after SN. However, it failed in chronic condition. These lead to deterioration of renal function and tubular injury. An imbalance between ET-1 and eNOS expression in chronic CKD model might play role in profound renal damage.

Ethanolic Extract of Centella asiatica Ameliorates Kidney Ischemia/reperfusion Injury Through Inhibition of Inflammatory Process

@#Introduction: Kidney ischemic-reperfusion injury (IRI) is the main cause of acute kidney injury (AKI) which leads to the inflammation epithelial apoptosis and interstitial fibrosis as the chronic consequenses. Centella asiatica (CeA) has been known to have various pharmacological effects such as, anti-inflammatory, antioxidant, anti-fibrosis, and, anti-apoptosis. We aimed to elucidate the role of CeA in inhibiting kidney injury and infammatory mediators due to kidney IRI. Methods: Kidney IRI were performed with bilateral renal pedicles clamping in Swiss background mice (3 months-old, 30-40 grams) for 30 minutes (IR group, n=6), then terminated at day 7 after operation. At the next day, the mice that have been underwent bilateral kidney IRI were administered per-orally with ethanolic extract of CeA (210 mg/kg of BW, CeA1 group, n=6, and 420 mg/kg of BW, CeA2 group, n=6). The Sham Operation (SO group, n=6) was used as control. At the day 7 after the surgery, the mice were sacrificed and the kidneys were harvested. The kidney was used to assess tubular injury, interstitial fibrosis, and macrophage number, and another kidney was used to assess the mRNA expression of TLR4. Data were quantified using SPSS 22. Results: Kidney IRI produced significantly higher tubular injury, interstitial fibrosis and macrophage number (p<0.05) compared to SO with upregulating TLR4 mRNA expression (p<0.05). CeA treatment attenuated the tubular injury, interstitial fibrosis, macrophage number, and TLR4 mRNA expression which obviously shown in higher-dose of CeA (p<0.05). Conclusion: CeA ameliorates tubular injury, kidney fibrosis, and inflammatory mediators due to kidney IRI.

Ethanol Extract of Centella asiatica (Gotu Kola) Attenuates Tubular Injury Through Inhibition of Inflammatory Cytokines and Enhancement of Anti-Fibrotic Factor in Mice with 5/6 Subtotal Nephrectomy

@#Background: Chronic kidney disease (CKD) leads to inflammation, fibrosis and destruction of the renal architecture. Centella asiatica (CeA) is an herbaceous plant with antiinflammatory effects. We aimed to elucidate the effect of CeA on inflammation, fibrosis, vascular remodelling and antifibrotic substances in a 5/6 subtotal nephrectomy (SN) model in mice. Methods: Mice were divided into three groups: sham operation (SO, n = 6), 5/6 SN for seven days (SN7, n = 7) and SN7 with oral CeA treatment (SN7-CeA, n = 7). At day 7, mice were euthanised, kidneys were harvested and stained with periodic-acid Schiff (for tubular injury and glomerulosclerosis) and sirius red (for fibrosis and vascular remodeling) staining. mRNA expression of prepro-endothelin-1, nephrin, E-cadherin, bone morphogenic protein-7 (BMP-7), toll-like receptor 4 (TLR4), tumour necrosis factor-α (TNFα) and hepatocyte growth factor (HGF) were quantified using reverse transcriptase-PCR. Results: SN group demonstrated significant higher interstitial fibrosis, vascular remodeling, tubular injury and glomerulosclerosis (P < 0.01) compared to SO group. Meanwhile, in SN7-CeA demonstrated attenuation of vascular remodeling as shown by significant higher lumen area with lower Wall/Lumen area ratio compared to SN7. RT-PCR analysis showed up-regulation of nephrin, BMP-7 and E-cadherin mRNA expression (P < 0.05) and down-regulation of ppET-1 in SN7-CeA group compared to SN7 group (P < 0.05). Conclusion: CeA may ameliorate renal injury in the SN model in mice.

Modulation of Renal Parenchyma in Response to Allogeneic Adipose-Derived Mesenchymal Stem Cells Transplantation in Acute Kidney Injury

BACKGROUND AND OBJECTIVES: In regenerative medicine, mesenchymal stem cells derived from adipose tissues (Ad-MSCs) are a very attractive target to treat many diseases. In relation to nephrology, the aim of the current study is to investigate the effects of Ad-MSCs for the amelioration of acute kidney injury and to explore the mechanism of renal parenchymal changes in response to allogeneic transplantation of Ad-MSCs. METHODS AND RESULTS: The nephrotoxicity was induced by cisplatin (CP) in balb/c mice according to RIFLE Class and AKIN Stage 3. PCR, qRT-PCR and fluorescent labeled cells infusion, histopathology, immunohistochemistry, functional analyses were used for genes and proteins expressions data acquisition respectively. We demonstrated that single intravenous infusion of 2.5×107/kg mAd-MSCs in mice pre-injected with CP recruited to the kidney, restored the renal structure, and function, which resulted in progressive survival of mice. The renal tissue morphology was recovered in terms of diminished necrosis or epithelial cells damage, protein casts formation, infiltration of inflammatory cells, tubular dilatation, and restoration of brush border protein; Megalin and decreased Kim-1 expressions in mAd-MSCs transplanted mice. Significant reduction in serum creatinine with slashed urea and urinary protein levels were observed. Anti-BrdU staining displayed enhanced tubular cells proliferation. Predominantly, downgrade expressions of TNF-α and TGF-β1 were observed post seven days in mAd-MSCs transplanted mice. CONCLUSIONS: Ad-MSCs exerts pro-proliferative, anti-inflammatory, and anti-fibrotic effects. Ad-MSCs transplantation without any chemical or genetic manipulation can provide the evidence of therapeutic strategy for the origin of regeneration and overall an improved survival of the system in functionally deprived failed kidneys.

Type I and Ⅱ Cardiorenal Syndrome-related Biomarkers / 中国实验方剂学杂志

The physiology and pathology of the heart and kidney are interdependent and interact with each other, and dysfunction of any one of them causes dysfunction of the other, namely cardiorenal syndrome, in which type I and type Ⅱ have the highest incidence rate and are the commonest in clinic. Traditional Chinese medicine has a long history of treating the cardiorenal syndrome. It believes that the disease is located in the heart and kidney, and Wenyang Yiqi, Huoxue Lishui and other methods shall be adopted to effectively improve the heart and kidney function of patients. However,the pathogenesis of cardiorenal syndrome is complicated, and the clinical manifestations are diverse, which makes it difficult to diagnose and treat in the early stage, and causes missing of the best intervention timing and a poor prognosis. Biomarkers play a vital role in predicting the occurrence and development of cardiorenal syndrome. Therefore, efforts shall be made to look for biomarkers with better specificity and sensitivity, accurately evaluate physiological and pathological changes in heart and kidney, so as to achieve early diagnosis and early intervention of cardiorenal syndrome, and improve the effect of disease diagnosis and treatment. At present, domestic and foreign scholars have studied and applied more markers mainly in renal tubular injury, including neutrophil gelatinase-associated lipocalin, kidney injury molecule-1 and urinary interleukin-18. In addition, other studies have found cell cycle arrest inducing factors, such as insulin-like growth factor binding protein 7, tissue inhibitor metallo proteinase-2, and fibroblast growth factor 23 associated with mineral metabolism. The increase of the content of these biomarkers in the body is earlier than the rise of serum creatinine, which can better predict the occurrence of early cardiorenal syndrome, and has a high application value and research value. After summarization of the biomarkers relating to type I and Ⅱ cardiorenal syndrome in domestic and foreign literatures, the research progress of several representative markers were reviewed to provide reference for related research.

Study on Expressions of HIF-1α and Renal Tubular Injury on Contrast-induced Nephropathy of Rats / 现代生物医学进展

Objective:To investigate the expression of HIF-1α in serum of rats with contrast induced nephropathy and its effect on renal tubular injury.Methods:45 SD rats were randomLy divided into three groups (n=15).The rats in the blank control group (group A)were treated with 12 h (Sodium Chloride Injection) for three 0.5 mL after fasting water for a period of about 15 minutes.Contrast nephropathy group (B group) rats after fasting 12 h,in the tail vein with 10 mg/kg injection ofindomethacin,15 minutes after the injection of 10 mg/kg nitro-L-arginine methyl ester (L-NAME),15 minutes after the injection ofiobitridol (3 G I/kg).Atorvastatin group (C group) rats in the first 3 days of the experiment started feeding atorvastatin calcium tablets,continuous feeding for 3 days,at a dose of 80 mg/kg/d,and fasting 12 h,making contrast nephropathy model,with the steps of contrast nephropathy group.The changes of renal function indexes (BUN,Cr),HIF-1α expression and renal tubular injury in three groups were observed and compared.Results:The level of BUN in rats with contrast induced nephropathy was lower than that in atorvastatin calcium group and blank control group,but the level of Scr was higher than that of atorvastatin calcium group and blank control group,the difference was statistically significant (P＜0.05).The level of BUN in atorvastatin calcium group was lower than that in blank control group,but Scr level was higher than that in blank control group,the difference was statistically significant (P＜0.05).Compared with the blank control group,the renal tubular injury in the rats with contrast induced nephropathy group was higher than that in atorvastatin calcium group and blank control group,the difference was statistically significant (P＜0.05).Compared with the control group,the expression of HIF-1 was significantly higher in rats with contrast induced nephropathy than that in atorvastatin calcium group and blank control group.The expression of HIF-1 was significantly higher than that in the control group (P＜0.05).Conclusions:It is suggested that the statins could prevent the contrast-induced nephropathy.However,the ending mechanism of statins should be further studied in the clinical practices.

Marcadores biológicos para necrose tubular aguda em pacientes com doença glomerular / Biological markers for acute tubular necrosis in patients with glomerular disease

INTRODUCTION: Acute kidney injury (AKI) is a common complication in patients with nephrotic syndrome (NS), and it is reported in 34% of adults with idiopathic nephrotic syndrome. Emergence of AKI in the course of nephrotic syndrome requires a prompt differential diagnosis between acute tubular necrosis (ATN) and proliferative glomerular lesions leading to rapidly progressive glomerulonephritis. Although clinical and conventional laboratory clues can be decisive in many cases, sometimes such distinctions rely on renal biopsy, which is an invasive procedure and is not available in many centers. Several new biomarkers have emerged, increasing the perspective on early diagnosis and the prognostic prediction of AKI. OBJECTIVES: In this work, we studied the use of tests based on the urinary concentrations of kidney injury molecule-1 (KIM-1)...

INTRODUÇÃO: A lesão renal aguda (LRA) é uma complicação frequente em pacientes com glomerulopatias, acomentendo até 34% dos adultos com síndrome nefrótica (SNO) idiopática. O diagnóstico diferencial de necrose tubular aguda (NTA) de glomeulonefrite proliferativa ou crescêntica em pacientes com SNO e LRA é fundamental, visto que a NTA pode mimetizar quadro de glomerulonefrite rapidamente progressiva. Dados clínicos e laboratoriais podem ser úteis no diagnóstico diferencial da LRA na SNO, entretanto a distinção entre NTA e glomerulonefrite proliferativa ou crescêntica é feito pela biópsia renal, procedimento invasivo e que não está disponível amplamente. Novos biomarcadores para diagnóstico precoce e preditores diagnósticos na LRA têm sido identificados. OBJETIVOS: Neste trabalho nós avaliamos o uso de testes baseados nas concentrações urinárias de kidney injury molecule-1 (KIM-1)...

Progress of the treatment on renal tubular injury / 国际儿科学杂志

A variety of kidney diseases have different degree of renal tubular injury,it even occur before the kidney damage.Since renal tubular injury still lack of effective therapy,it usually recovered by renal tubular epithelial cell itself.But the self-recover process is too long.If it is unable to spend this period,renal damage may not be reversible.So effective therapy is an emergency problem to be solved in clinical.

The Efficacy of N-acety1-beta-D-glucosaminidase as a Prognostic Factor of Glomerulonephritis / 대한신장학회잡지

BACKGROUNDS: N-acetyl-beta-D-glucosaminidase (NAG) is one of many enzymes that exist in the renal proximal tubular cells. It is said that functional impairment of renal tubule can be detected by checking NAG in the urine. But, it has never been known whether urinary NAG value can be used as a predictor for the prognosis of patients with glomerulonephritis. In this study, we evaluated the relationship between urinary NAG level and the degree of injury in cortical interstitium which has been known to influence the prognosis of renal function in glomerulonephritis closely. METHODS: Before renal biopsy was performed in each patient, urinary NAG(isoenzyme A and B), urinary beta2-microglobulin, serum blood urea nitrogen (BUN), serum creatinine, serum albumin, creatinine clearance and 24 hour urinary protein excretion were measured. Then, we calculated volume density of cortical interstitium [Vv(i/c)] in each specimen using point count morphometry method after getting a confirmative diagnosis from pathologist. Simple correlation analysis and multivariate regression analysis were carried out. RESULTS: The number of total patients was 32(male:16), whose median age was 60(32-80). Vv (i/c) had significant correlation with serum creatinine, creatinine clearance and serum BUN. But it was not correlated well with urinary NAG and urinary beta2-microglobulin. Urinary NAG concentration(2.131 2.549unit/mmol Cr) was higher than that of normal control and showed significant correlation with urinary beta2-microglobulin, serum albumin and 24 hour urinary protein excretion in patients. CONCLUSION: Urinary NAG had no significant correlation with Vv(i/c) that has been known as an important prognostic factor for the renal function in glomerulonephritis, but had significant correlation with urinary protein excretion. We concluded that urinary NAG was not regarded to be an appropriate marker for predicting the prognosis of renal function in patient with glomerulonephritis.