|
1
|
Herath S, Au AYM, Taylor KM, Kapoor-Kaushik N, Endre ZH, Erlich JH. Long-Term Functional and Structural Renoprotection After Experimental Acute Kidney Injury in Subclinical Chronic Kidney Disease In Vivo. Int J Mol Sci 2025; 26:4616. [PMID: 40429761 PMCID: PMC12111561 DOI: 10.3390/ijms26104616] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2025] [Revised: 05/02/2025] [Accepted: 05/07/2025] [Indexed: 05/29/2025] Open
Abstract
Subclinical chronic kidney disease (sCKD) predisposes one to acute kidney injury (AKI) and chronic kidney disease (CKD). Reduced kidney functional reserve (KFR) detects sCKD in preclinical studies and predicts AKI after cardiac surgery. We evaluated renal protection in a rat model of kidney injury where ischaemia-reperfusion injury (IRI) was induced after sCKD. Dual treatment boosting nicotinamide adenine dinucleotide (NAD) by nicotinamide riboside (NR) combined with the mitochondria-targeted antioxidant SkQR1 protected the KFR and reduced structural kidney damage, including markers of vascular integrity and the relative blood volume (rBV). The dual treatment upregulated Sirt1 and Nrf2, increased the nuclear localisation of the mitochondrial biogenesis regulator PGC-1α and the mitochondrial protein marker COX4, and upregulated the antioxidant gene NOQ1. These observations suggest mitochondrial protection and modulation of the cellular redox state provided long-term structural and functional protection against kidney injury superimposed on background sCKD.
Collapse
Affiliation(s)
- Sanjeeva Herath
- School of Clinical Medicine, Faculty of Medicine and Health, University of New South Wales, Sydney, NSW 2052, Australia; (S.H.); (A.Y.M.A.); (J.H.E.)
| | - Amy Y. M. Au
- School of Clinical Medicine, Faculty of Medicine and Health, University of New South Wales, Sydney, NSW 2052, Australia; (S.H.); (A.Y.M.A.); (J.H.E.)
- Department of Nephrology, Prince of Wales Hospital, Sydney, NSW 2031, Australia
| | - Kylie M. Taylor
- School of Clinical Medicine, Faculty of Medicine and Health, University of New South Wales, Sydney, NSW 2052, Australia; (S.H.); (A.Y.M.A.); (J.H.E.)
| | - Natasha Kapoor-Kaushik
- Electron Microscopy Unit, Mark Wainwright Analytical Centre, University of New South Wales, Sydney, NSW 2052, Australia
| | - Zoltán H. Endre
- School of Clinical Medicine, Faculty of Medicine and Health, University of New South Wales, Sydney, NSW 2052, Australia; (S.H.); (A.Y.M.A.); (J.H.E.)
- Department of Nephrology, Prince of Wales Hospital, Sydney, NSW 2031, Australia
| | - Jonathan H. Erlich
- School of Clinical Medicine, Faculty of Medicine and Health, University of New South Wales, Sydney, NSW 2052, Australia; (S.H.); (A.Y.M.A.); (J.H.E.)
- Department of Nephrology, Prince of Wales Hospital, Sydney, NSW 2031, Australia
| |
Collapse
|
|
2
|
Jiang W, Shi K, Shao J, Song L, Shi Y, Wang H, Zhou L, Li L, Feng Y, Yu J, Zheng R. Protective effect of intravenous amino acid on kidney function: A systematic review and meta-analysis of randomized controlled trials. J Crit Care 2025; 85:154937. [PMID: 39447274 DOI: 10.1016/j.jcrc.2024.154937] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2024] [Revised: 10/07/2024] [Accepted: 10/08/2024] [Indexed: 10/26/2024]
Abstract
BACKGROUND Acute kidney injury (AKI) is a common complication in critically ill and cardiac surgery patients. Intravenous amino acids can increase renal perfusion and replenish renal functional reserves. However, the exact therapeutic efficacy of intravenous amino acids in reducing the incidence of AKI remains uncertain. Therefore, this study aims to comprehensively review the existing evidence to assess the potential of intravenous amino acids in kidney protection. METHODS EMBASE, PubMed, MEDLINE, and the Cochrane Library were searched for randomized controlled trials published on or before July 2, 2024, that examined the relationship between Intravenous amino acids and renal function. We extracted population characteristics and outcome variables related to renal function from randomized controlled trials comparing intravenous amino acid supplementation with no supplementation. We assessed this evidence using the Risk of Bias 2 (RoB2) tool for randomized controlled trials. Data were synthesized using a random-effects model. RESULTS This review included 7 randomized controlled trials with a total of 505 patients. The results showed that compared with the control group, intravenous amino acid administration significantly reduced the incidence of AKI (RR: 0.81, 95 % CI: 0.68-0.97, P = 0.02) and increased urine output (MD: 308.87, 95 % CI: 168.68-449.06, P < 0.0001). However, intravenous amino acids did not reduce mortality or the incidence of kidney replacement therapy, with no statistical difference in 30-day mortality (RR: 0.93, 95 % CI: 0.65-1.34, P = 0.71), 90-day mortality (RR:1.00, 95 % CI: 0.77-1.29, P = 0.98), or need for kidney replacement therapy (RR: 0.92, 95 % CI: 0.41-2.06, P = 0.83). Subgroup analysis suggested that, regardless of sample size, intravenous amino acid administration reduced the incidence of AKI and was particularly significant in patients undergoing cardiac and major vascular surgery. Furthermore, intraoperative intravenous amino acid therapy demonstrated a significant reduction in the incidence of AKI compared to postoperative administration. CONCLUSIONS Intravenous amino acids protect renal function in patients at high risk of AKI, particularly after cardiac surgery. It reduces the incidence of AKI and increases urine output, but has no significant effect on KRT and mortality.
Collapse
Affiliation(s)
- Wei Jiang
- Department of Critical Care Medicine, Northern Jiangsu People's Hospital Affiliated to Yangzhou University, Yangzhou 225001, China; Department of Critical Care Medicine, Northern Jiangsu People's Hospital, Yangzhou 225001, China
| | - Keran Shi
- Department of Critical Care Medicine, Northern Jiangsu People's Hospital Affiliated to Yangzhou University, Yangzhou 225001, China; Department of Critical Care Medicine, Northern Jiangsu People's Hospital, Yangzhou 225001, China
| | - Jun Shao
- Department of Critical Care Medicine, Northern Jiangsu People's Hospital Affiliated to Yangzhou University, Yangzhou 225001, China; Department of Critical Care Medicine, Northern Jiangsu People's Hospital, Yangzhou 225001, China
| | - Lin Song
- Department of Critical Care Medicine, Northern Jiangsu People's Hospital Affiliated to Yangzhou University, Yangzhou 225001, China; Department of Critical Care Medicine, Northern Jiangsu People's Hospital, Yangzhou 225001, China
| | - Ying Shi
- Department of Critical Care Medicine, Northern Jiangsu People's Hospital Affiliated to Yangzhou University, Yangzhou 225001, China; Department of Critical Care Medicine, Northern Jiangsu People's Hospital, Yangzhou 225001, China
| | - Haoran Wang
- Department of Critical Care Medicine, Northern Jiangsu People's Hospital Affiliated to Yangzhou University, Yangzhou 225001, China; Department of Critical Care Medicine, Northern Jiangsu People's Hospital, Yangzhou 225001, China
| | - Lulun Zhou
- Department of Critical Care Medicine, Northern Jiangsu People's Hospital Affiliated to Yangzhou University, Yangzhou 225001, China; Department of Critical Care Medicine, Northern Jiangsu People's Hospital, Yangzhou 225001, China
| | - Luanluan Li
- Department of Critical Care Medicine, Northern Jiangsu People's Hospital Affiliated to Yangzhou University, Yangzhou 225001, China; Department of Critical Care Medicine, Northern Jiangsu People's Hospital, Yangzhou 225001, China
| | - Yunfan Feng
- Department of Critical Care Medicine, Northern Jiangsu People's Hospital Affiliated to Yangzhou University, Yangzhou 225001, China; Department of Critical Care Medicine, Northern Jiangsu People's Hospital, Yangzhou 225001, China
| | - Jiangquan Yu
- Department of Critical Care Medicine, Northern Jiangsu People's Hospital Affiliated to Yangzhou University, Yangzhou 225001, China; Department of Critical Care Medicine, Northern Jiangsu People's Hospital, Yangzhou 225001, China
| | - Ruiqiang Zheng
- Department of Critical Care Medicine, Northern Jiangsu People's Hospital Affiliated to Yangzhou University, Yangzhou 225001, China; Department of Critical Care Medicine, Northern Jiangsu People's Hospital, Yangzhou 225001, China.
| |
Collapse
|
|
3
|
Aly R, Dogan YE, Bala N, Lugo C, Darwish S, Shoemaker L, Alli AA. Dysregulation of kidney proteases in the pathogenesis of hypertension following unilateral nephrectomy in juvenile mice. Am J Transl Res 2024; 16:544-556. [PMID: 38463588 PMCID: PMC10918144] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2023] [Accepted: 08/12/2023] [Indexed: 03/12/2024]
Abstract
BACKGROUND Unliteral nephrectomy (UNX) results in the reduction of kidney mass. The remaining kidney undergoes compensatory renal growth via hypertrophy of the glomeruli and renal tubules to maintain a normal glomerular filtration rate (GFR). These compensatory mechanisms result in increased capillary pressure and glomerular hyperfiltration to increase single nephron GFR. Over time, hyperfiltration may lead to kidney scarring and the development of hypertension. OBJECTIVES The first objective of this study was to test the hypothesis that a 50% reduction in functioning nephrons in juvenile mice leads to increased blood pressure over a 24-hour phase. The second objective was to test the hypothesis that UNX leads to changes in the expression and activity of kidney proteases in juvenile mice. METHODS Eight male C57B6 juvenile wild-type mice were subject to UNX and an equal number of mice were subject to sham (SH) surgery. Metabolic cage studies were performed for 5 weeks to collect urine produced during the inactive and active phases. Blood pressure was measured using the tail cuff method twice weekly and tail blood was collected on different days during the inactive or active phase of each animal. The mice were euthanized at the age of 9 weeks. Western blotting and immunohistochemistry were performed to investigate changes in renal protein expression of various cathepsins and renal kallikrein 1 (KLK1) between the two groups. Protease activity assays were performed using kidney lysates and urine samples from each group. RESULTS Compared to the SH group, UNX mice showed a persistent increase in blood pressure at week 3 which progressed toward the end of the study at week 5 of age. Cathepsin B, D, and S expression and activity were up-regulated in kidney cortex lysates from UNX mice compared to the SH control group. KLK1 protein expression was down-regulated and urinary nitric oxide excretion was decreased in UNX mice compared to the SH control group. CONCLUSION UNX results in the development of persistent and progressive hypertension. Down-regulation of KLK1 and up-regulation of various cathepsins may contribute to the development of hypertension via multiple mechanisms including a decrease in nitric oxide (NO) production.
Collapse
Affiliation(s)
- Rasha Aly
- Department of Pediatrics, Division of Pediatric Nephrology, University of Florida College of MedicineGainesville, FL, USA
| | - Yunus E Dogan
- Department of Physiology and Aging, University of Florida College of MedicineGainesville, FL, USA
- Department of Pediatrics, Faculty of Medicine, Erciyes UniversityKayseri, Turkey
| | - Niharika Bala
- Department of Physiology and Aging, University of Florida College of MedicineGainesville, FL, USA
| | - Carlos Lugo
- Department of Physiology and Aging, University of Florida College of MedicineGainesville, FL, USA
| | - Seena Darwish
- Department of Physiology and Aging, University of Florida College of MedicineGainesville, FL, USA
| | - Lawrence Shoemaker
- Department of Pediatrics, Division of Pediatric Nephrology, University of Florida College of MedicineGainesville, FL, USA
| | - Abdel A Alli
- Department of Pediatrics, Division of Pediatric Nephrology, University of Florida College of MedicineGainesville, FL, USA
- Department of Physiology and Aging, University of Florida College of MedicineGainesville, FL, USA
- Department of Medicine, Division of Nephrology, Hypertension and Renal Transplantation, University of Florida College of MedicineGainesville, FL, USA
| |
Collapse
|
|
4
|
Weiss R, Meersch M, Gerke M, Wempe C, Schäfers M, Kellum JA, Zarbock A. Effect of Glutamine Administration After Cardiac Surgery on Kidney Damage in Patients at High Risk for Acute Kidney Injury: A Randomized Controlled Trial. Anesth Analg 2023; 137:1029-1038. [PMID: 36730070 DOI: 10.1213/ane.0000000000006288] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
BACKGROUND Acute kidney injury (AKI) is a common complication after cardiac surgery and is associated with increased morbidity and mortality. However, no specific treatment options are available, emphasizing the need for preventive measures. The aim of this study was to clarify the effect of glutamine on [TIMP2]*[IGFBP7] levels at the end of the intervention period. METHODS In a randomized clinical, double-blind pilot study, 64 eligible cardiac surgery patients at high risk for AKI identified by high urinary [TIMP2]*[IGFBP7] were randomized, and body weight-adapted intravenous glutamine or saline-control was administered continuously for 12 hours postoperatively. The primary outcome was urinary [TIMP2]*[IGFBP7] at the end of the 12-hour study period. Secondary outcomes included kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) at 12 hours, overall AKI rates at 72 hours, free days through day 28 of mechanical ventilation and vasoactive medication, renal recovery at day 90, requirement of renal replacement therapy and mortality each at days 30, 60, and 90, length of intensive care unit (ICU) and hospital stay, and major adverse kidney events consisting of mortality, dialysis dependency, and persistent renal dysfunction (serum creatinine ≥2× compared to baseline value) at day 90 (major adverse kidney event; MAKE 90 ). RESULTS Sixty-four patients (mean age, 68.38 [standard deviation {SD} ± 10.48] years; 10 of 64 women) were enrolled and randomized. Patients received coronary artery bypass graft surgery (32/64), valve surgery (18/64), coronary artery bypass graft and valve surgery (6/64), or other procedures (8/64). Mean on-pump time was 68.38 (standard deviation ± 10.48) minutes. After glutamine administration, urinary [TIMP-2]*[IGFBP7] was significantly lower in the glutamine compared to the control group (primary end point, intervention: median, 0.18 [Q1, Q3; 0.09, 0.29], controls: median, 0.44 [Q1, Q3; 0.14, 0.79]; P = .01). In addition, [KIM-1] and [NGAL] were also significantly lower in the glutamine group. The overall AKI rate within 72 hours was not different among groups: (intervention 11/31 [35.5%] versus control 8/32 [25.0%]; P = .419; relative risk [RR], 0.86% [95% confidence interval {CI}, 0.62-1.20]). There were no differences regarding secondary end points. CONCLUSIONS Glutamine significantly decreased markers of kidney damage in cardiac surgery patients at high risk for AKI. Future trials have to be performed to investigate whether the administration of glutamine might be able to reduce the occurrence of AKI after cardiac surgery.
Collapse
Affiliation(s)
- Raphael Weiss
- From the Departments of Anesthesiology, Intensive Care and Pain Medicine
| | - Melanie Meersch
- From the Departments of Anesthesiology, Intensive Care and Pain Medicine
| | - Mena Gerke
- From the Departments of Anesthesiology, Intensive Care and Pain Medicine
| | - Carola Wempe
- From the Departments of Anesthesiology, Intensive Care and Pain Medicine
| | | | - John A Kellum
- Center for Critical Care Nephrology, Department of Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Alexander Zarbock
- From the Departments of Anesthesiology, Intensive Care and Pain Medicine
| |
Collapse
|
|
5
|
Martins L, Amorim WW, Gregnani MF, de Carvalho Araújo R, Qadri F, Bader M, Pesquero JB. Kinin receptors regulate skeletal muscle regeneration: differential effects for B1 and B2 receptors. Inflamm Res 2023; 72:1583-1601. [PMID: 37464053 PMCID: PMC10499706 DOI: 10.1007/s00011-023-01766-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2023] [Revised: 06/20/2023] [Accepted: 07/02/2023] [Indexed: 07/20/2023] Open
Abstract
OBJECTIVE AND DESIGN After traumatic skeletal muscle injury, muscle healing is often incomplete and produces extensive fibrosis. Bradykinin (BK) reduces fibrosis in renal and cardiac damage models through the B2 receptor. The B1 receptor expression is induced by damage, and blocking of the kallikrein-kinin system seems to affect the progression of muscular dystrophy. We hypothesized that both kinin B1 and B2 receptors could play a differential role after traumatic muscle injury, and the lack of the B1 receptor could produce more cellular and molecular substrates for myogenesis and fewer substrates for fibrosis, leading to better muscle healing. MATERIAL AND METHODS To test this hypothesis, tibialis anterior muscles of kinin receptor knockout animals were subjected to traumatic injury. Myogenesis, angiogenesis, fibrosis, and muscle functioning were evaluated. RESULTS Injured B1KO mice showed a faster healing progression of the injured area with a larger amount of central nucleated fiber post-injury when compared to control mice. In addition, they exhibited higher neovasculogenic capacity, maintaining optimal tissue perfusion for the post-injury phase; had higher amounts of myogenic markers with less inflammatory infiltrate and tissue destruction. This was followed by higher amounts of SMAD7 and lower amounts of p-SMAD2/3, which resulted in less fibrosis. In contrast, B2KO and B1B2KO mice showed more severe tissue destruction and excessive fibrosis. B1KO animals had better results in post-injury functional tests compared to control animals. CONCLUSIONS We demonstrate that injured skeletal muscle tissues have a better repair capacity with less fibrosis in the presence of B2 receptor and absence of B1 receptor, including better performances in functional tests.
Collapse
Affiliation(s)
- Leonardo Martins
- Division of Medical Sciences, Laboratory of Transcriptional Regulation, Institute of Medical Biology of Polish Academy of Sciences (IMB-PAN), 3a Tylna St., 90-364, Łódź, Poland.
- Center for Research and Molecular Diagnosis of Genetic Diseases, Federal University of São Paulo, Rua Pedro de Toledo 669, 9th Floor, São Paulo, 04039032, Brazil.
- Department of Biochemistry and Molecular Biology, Federal University of São Paulo, Rua Três de Maio 100, 4th Floor, São Paulo, 04044-020, Brazil.
| | - Weslley Wallace Amorim
- Center for Research and Molecular Diagnosis of Genetic Diseases, Federal University of São Paulo, Rua Pedro de Toledo 669, 9th Floor, São Paulo, 04039032, Brazil
| | - Marcos Fernandes Gregnani
- Laboratory of Exercise Genetics and Metabolism, Federal University of São Paulo, Rua Pedro de Toledo 669, 9th Floor, São Paulo, 04039032, Brazil
| | - Ronaldo de Carvalho Araújo
- Laboratory of Exercise Genetics and Metabolism, Federal University of São Paulo, Rua Pedro de Toledo 669, 9th Floor, São Paulo, 04039032, Brazil
| | - Fatimunnisa Qadri
- Max-Delbrück Center for Molecular Medicine (MDC), Robert-Rössle-Str. 10, 13125, Berlin, Germany
| | - Michael Bader
- Max-Delbrück Center for Molecular Medicine (MDC), Robert-Rössle-Str. 10, 13125, Berlin, Germany
- Institute for Biology, University of Lübeck, Ratzeburger Allee 160, 23562, Lübeck, Germany
- Charité University Medicine Berlin, Charitéplatz 1, 10117, Berlin, Germany
- German Center for Cardiovascular Research (DZHK), Potsdamer Str. 58, 10785, Berlin, Germany
| | - João Bosco Pesquero
- Center for Research and Molecular Diagnosis of Genetic Diseases, Federal University of São Paulo, Rua Pedro de Toledo 669, 9th Floor, São Paulo, 04039032, Brazil.
- Department of Biophysics, Federal University of São Paulo, Rua Botucatu 862, 6th Floor, São Paulo, 04023-062, Brazil.
| |
Collapse
|
|
6
|
Cui H, Ren G, Hu X, Xu B, Li Y, Niu Z, Mu L. Suppression of lncRNA GAS6-AS2 alleviates sepsis-related acute kidney injury through regulating the miR-136-5p/OXSR1 axis in vitro and in vivo. Ren Fail 2022; 44:1070-1082. [PMID: 35793478 PMCID: PMC9272941 DOI: 10.1080/0886022x.2022.2092001] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
Acute kidney injury (AKI) is a common complication of sepsis and increase morbidity and mortality. Long non-coding RNA (LncRNA) GAS6-AS2 was related to inflammation and apoptosis in different diseases by regulating miRNAs and downstream genes, but its role in AKI remains unclear. Thus, we speculated that GAS6-AS2 might function in sepsis-related AKI via regulating target genes. Here, LPS or CLP was used to establish in vitro or in vivo sepsis-related AKI model. The interactions between GAS6-AS2 and miR-136-5p, and miR-136-5p and OXSR1, were validated by luciferase reporter assay, RNA pull-down, or RIP assay. Cell apoptosis was determined by flow cytometry, Western blotting, or IHC. The kidney injury was evaluated by H&E staining. The expression of GAS6-AS2, miR-136-5p, and OXSR1 was determined by qRT-PCR or Western blotting. We found that GAS6-AS2 was up-regulated in LPS-treated HK2 cells and the CLP-induced rat model. In vitro, GAS6-AS2 knockdown decreased cleaved caspase-3 and bax expression and increased bcl-2 expression. The levels of TNF-α, IL-1β, and IL-6 were reduced by GAS6-AS2 down-regulation. GAS6-AS2 knockdown ameliorated oxidative stress in the cells, as indicated by the reduced ROS and MDA levels and the elevated SOD level. In vivo, GAS6-AS2 down-regulation decreased urinary NGAL and Kim-1 levels and serum sCr and BUN levels, and H&E proved that the kidney injury was alleviated. GAS6-AS2 knockdown also reduced apoptosis, inflammation, and oxidation induced by CLP in vivo. Mechanically, GAS6-AS2 sponged miR-136-5p which targeted OXSR1. Overall, lncRNA GAS6-AS2 knockdown has the potential to ameliorate sepsis-related AKI, and the mechanism is related to miR-136-5p/OXSR1 axis.
Collapse
Affiliation(s)
- Hongrui Cui
- Department of Nephrology, The First Hospital of Hebei Medical University, Shijiazhuang, PR China
| | - Guangwei Ren
- Department of Nephrology, The First Hospital of Hebei Medical University, Shijiazhuang, PR China
| | - Xiuhong Hu
- Department of Nephrology, The First Hospital of Hebei Medical University, Shijiazhuang, PR China
| | - Baozhen Xu
- Department of Nephrology, The First Hospital of Hebei Medical University, Shijiazhuang, PR China
| | - Yuping Li
- Department of Nephrology, The First Hospital of Hebei Medical University, Shijiazhuang, PR China
| | - Zheli Niu
- Department of Nephrology, The First Hospital of Hebei Medical University, Shijiazhuang, PR China
| | - Liqin Mu
- Department of General Practice, The First Hospital of Hebei Medical University, Shijiazhuang, PR China
| |
Collapse
|
|
7
|
Thomas K, Zondler L, Ludwig N, Kardell M, Lüneburg C, Henke K, Mersmann S, Margraf A, Spieker T, Tekath T, Velic A, Holtmeier R, Hermann J, Jankowski V, Meersch M, Vestweber D, Westphal M, Roth J, Schäfers MA, Kellum JA, Lowell CA, Rossaint J, Zarbock A. Glutamine prevents acute kidney injury by modulating oxidative stress and apoptosis in tubular epithelial cells. JCI Insight 2022; 7:163161. [PMID: 36107633 PMCID: PMC9675453 DOI: 10.1172/jci.insight.163161] [Citation(s) in RCA: 55] [Impact Index Per Article: 18.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2022] [Accepted: 09/12/2022] [Indexed: 12/15/2022] Open
Abstract
Acute kidney injury (AKI) represents a common complication in critically ill patients that is associated with increased morbidity and mortality. In a murine AKI model induced by ischemia/reperfusion injury (IRI), we show that glutamine significantly decreases kidney damage and improves kidney function. We demonstrate that glutamine causes transcriptomic and proteomic reprogramming in murine renal tubular epithelial cells (TECs), resulting in decreased epithelial apoptosis, decreased neutrophil recruitment, and improved mitochondrial functionality and respiration provoked by an ameliorated oxidative phosphorylation. We identify the proteins glutamine gamma glutamyltransferase 2 (Tgm2) and apoptosis signal-regulating kinase (Ask1) as the major targets of glutamine in apoptotic signaling. Furthermore, the direct modulation of the Tgm2-HSP70 signalosome and reduced Ask1 activation resulted in decreased JNK activation, leading to diminished mitochondrial intrinsic apoptosis in TECs. Glutamine administration attenuated kidney damage in vivo during AKI and TEC viability in vitro under inflammatory or hypoxic conditions.
Collapse
Affiliation(s)
- Katharina Thomas
- Department of Anesthesiology, Intensive Care and Pain Medicine, University Hospital Münster, Münster, Germany
| | - Lisa Zondler
- Department of Anesthesiology, Intensive Care and Pain Medicine, University Hospital Münster, Münster, Germany
| | - Nadine Ludwig
- Department of Anesthesiology, Intensive Care and Pain Medicine, University Hospital Münster, Münster, Germany
| | - Marina Kardell
- Department of Anesthesiology, Intensive Care and Pain Medicine, University Hospital Münster, Münster, Germany
| | - Corinna Lüneburg
- Department of Anesthesiology, Intensive Care and Pain Medicine, University Hospital Münster, Münster, Germany
| | - Katharina Henke
- Department of Anesthesiology, Intensive Care and Pain Medicine, University Hospital Münster, Münster, Germany
| | - Sina Mersmann
- Department of Anesthesiology, Intensive Care and Pain Medicine, University Hospital Münster, Münster, Germany
| | - Andreas Margraf
- Department of Anesthesiology, Intensive Care and Pain Medicine, University Hospital Münster, Münster, Germany
| | - Tilmann Spieker
- Institute for Pathology, St. Franziskus Hospital Münster, Münster, Germany
| | - Tobias Tekath
- Institute of Medical Informatics, University of Münster, Münster, Germany
| | - Ana Velic
- Department of Quantitative Proteomics, University of Tübingen, Tübingen, Germany
| | - Richard Holtmeier
- Institute of Clinical Radiology, University Hospital Münster, Münster, Germany
| | - Juliane Hermann
- Institute for Molecular Cardiovascular Research, RWTH Aachen University Hospital, Aachen, Germany
| | - Vera Jankowski
- Institute for Molecular Cardiovascular Research, RWTH Aachen University Hospital, Aachen, Germany
| | - Melanie Meersch
- Department of Anesthesiology, Intensive Care and Pain Medicine, University Hospital Münster, Münster, Germany
| | | | - Martin Westphal
- Department of Anesthesiology, Intensive Care and Pain Medicine, University Hospital Münster, Münster, Germany.,Fresenius Kabi AG, Bad Homburg, Germany
| | - Johannes Roth
- Institute for Immunology, University of Münster, Münster
| | - Michael A. Schäfers
- European Institute for Molecular Imaging, University Hospital Münster, Münster, Germany
| | - John A. Kellum
- Center for Critical Care Nephrology, Department of Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
| | - Clifford A. Lowell
- Department of Laboratory Medicine, University of California, San Francisco, San Francisco, California, USA
| | - Jan Rossaint
- Department of Anesthesiology, Intensive Care and Pain Medicine, University Hospital Münster, Münster, Germany
| | - Alexander Zarbock
- Department of Anesthesiology, Intensive Care and Pain Medicine, University Hospital Münster, Münster, Germany
| |
Collapse
|
|
8
|
Exogenous pancreatic kininogenase protects against tacrolimus-induced renal injury by inhibiting PI3K/AKT signaling: The role of bradykinin receptors. Int Immunopharmacol 2022; 105:108547. [DOI: 10.1016/j.intimp.2022.108547] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2020] [Revised: 01/12/2022] [Accepted: 01/13/2022] [Indexed: 11/20/2022]
|
|
9
|
Xiao M, Zheng L, Zhang X, Duan X, Hang T, Lu S, Liu S, Lin H. Renal-on-Chip Microfluidic Platform with a Force-Sensitive Resistor (ROC-FS) for Molecular Pathogenesis Analysis of Hydronephrosis. Anal Chem 2021; 94:748-757. [PMID: 34951537 DOI: 10.1021/acs.analchem.1c03155] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Hydronephrosis is one of the most common diseases in urology. However, due to the difficulties in clinical trials and the lack of reliable in vitro platforms, the surgical indicators are not clear. Herein, the renal-on-chip with a force-sensitive resistor microfluidic platform was established to simulate the state of hydronephrosis. Cell counting kit-8 (CCK-8) and tight junction protein claudin-2 were detected on a renal-on-chip microfluidic platform with a force-sensitive resistor (ROC-FS). The results indicated that the ROC-FS had normal physiological functions and the cell viability on ROC-FS declined to around 40% after 48 h of hydronephrosis-simulated treatment. In addition, proteomics analysis of 15 clinical ureteropelvic junction obstruction (UPJO) samples showed that compared with normal children, a total of 50 common proteins were differentially expressed in UPJO children (P < 0.05, |log2fold change| ≥ 1). Metabolomic analysis of 39 clinical UPJO samples showed that a total of 241 metabolisms were dysregulated. Subsequent immunofluorescence and enzyme-linked immunosorbent assay (ELISA) analysis using ROC-FS were performed to identify the clinical multi-omics results for screening. All results pointed out that the TGF-β-related signaling pathways and arginine-related metabolism signaling pathways were dysregulated and α-SMA, AGT, and AGA might be the potential biomarkers of hydronephrosis. In addition, correlation analysis of AGT and KLK1 with differential renal function (DRF) from clinical samples indicated good correlation coefficients (R2 0.923, 0.8742, 0.6412, and 0.8347). This demonstrates the state of hydronephrosis could be significantly correlated with the biomarkers. These findings could provide a reliable reference for determining surgical biomarkers clinically, and ROC could be further used in the analysis of other kidney diseases.
Collapse
Affiliation(s)
- Mingming Xiao
- Shanghai Key Laboratory of Atmospheric Particle Pollution Prevention (LAP3), Department of Environmental Science & Engineering, Fudan University, 2005 Songhu Road, Shanghai 200438, China
| | - Lulu Zheng
- Engineering Research Center of Optical Instrument and System, Ministry of Education, Shanghai Key Lab of Modern Optical System, University of Shanghai for Science and Technology, 526 Jugong Road, Shanghai 200093, China
| | - Xinlian Zhang
- Shanghai Key Laboratory of Atmospheric Particle Pollution Prevention (LAP3), Department of Environmental Science & Engineering, Fudan University, 2005 Songhu Road, Shanghai 200438, China
| | - Xiaoxiao Duan
- Shanghai Key Laboratory of Atmospheric Particle Pollution Prevention (LAP3), Department of Environmental Science & Engineering, Fudan University, 2005 Songhu Road, Shanghai 200438, China
| | - Tian Hang
- Department of Pediatric Surgery, Jiaxing Women and Children Hospital Affiliated to Jiaxing University, 2468 East Zhonghuan Road, Jiaxing, Zhejiang 314050, China
| | - Shijiao Lu
- Department of Pediatric Surgery, Jiaxing Women and Children Hospital Affiliated to Jiaxing University, 2468 East Zhonghuan Road, Jiaxing, Zhejiang 314050, China
| | - Sixiu Liu
- Shanghai Key Laboratory of Atmospheric Particle Pollution Prevention (LAP3), Department of Environmental Science & Engineering, Fudan University, 2005 Songhu Road, Shanghai 200438, China
| | - Houwei Lin
- Department of Pediatric Surgery, Jiaxing Women and Children Hospital Affiliated to Jiaxing University, 2468 East Zhonghuan Road, Jiaxing, Zhejiang 314050, China
| |
Collapse
|
|
10
|
Li YM, Zhang J, Su LJ, Kellum JA, Peng ZY. Downregulation of TIMP2 attenuates sepsis-induced AKI through the NF-κb pathway. Biochim Biophys Acta Mol Basis Dis 2019; 1865:558-569. [PMID: 30562560 DOI: 10.1016/j.bbadis.2018.10.041] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2018] [Revised: 10/03/2018] [Accepted: 10/31/2018] [Indexed: 12/13/2022]
Abstract
Acute kidney injury (AKI) is a frequent complication of sepsis and contributes to increased morbidity and mortality. Urinary tissue inhibitor of metalloproteinases-2 (TIMP2) has been recently recognized as an early biomarker to predict AKI in critically ill patients. However, the biological functions of TIMP2 remain largely unknown. In this study, we investigated the role of TIMP2 in mediating inflammation and tubular cell apoptosis in AKI. In kidney tissue taken from mice exposed to cecal ligation and puncture (CLP) and in human kidney 2 (HK-2) cells exposed to lipopolysaccharide (LPS) in culture, TIMP2 expression was significantly upregulated. The expression of TIMP2 in the kidney tissue correlated with the severity of AKI in vivo. In cultured HK-2 cells, LPS challenge markedly induced cytokine release, and recombinant cytokines promoted TIMP2 expression and apoptosis. However, TIMP2 silencing ameliorated LPS-induced cytokine release, apoptosis, and cell injury. We further found that the effects of downregulation of TIMP2 on a suppression of release of inflammatory cytokines were mediated by p-P65. Stable, kidney-specific TIMP2 knockdown mice were transduced by injecting the TIMP2 knockdown lentiviral vector into kidney parenchyma. TIMP2 silencing ameliorated CLP-induced proinflammatory cytokines, kidney dysfunction as measured by serum creatinine level, and histopathological changes. Downregulation of TIMP2 showed renoprotective effects on endotoxin-induced AKI, which was associated with the anti-inflammatory activity through inhibition of the nuclear factor (NF)-κB pathway. Collectively, our results indicate that TIMP2 plays an important role in mediating sepsis-induced AKI through regulation of NF-κB. These findings reveal the pathogenic role of TIMP2 in AKI and suggest a novel target for the treatment of AKI.
Collapse
Affiliation(s)
- Yi-Ming Li
- Dept. of Critical Care Medicine, Zhongnan Hospital of Wuhan University, Wuhan 430071, Hubei, China
| | - Jing Zhang
- Dept. of Critical Care Medicine, Zhongnan Hospital of Wuhan University, Wuhan 430071, Hubei, China
| | - Lian-Jiu Su
- Dept. of Critical Care Medicine, Zhongnan Hospital of Wuhan University, Wuhan 430071, Hubei, China
| | - John A Kellum
- Center of Critical Care Nephrology, Department of Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
| | - Zhi-Yong Peng
- Dept. of Critical Care Medicine, Zhongnan Hospital of Wuhan University, Wuhan 430071, Hubei, China; Center of Critical Care Nephrology, Department of Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
| |
Collapse
|
|
11
|
Cheng Y, Liu C, Cui Y, Lv T, Guo Y, Liang J, Qian H. Sporidiobolus pararoseuswall-broken powder ameliorates oxidative stress in diabetic nephropathy in type-2 diabetic mice by activating the Nrf2/ARE pathway. RSC Adv 2019; 9:8394-8403. [PMID: 35518685 PMCID: PMC9061701 DOI: 10.1039/c8ra10484k] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2018] [Accepted: 01/22/2019] [Indexed: 11/21/2022] Open
Abstract
STZ-induced diabetic mice are given a high-fat diet and SPP, which is a rich source of β-carotene, γ-carotene, torulene and torularhodin. The result indicated SPP can ameliorate diabetic nephropathyviaactivating Nrf2/ARE pathway.
Collapse
Affiliation(s)
- Yuliang Cheng
- School of Food Science and Technology
- Jiangnan University
- Wuxi
- P. R. China
- Synergetic Innovation Center for Food Safety and Nutrition
| | - Chang Liu
- School of Food Science and Technology
- Jiangnan University
- Wuxi
- P. R. China
- Synergetic Innovation Center for Food Safety and Nutrition
| | - Yan Cui
- Institute of Agricultural Products Processing
- Key Laboratory of Preservation Engineering of Agricultural Products
- Ningbo Academy of Agricultural Sciences
- Ningbo 315040
- China
| | - Tianqi Lv
- School of Food Science and Technology
- Jiangnan University
- Wuxi
- P. R. China
- Synergetic Innovation Center for Food Safety and Nutrition
| | - Yahui Guo
- School of Food Science and Technology
- Jiangnan University
- Wuxi
- P. R. China
- Synergetic Innovation Center for Food Safety and Nutrition
| | - Jun Liang
- Guangzhou GRE Metrology & Test Co., Ltd
- Guangzhou
- PR China
| | - He Qian
- School of Food Science and Technology
- Jiangnan University
- Wuxi
- P. R. China
- Synergetic Innovation Center for Food Safety and Nutrition
| |
Collapse
|
|
12
|
Devetzi M, Goulielmaki M, Khoury N, Spandidos DA, Sotiropoulou G, Christodoulou I, Zoumpourlis V. Genetically‑modified stem cells in treatment of human diseases: Tissue kallikrein (KLK1)‑based targeted therapy (Review). Int J Mol Med 2018; 41:1177-1186. [PMID: 29328364 PMCID: PMC5819898 DOI: 10.3892/ijmm.2018.3361] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2017] [Accepted: 01/02/2018] [Indexed: 12/12/2022] Open
Abstract
The tissue kallikrein-kinin system (KKS) is an endogenous multiprotein metabolic cascade which is implicated in the homeostasis of the cardiovascular, renal and central nervous system. Human tissue kallikrein (KLK1) is a serine protease, component of the KKS that has been demonstrated to exert pleiotropic beneficial effects in protection from tissue injury through its anti-inflammatory, anti-apoptotic, anti-fibrotic and anti-oxidative actions. Mesenchymal stem cells (MSCs) or endothelial progenitor cells (EPCs) constitute populations of well-characterized, readily obtainable multipotent cells with special immunomodulatory, migratory and paracrine properties rendering them appealing potential therapeutics in experimental animal models of various diseases. Genetic modification enhances their inherent properties. MSCs or EPCs are competent cellular vehicles for drug and/or gene delivery in the targeted treatment of diseases. KLK1 gene delivery using adenoviral vectors or KLK1 protein infusion into injured tissues of animal models has provided particularly encouraging results in attenuating or reversing myocardial, renal and cerebrovascular ischemic phenotype and tissue damage, thus paving the way for the administration of genetically modified MSCs or EPCs with the human tissue KLK1 gene. Engraftment of KLK1-modified MSCs and/or KLK1-modified EPCs resulted in advanced beneficial outcome regarding heart and kidney protection and recovery from ischemic insults. Collectively, findings from pre-clinical studies raise the possibility that tissue KLK1 may be a novel future therapeutic target in the treatment of a wide range of cardiovascular, cerebrovascular and renal disorders.
Collapse
Affiliation(s)
- Marina Devetzi
- Biomedical Applications Unit, Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, 11635 Athens, Greece
| | - Maria Goulielmaki
- Biomedical Applications Unit, Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, 11635 Athens, Greece
| | - Nicolas Khoury
- Biomedical Applications Unit, Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, 11635 Athens, Greece
| | - Demetrios A Spandidos
- Laboratory of Clinical Virology, Medical School, University of Crete, 71003 Heraklion, Greece
| | | | - Ioannis Christodoulou
- Biomedical Applications Unit, Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, 11635 Athens, Greece
| | - Vassilis Zoumpourlis
- Biomedical Applications Unit, Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, 11635 Athens, Greece
| |
Collapse
|
|
13
|
Acuña MJ, Salas D, Córdova-Casanova A, Cruz-Soca M, Céspedes C, Vio CP, Brandan E. Blockade of Bradykinin receptors worsens the dystrophic phenotype of mdx mice: differential effects for B1 and B2 receptors. J Cell Commun Signal 2017; 12:589-601. [PMID: 29250740 DOI: 10.1007/s12079-017-0439-x] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2017] [Accepted: 12/04/2017] [Indexed: 02/06/2023] Open
Abstract
The Kallikrein Kinin System (KKS) is a vasoactive peptide system with known functions in the maintenance of tissue homeostasis, renal function and blood pressure. The main effector peptide of KKS is Bradykinin (BK). This ligand has two receptors: a constitutive B2 receptor (B2R), which has been suggested to have anti-fibrotic effects in renal and cardiac models of fibrosis; and the inducible B1 receptor (B1R), whose expression is induced by damage and inflammation. Inflammation and fibrosis are hallmarks of Duchenne muscular dystrophy (DMD), therefore we hypothesized that the KKS may play a role in this disease. To evaluate this hypothesis we used the mdx mouse a model for DMD. We blocked the endogenous activity of the KKS by treating mdx mice with B2R antagonist (HOE-140) or B1R antagonist (DesArgLeu8BK (DALBK)) for four weeks. Both antagonists increased damage, fibrosis, TGF-β and Smad-dependent signaling, CTGF/CCN-2 levels as well as the number of CD68 positive inflammatory cells. B2R blockade also reduced isolated muscle contraction force. These results indicate that the endogenous KKS has a protective role in the dystrophic muscle. The KKS may be a new target for future therapies to reduce inflammation and fibrosis in dystrophic muscle.
Collapse
Affiliation(s)
- María José Acuña
- Centro de Envejecimiento y Regeneración, CARE Chile UC y Departamento de Biología Celular y Molecular, Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile.,Centro de Biología y Química Aplicada (CIBQA), Universidad Bernardo O Higgins, Santiago, Chile
| | - Daniela Salas
- Centro de Envejecimiento y Regeneración, CARE Chile UC y Departamento de Biología Celular y Molecular, Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Adriana Córdova-Casanova
- Centro de Envejecimiento y Regeneración, CARE Chile UC y Departamento de Biología Celular y Molecular, Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Meilyn Cruz-Soca
- Centro de Envejecimiento y Regeneración, CARE Chile UC y Departamento de Biología Celular y Molecular, Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Carlos Céspedes
- Centro de Envejecimiento y Regeneración, CARE Chile UC y Departamento de Biología Celular y Molecular, Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Carlos P Vio
- Centro de Envejecimiento y Regeneración, CARE Chile UC y Departamento de Biología Celular y Molecular, Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile. .,Centro de Excelencia en Biomedicina de Magallanes (CEBIMA), Universidad de Magallanes, Punta Arenas, Chile. .,Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Libertador Bernardo O'Higgins 340, 8331150, Santiago, Chile.
| | - Enrique Brandan
- Centro de Envejecimiento y Regeneración, CARE Chile UC y Departamento de Biología Celular y Molecular, Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile. .,Centro de Excelencia en Biomedicina de Magallanes (CEBIMA), Universidad de Magallanes, Punta Arenas, Chile. .,Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Libertador Bernardo O'Higgins 340, 8331150, Santiago, Chile.
| |
Collapse
|
|
14
|
Guo Y, Li P, Gao L, Zhang J, Yang Z, Bledsoe G, Chang E, Chao L, Chao J. Kallistatin reduces vascular senescence and aging by regulating microRNA-34a-SIRT1 pathway. Aging Cell 2017; 16:837-846. [PMID: 28544111 PMCID: PMC5506400 DOI: 10.1111/acel.12615] [Citation(s) in RCA: 76] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/16/2017] [Indexed: 01/13/2023] Open
Abstract
Kallistatin, an endogenous protein, protects against vascular injury by inhibiting oxidative stress and inflammation in hypertensive rats and enhancing the mobility and function of endothelial progenitor cells (EPCs). We aimed to determine the role and mechanism of kallistatin in vascular senescence and aging using cultured EPCs, streptozotocin (STZ)‐induced diabetic mice, and Caenorhabditis elegans (C. elegans). Human kallistatin significantly decreased TNF‐α‐induced cellular senescence in EPCs, as indicated by reduced senescence‐associated β‐galactosidase activity and plasminogen activator inhibitor‐1 expression, and elevated telomerase activity. Kallistatin blocked TNF‐α‐induced superoxide levels, NADPH oxidase activity, and microRNA‐21 (miR‐21) and p16INK4a synthesis. Kallistatin prevented TNF‐α‐mediated inhibition of SIRT1, eNOS, and catalase, and directly stimulated the expression of these antioxidant enzymes. Moreover, kallistatin inhibited miR‐34a synthesis, whereas miR‐34a overexpression abolished kallistatin‐induced antioxidant gene expression and antisenescence activity. Kallistatin via its active site inhibited miR‐34a, and stimulated SIRT1 and eNOS synthesis in EPCs, which was abolished by genistein, indicating an event mediated by tyrosine kinase. Moreover, kallistatin administration attenuated STZ‐induced aortic senescence, oxidative stress, and miR‐34a and miR‐21 synthesis, and increased SIRT1, eNOS, and catalase levels in diabetic mice. Furthermore, kallistatin treatment reduced superoxide formation and prolonged wild‐type C. elegans lifespan under oxidative or heat stress, although kallistatin's protective effect was abolished in miR‐34 or sir‐2.1 (SIRT1 homolog) mutant C. elegans. Kallistatin inhibited miR‐34, but stimulated sir‐2.1 and sod‐3 synthesis in C. elegans. These in vitro and in vivo studies provide significant insights into the role and mechanism of kallistatin in vascular senescence and aging by regulating miR‐34a‐SIRT1 pathway.
Collapse
Affiliation(s)
- Youming Guo
- Department of Biochemistry and Molecular Biology; Medical University of South Carolina; Charleston South Carolina
| | - Pengfei Li
- Department of Biochemistry and Molecular Biology; Medical University of South Carolina; Charleston South Carolina
| | - Lin Gao
- Department of Biochemistry and Molecular Biology; Medical University of South Carolina; Charleston South Carolina
| | - Jingmei Zhang
- Department of Biochemistry and Molecular Biology; Medical University of South Carolina; Charleston South Carolina
| | - Zhirong Yang
- Department of Biochemistry and Molecular Biology; Medical University of South Carolina; Charleston South Carolina
| | - Grant Bledsoe
- Division of Molecular Biology and Biochemistry; School of Biological Sciences; University of Missouri-Kansas City; Kansas City Missouri
| | - Eugene Chang
- Department of Obstetrics and Gynecology; College of Medicine; Medical University of South Carolina; Charleston South Carolina
| | - Lee Chao
- Department of Biochemistry and Molecular Biology; Medical University of South Carolina; Charleston South Carolina
| | - Julie Chao
- Department of Biochemistry and Molecular Biology; Medical University of South Carolina; Charleston South Carolina
| |
Collapse
|
|
15
|
Parrish AR. Matrix Metalloproteinases in Kidney Disease: Role in Pathogenesis and Potential as a Therapeutic Target. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2017; 148:31-65. [PMID: 28662825 DOI: 10.1016/bs.pmbts.2017.03.001] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Matrix metalloproteinases (MMPs) are large family of proteinases. In addition to a fundamental role in the remodeling of the extracellular matrix, they also cleave a number of cell surface proteins and are involved in multiple cellular processes. MMP activity is regulated via numerous mechanisms, including inhibition by endogenous tissue inhibitors of metalloproteinases (TIMPs). Similar to MMPs, a role for TIMPs has been established in multiple cell signaling pathways. Aberrant expression of MMPs and TIMPS in renal pathophysiology has long been recognized, and with the generation of specific knockout mice, the mechanistic role of several MMPs and TIMPs is becoming more understood and has revealed both pathogenic and protective roles. This chapter will focus on the expression and localization of MMPs and TIMPs in the kidney, as well as summarizing the current information linking these proteins to acute kidney injury and chronic kidney disease. In addition, we will summarize studies suggesting that MMPs and TIMPs may be biomarkers of renal dysfunction and represent novel therapeutic targets to attenuate kidney disease.
Collapse
Affiliation(s)
- Alan R Parrish
- School of Medicine, University of Missouri, Columbia, MO, United States.
| |
Collapse
|
|
16
|
Bivol LM, Iversen BM, Hultström M, Wallace PW, Reed RK, Wiig H, Tenstad O. Unilateral renal ischaemia in rats induces a rapid secretion of inflammatory markers to renal lymph and increased capillary permeability. J Physiol 2015; 594:1709-26. [PMID: 26584508 DOI: 10.1113/jp271578] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2015] [Accepted: 11/11/2015] [Indexed: 12/14/2022] Open
Abstract
A better understanding of the inflammatory process associated with renal ischaemia-reperfusion (IR) injury may be clinically important. In this study we examined the role of the kidney in production of inflammatory mediators by analysing renal lymph after 30 min unilateral occlusion of renal artery followed by 120 min reperfusion, as well as the effect of IR on size selectivity for proteins in both glomerular and peritubular capillaries. All measured mediators increased dramatically in renal hilar lymph, plasma and renal cortical tissue samples and returned to control levels after 120 min reperfusion. The responses were differentiated; interleukin-1β, monocyte chemoattractant protein-1 and leptin were markedly increased in plasma before reperfusion, reflecting an extrarenal response possibly induced by afferent renal nerve activity from the ischaemic kidney. Tumour necrosis factor-α was the only mediator showing elevated lymph-to-plasma ratio following 30 min reperfusion, indicating that most cytokines were released directly into the bloodstream. The IR-induced rise in cytokine levels was paralleled by a significant increase in high molecular weight plasma proteins in both lymph and urine. The latter was shown as a 14- to 166-fold increase in glomerular sieving coefficient of plasma proteins assessed by a novel proteomic approach, and indicated a temporarily reduced size selectivity of both glomerular and peritubular capillaries. Collectively, our data suggest that cytokines from the ischaemic kidney explain most of the rise in plasma concentration, and that the locally produced substances enter the systemic circulation through transport directly to plasma and not via the interstitium to lymph.
Collapse
Affiliation(s)
| | - Bjarne Magnus Iversen
- Department of Clinical Science, University of Bergen, Norway.,Haukeland University Hospital, Norway
| | - Michael Hultström
- Department of Clinical Science, University of Bergen, Norway.,Haukeland University Hospital, Norway
| | | | - Rolf Kåre Reed
- Department of Biomedicine, University of Bergen, Norway.,Centre for Cancer Biomarkers (CCBIO), University of Bergen, Norway
| | - Helge Wiig
- Department of Biomedicine, University of Bergen, Norway
| | - Olav Tenstad
- Department of Biomedicine, University of Bergen, Norway
| |
Collapse
|
|
17
|
Zhu P, Yu H, Huang S, Xiang H, Li F, Zheng W. Synergistic effect of a tissue kallikrein 1 and tissue inhibitor of matrix metalloproteinase 1 co‑expression vector on the proliferation of rat vascular smooth muscle cells. Mol Med Rep 2015; 12:5671-8. [PMID: 26252163 PMCID: PMC4581792 DOI: 10.3892/mmr.2015.4198] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2014] [Accepted: 06/22/2015] [Indexed: 12/05/2022] Open
Abstract
Tissue kallikrein 1 (TK1) and tissue inhibitor of matrix metalloproteinase 1 (TIMP1) are important in inhibiting vascular smooth muscle cell (VSMC) proliferation and improving vascular remodeling, respectively. It was hypothesized that a combination of TK1 and TIMP1 genes, mediated by an adenovirus vector could augment or act in synergy to enhance the inhibitory effects. The promoter, mCMV carrying hTIMP1 cDNA was subcloned into pDC316-hTK1 to construct a recombinant plasmid carrying hTK1 and hTIMP1 genes. Subsequently, the double gene plasmid and adenovirus backbone plasmid were packaged into HEK293A cells. Gene transcription and protein expression were examined, respectively using reverse transcription-quantitative polymerase chain reaction (PCR) and western blotting assays. VSMC proliferation was assessed using cell counting and methyl-thiazolyl-tetrazoliuin methods. The constructed plasmid containing hTK1 and hTIMP1 genes was correctly identified by means of PCR, double digestion and sequencing analysis. The co-expression vector, Ad-hTK1-hTIMP1 was successfully constructed and packaged into HEK293A cells. When VSMCs were transfected with the co-expression vector, the mRNA transcription and protein expression of hTK1 and hTIMP1 exhibited abundant expression in a concentration-dependent and time-dependent manner, independently. In conclusion, the co-expression vector synergistically inhibited the cell growth and proliferation induced by platelet-derived growth factor-BB compared with the single gene vector.
Collapse
Affiliation(s)
- Pengli Zhu
- Department of Geriatrics, Fujian Provincial Hospital Key Laboratory of Geriatrics, Fujian Medical University, Fuzhou, Fujian 350001, P.R. China
| | - Huizhen Yu
- Department of Geriatrics, Fujian Provincial Hospital Key Laboratory of Geriatrics, Fujian Medical University, Fuzhou, Fujian 350001, P.R. China
| | - Shujie Huang
- Department of Cardiology, Fujian Provincial Hospital, Fuzhou, Fujian 350001, P.R. China
| | - Hong Xiang
- Department of Geriatrics, Fujian Provincial Hospital Key Laboratory of Geriatrics, Fujian Medical University, Fuzhou, Fujian 350001, P.R. China
| | - Feng Li
- Department of Geriatrics, Fujian Provincial Hospital Key Laboratory of Geriatrics, Fujian Medical University, Fuzhou, Fujian 350001, P.R. China
| | - Weiping Zheng
- Department of Geriatrics, Fujian Provincial Hospital Key Laboratory of Geriatrics, Fujian Medical University, Fuzhou, Fujian 350001, P.R. China
| |
Collapse
|
|
18
|
Chao J, Bledsoe G, Chao L. Kallikrein-kinin in stem cell therapy. World J Stem Cells 2014; 6:448-457. [PMID: 25258666 PMCID: PMC4172673 DOI: 10.4252/wjsc.v6.i4.448] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/22/2014] [Revised: 08/27/2014] [Accepted: 09/01/2014] [Indexed: 02/06/2023] Open
Abstract
The tissue kallikrein-kinin system exerts a wide spectrum of biological activities in the cardiovascular, renal and central nervous systems. Tissue kallikrein-kinin modulates the proliferation, viability, mobility and functional activity of certain stem cell populations, namely mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), mononuclear cell subsets and neural stem cells. Stimulation of these stem cells by tissue kallikrein-kinin may lead to protection against renal, cardiovascular and neural damage by inhibiting apoptosis, inflammation, fibrosis and oxidative stress and promoting neovascularization. Moreover, MSCs and EPCs genetically modified with tissue kallikrein are resistant to hypoxia- and oxidative stress-induced apoptosis, and offer enhanced protective actions in animal models of heart and kidney injury and hindlimb ischemia. In addition, activation of the plasma kallikrein-kinin system promotes EPC recruitment to the inflamed synovium of arthritic rats. Conversely, cleaved high molecular weight kininogen, a product of plasma kallikrein, reduces the viability and vasculogenic activity of EPCs. Therefore, kallikrein-kinin provides a new approach in enhancing the efficacy of stem cell therapy for human diseases.
Collapse
|
|
19
|
Parvin AA, Pranap RA, Shalini U, Devendran A, Baker JE, Dhanasekaran A. Erythropoietin protects cardiomyocytes from cell death during hypoxia/reperfusion injury through activation of survival signaling pathways. PLoS One 2014; 9:e107453. [PMID: 25237819 PMCID: PMC4169563 DOI: 10.1371/journal.pone.0107453] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2014] [Accepted: 05/29/2014] [Indexed: 12/17/2022] Open
Abstract
Hypoxia/Reoxygenation (H/R) cardiac injury is of great importance in understanding Myocardial Infarctions, which affect a major part of the working population causing debilitating side effects and often-premature mortality. H/R injury primarily consists of apoptotic and necrotic death of cardiomyocytes due to a compromise in the integrity of the mitochondrial membrane. Major factors associated in the deregulation of the membrane include fluctuating reactive oxygen species (ROS), deregulation of mitochondrial permeability transport pore (MPTP), uncontrolled calcium (Ca2+) fluxes, and abnormal caspase-3 activity. Erythropoietin (EPO) is strongly inferred to be cardioprotective and acts by inhibiting the above-mentioned processes. Surprisingly, the underlying mechanism of EPO's action and H/R injury is yet to be fully investigated and elucidated. This study examined whether EPO maintains Ca2+ homeostasis and the mitochondrial membrane potential (ΔΨm) in cardiomyocytes when subjected to H/R injury and further explored the underlying mechanisms involved. H9C2 cells were exposed to different concentrations of EPO post-H/R, and 20 U/ml EPO was found to significantly increase cell viability by inhibiting the intracellular production of ROS and caspase-3 activity. The protective effect of EPO was abolished when H/R-induced H9C2 cells were treated with Wortmannin, an inhibitor of Akt, suggesting the mechanism of action through the activation Akt, a major survival pathway.
Collapse
Affiliation(s)
- Asiya A Parvin
- Centre for Biotechnology, Anna University, Chennai, Tamil Nadu, India
| | - Raj A Pranap
- Centre for Biotechnology, Anna University, Chennai, Tamil Nadu, India
| | - U Shalini
- Centre for Biotechnology, Anna University, Chennai, Tamil Nadu, India
| | - Ajay Devendran
- Centre for Biotechnology, Anna University, Chennai, Tamil Nadu, India
| | - John E Baker
- Department of Surgery, Division of Cardiothoracic Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America
| | | |
Collapse
|
|
20
|
The attenuation of Moutan Cortex on oxidative stress for renal injury in AGEs-induced mesangial cell dysfunction and streptozotocin-induced diabetic nephropathy rats. OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2014; 2014:463815. [PMID: 24876912 PMCID: PMC4021834 DOI: 10.1155/2014/463815] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/13/2013] [Revised: 03/25/2014] [Accepted: 04/02/2014] [Indexed: 02/07/2023]
Abstract
Oxidative stress (OS) has been regarded as one of the major pathogeneses of diabetic nephropathy (DN) through damaging kidney which is associated with renal cells dysfunction. The aim of this study was to investigate whether Moutan Cortex (MC) could protect kidney function against oxidative stress in vitro or in vivo. The compounds in MC extract were analyzed by HPLC-ESI-MS. High-glucose-fat diet and STZ (30 mg kg−1) were used to induce DN rats model, while 200 μg mL−1 AGEs were for HBZY-1 mesangial cell damage. The treatment with MC could significantly increase the activity of SOD, glutathione peroxidase (GSH-PX), and catalase (CAT). However, lipid peroxidation malondialdehyde (MDA) was reduced markedly in vitro or in vivo. Furthermore, MC decreased markedly the levels of blood glucose, serum creatinine, and urine protein in DN rats. Immunohistochemical assay showed that MC downregulated significantly transforming growth factor beta 2 (TGF-β2) protein expression in renal tissue. Our data provided evidence to support this fact that MC attenuated OS in AGEs-induced mesangial cell dysfunction and also in high-glucose-fat diet and STZ-induced DN rats.
Collapse
|
|
21
|
Chao J, Bledsoe G, Chao L. Tissue kallikrein-kinin therapy in hypertension and organ damage. PROGRESS IN DRUG RESEARCH. FORTSCHRITTE DER ARZNEIMITTELFORSCHUNG. PROGRES DES RECHERCHES PHARMACEUTIQUES 2014; 69:37-57. [PMID: 25130039 DOI: 10.1007/978-3-319-06683-7_3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/03/2023]
Abstract
Tissue kallikrein is a serine proteinase that cleaves low molecular weight kininogen to produce kinin peptides, which in turn activate kinin receptors to trigger multiple biological functions. In addition to its kinin-releasing activity, tissue kallikrein directly interacts with the kinin B2 receptor, protease-activated receptor-1, and gamma-epithelial Na channel. The tissue kallikrein-kinin system (KKS) elicits a wide spectrum of biological activities, including reducing hypertension, cardiac and renal damage, restenosis, ischemic stroke, and skin wound injury. Both loss-of-function and gain-of-function studies have shown that the KKS plays an important endogenous role in the protection against health pathologies. Tissue kallikrein/kinin treatment attenuates cardiovascular, renal, and brain injury by inhibiting oxidative stress, apoptosis, inflammation, hypertrophy, and fibrosis and promoting angiogenesis and neurogenesis. Approaches that augment tissue kallikrein-kinin activity might provide an effective strategy for the treatment of hypertension and associated organ damage.
Collapse
|
|
22
|
Shao X, Yang R, Yan M, Li Y, Du Y, Raman I, Zhang B, Wakeland EK, Igarashi P, Mohan C, Li QZ. Inducible expression of kallikrein in renal tubular cells protects mice against spontaneous lupus nephritis. ARTHRITIS AND RHEUMATISM 2013; 65:780-791. [PMID: 23280471 DOI: 10.1002/art.37798] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/20/2011] [Accepted: 11/08/2012] [Indexed: 01/06/2023]
Abstract
OBJECTIVE To ascertain whether engineered expression of kallikreins within the kidneys, using an inducible Cre/loxP system, can ameliorate murine lupus nephritis. METHODS In mice with a lupus-prone genetic background, we engineered the expression of tamoxifen-inducible Cre recombinase under the control of a kidney-specific promoter whose activation initiates murine kallikrein-1 expression within the kidneys. These transgenic mice were injected with either tamoxifen or vehicle at age 2 months and then were monitored for 8 months for kallikrein expression and disease. RESULTS Elevated expression of kallikrein was detected in the kidney and urine of tamoxifen-injected mice but not in controls. At age 10 months, all vehicle-injected mice developed severe lupus nephritis, as evidenced by increased proteinuria (mean ± SD 13.43 ± 5.65 mg/24 hours), increased blood urea nitrogen (BUN) and serum creatinine levels (39.86 ± 13.45 mg/dl and 15.23 ± 6.89 mg/dl, respectively), and severe renal pathology. In contrast, the tamoxifen-injected mice showed significantly reduced proteinuria (6.6 ± 4.12 mg/24 hours), decreased BUN and serum creatinine levels (15.71 ± 8.17 mg/dl and 6.64 ± 3.39 mg/dl, respectively), and milder renal pathology. Tamoxifen-induced up-regulation of renal kallikrein expression increased nitric oxide production and dampened renal superoxide production and inflammatory cell infiltration, alluding to some of the pathways through which kallikreins may be operating within the kidneys. CONCLUSION Local expression of kallikreins within the kidney has the capacity to dampen lupus nephritis, possibly by modulating inflammation and oxidative stress.
Collapse
Affiliation(s)
- Xinli Shao
- University of Texas-Southwestern Medical Center, Dallas, TX 75235, USA
| | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
23
|
Liu Y, Bledsoe G, Hagiwara M, Shen B, Chao L, Chao J. Depletion of endogenous kallistatin exacerbates renal and cardiovascular oxidative stress, inflammation, and organ remodeling. Am J Physiol Renal Physiol 2012; 303:F1230-8. [PMID: 22811485 DOI: 10.1152/ajprenal.00257.2012] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
Kallistatin (KS) levels are reduced in the kidney and blood vessels under oxidative stress conditions. To determine the function of endogenous KS in the renal and cardiovascular systems, KS levels were depleted by daily injection of anti-rat KS antibody into DOCA-salt hypertensive rats for 10 days. Administration of anti-KS antibody resulted in reduced KS levels in the circulation but increased levels of serum thiobarbituric acid reactive substances (an indicator of lipid peroxidation) as well as superoxide formation in the aorta. Moreover, anti-KS antibody injection resulted in increased NADH oxidase activity and superoxide production but decreased nitric oxide levels in the kidney and heart. Endogenous KS blockade aggravated renal dysfunction, damage, hypertrophy, inflammation, and fibrosis as evidenced by decreased creatinine clearance and increased serum creatinine, blood urea nitrogen and urinary protein levels, tubular dilation, protein cast formation, glomerulosclerosis, glomerular enlargement, inflammatory cell accumulation, and collagen deposition. In addition, rats receiving anti-KS antibody had enhanced cardiac injury as indicated by cardiomyocyte hypertrophy, inflammation, myofibroblast accumulation, and fibrosis. Renal and cardiac injury caused by endogenous KS depletion was accompanied by increases in the expression of the proinflammatory genes tumor necrosis factor-α and intercellular adhesion molecule-1 and the profibrotic genes collagen I and III, transforming growth factor-β, and tissue inhibitor of metalloproteinase-1. Taken together, these results implicate an important role for endogenous KS in protection against salt-induced renal and cardiovascular injury in rats by suppressing oxidative stress, inflammation, hypertrophy, and fibrosis.
Collapse
Affiliation(s)
- Yuying Liu
- Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Ave, Charleston, SC 29425-2211, USA.
| | | | | | | | | | | |
Collapse
|
|
24
|
Patel AB, Chao J, Palmer LG. Tissue kallikrein activation of the epithelial Na channel. Am J Physiol Renal Physiol 2012; 303:F540-50. [PMID: 22622459 DOI: 10.1152/ajprenal.00133.2012] [Citation(s) in RCA: 64] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Epithelial Na Channels (ENaC) are responsible for the apical entry of Na(+) in a number of different epithelia including the renal connecting tubule and cortical collecting duct. Proteolytic cleavage of γ-ENaC by serine proteases, including trypsin, furin, elastase, and prostasin, has been shown to increase channel activity. Here, we investigate the ability of another serine protease, tissue kallikrein, to regulate ENaC. We show that excretion of tissue kallikrein, which is secreted into the lumen of the connecting tubule, is stimulated following 5 days of a high-K(+) or low-Na(+) diet in rats. Urinary proteins reconstituted in a low-Na buffer activated amiloride-sensitive currents (I(Na)) in ENaC-expressing oocytes, suggesting an endogenous urinary protease can activate ENaC. We next tested whether tissue kallikrein can directly cleave and activate ENaC. When rat ENaC-expressing oocytes were exposed to purified tissue kallikrein from rat urine (RTK), ENaC currents increased threefold in both the presence and absence of a soybean trypsin inhibitor (SBTI). RTK and trypsin both decreased the apparent molecular mass of cleaved cell-surface γ-ENaC, while immunodepleted RTK produced no shift in apparent molecular mass, demonstrating the specificity of the tissue kallikrein. A decreased effect of RTK on Xenopus ENaC, which has variations in the putative prostasin cleavage sites in γ-ENaC, suggests these sites are important in RTK activation of ENaC. Mutating the prostasin site in mouse γ-ENaC (γRKRK186QQQQ) abolished ENaC activation and cleavage by RTK while wild-type mouse ENaC was activated and cleaved similar to that of the rat. We conclude that tissue kallikrein can be a physiologically relevant regulator of ENaC activity.
Collapse
Affiliation(s)
- Ankit B Patel
- Department of Physiology and Biophysics, Weill Cornell Medical College, 1300 York Ave., New York, NY 10065, USA
| | | | | |
Collapse
|