https://cdr.lib.unc.edu/downloads/8049g9405?locale=en
Vitamin B12 Protects Against Hypoxia/Reperfusion Injury in Mouse Proximal Tubule Cells
Abstract
Acute kidney injury (AKI) is a common syndrome characterized by a sudden decline in kidney function that can potentially lead to death. Ischemia/reperfusion injury (IRI) is the leading cause of AKI and is inevitable during kidney transplants. There is no effective treatment available to treat IRI. Pathways involved in IRI are evidenced to lead to reactive oxygen species (ROS), inflammation, fibrosis, apoptosis, DNA damage response (DDR) and autophagy. Vitamin B12 (B12) or cobalamin, is essential for the human body and is pharmacologically known to scavenge ROS, suppressing inflammation and reverse impaired autophagy that occurs in B12 deficient conditions.
To test whether B12 has beneficial effects in IRI, I subjected cultured mouse proximal tubule cells (BU.MPT) to a hypoxia/reperfusion (H/R) procedure and measured transcription of markers for inflammation (Mcp1, Il6, Nos2) and fibrosis (fibronectin), protein markers for apoptosis(Tgf1, c-cap3), and DDR (p.H2AX) induced by hypoxia/reperfusion (H/R). Presence of B12 during the H/R procedure at 0.3µM dramatically inhibited the upregulation of these markers studied and to an increased cell survival. Together, my findings suggest that B12 is a highly promising molecule to prevent/treat AKI.
Introduction
Acute kidney injury (AKI) is a clinical condition that affects roughly 200,000 people every year in the United States1 . It is characterized by the sudden decline of renal function, leading to the dangerous accumulation of waste products and chemical imbalance in one’s blood2 . The disease progresses from cellular damage to decreased glomerular filtration rate, leading to kidney failure and in severe situations, it results in death2 . Despite its frequency and severity, there is limited treatment of AKI and most are still in the development stage. For example, animal studies of α-melanocyte– stimulating hormone (α-MSH) have confirmed its anti-inflammatory and anti-apoptotic activities to protect from AKI1 . However, because its reduction of serum creatinine is not considered an acceptable endpoint for FDA requirements of drug registration, it is not yet an approved treatment1 . Many causes can lead to AKI such as severe dehydration, but the leading cause of AKI is ischemia renal injury (IRI) or renal ischemia/reperfusion injury which is unavoidable during kidney transplant3,4. During IRI, there are two distinct stages that lead to cell damage. The ischemia phase can be characterized as the inhibition of oxygen flow to the organ, which leads to the accumulation of metabolic intermediates. When reperfusion occurs and oxygen flow is restored to the organ, these intermediates react with oxygen to produce oxygen radicals, namely superoxide (O2 ˉ ) and hydrogen peroxide (H2O2) which leads to uncontrolled oxidation of cells. For example, during ischemia, ATP is catabolized into ADP and AMP, which leads to an abnormally high accumulation of hypoxanthine. When hypoxanthine reacts with oxygen of reperfusion, xanthine oxidase catabolizes hypoxanthine to xanthine which generates O2 ˉ , a free radical. Xanthine is further catabolized to uric acid by xanthine oxidase and more O2 ˉ is also generated, which causes excessive oxidative stress on cells5 . IRI can be reduced if certain reagents can suppress free radical generation or scavenge them. Allopurinol, an inhibitor of xanthine oxidase, which was used mainly for clinical treatment of gout may also have beneficial effects on ischemia-reperfused kidneys5, 6 . However, Bussmann et al. reported that Allopurinol did not exert protective effects on the kidneys of rats subjected to ischemia-reperfusion injury7 , Therefore, there is yet to be a universally successful result of using allopurinol to treat IRI. Superoxide dismutase (SOD), however, could be more effective in treating renal IRI, as it is an O2ˉ scavenger. In 1993, Pollak et al. found that post-operative renal function did not change when human recombinant SOD was administered immediately prior to renal allograft and 1 hour after it in a randomized double-blind trial8 . In 2001, Yin et al reported that Sod gene transduction minimized ischemia-reperfusion–induced acute renal failure9 . Therefore, SOD plays an inclusive role in renal IRI, as almost no findings have been published regarding the topic since. This also indicates the urgency of finding AKI interventions, as hundreds of thousands of patients experience this life-threatening illness every year. Vitamin B12 (B12, cobalamin) has been shown to exhibit a SOD mimetic activity which scavenges superoxide free radicals10. In a reverse direction, B12 deficiency has been found to result in lower SOD activity in livers of C57BL/6 mice11. In addition to its antioxidant effect, B12 has anti-inflammation and autophagy properties, as it is involved in the production of S-Adenosyl Methionine (SAM) 12, the universal donor to over 100 DNA, protein, and lipid methylation reactions13. More specifically, B12 is a cofactor for the conversion of homocysteine to methionine, which is then converted to SAM. Therefore, the addition of B12 reverses impaired autophagy caused by high concentration of homocysteine resulting from B12 deficiency in mouse astrocytes15 . SAM additionally inhibits the expression of tumor necrosis factor-alpha (TNFα), an inflammatory protein induced by lipopolysaccharides (LPS) in human leukocytes. Based on these previous findings, the first objective of my study was to test whether B12 has a beneficial effect on hypoxia/reperfusion injury and the mechanism(s) involved in the effect of this molecule in vitro."
Vitamin B12 Protects Against Hypoxia/Reperfusion Injury in Mouse Proximal Tubule Cells
Abstract
Acute kidney injury (AKI) is a common syndrome characterized by a sudden decline in kidney function that can potentially lead to death. Ischemia/reperfusion injury (IRI) is the leading cause of AKI and is inevitable during kidney transplants. There is no effective treatment available to treat IRI. Pathways involved in IRI are evidenced to lead to reactive oxygen species (ROS), inflammation, fibrosis, apoptosis, DNA damage response (DDR) and autophagy. Vitamin B12 (B12) or cobalamin, is essential for the human body and is pharmacologically known to scavenge ROS, suppressing inflammation and reverse impaired autophagy that occurs in B12 deficient conditions.
To test whether B12 has beneficial effects in IRI, I subjected cultured mouse proximal tubule cells (BU.MPT) to a hypoxia/reperfusion (H/R) procedure and measured transcription of markers for inflammation (Mcp1, Il6, Nos2) and fibrosis (fibronectin), protein markers for apoptosis(Tgf1, c-cap3), and DDR (p.H2AX) induced by hypoxia/reperfusion (H/R). Presence of B12 during the H/R procedure at 0.3µM dramatically inhibited the upregulation of these markers studied and to an increased cell survival. Together, my findings suggest that B12 is a highly promising molecule to prevent/treat AKI.
Introduction
Acute kidney injury (AKI) is a clinical condition that affects roughly 200,000 people every year in the United States1 . It is characterized by the sudden decline of renal function, leading to the dangerous accumulation of waste products and chemical imbalance in one’s blood2 . The disease progresses from cellular damage to decreased glomerular filtration rate, leading to kidney failure and in severe situations, it results in death2 . Despite its frequency and severity, there is limited treatment of AKI and most are still in the development stage. For example, animal studies of α-melanocyte– stimulating hormone (α-MSH) have confirmed its anti-inflammatory and anti-apoptotic activities to protect from AKI1 . However, because its reduction of serum creatinine is not considered an acceptable endpoint for FDA requirements of drug registration, it is not yet an approved treatment1 . Many causes can lead to AKI such as severe dehydration, but the leading cause of AKI is ischemia renal injury (IRI) or renal ischemia/reperfusion injury which is unavoidable during kidney transplant3,4. During IRI, there are two distinct stages that lead to cell damage. The ischemia phase can be characterized as the inhibition of oxygen flow to the organ, which leads to the accumulation of metabolic intermediates. When reperfusion occurs and oxygen flow is restored to the organ, these intermediates react with oxygen to produce oxygen radicals, namely superoxide (O2 ˉ ) and hydrogen peroxide (H2O2) which leads to uncontrolled oxidation of cells. For example, during ischemia, ATP is catabolized into ADP and AMP, which leads to an abnormally high accumulation of hypoxanthine. When hypoxanthine reacts with oxygen of reperfusion, xanthine oxidase catabolizes hypoxanthine to xanthine which generates O2 ˉ , a free radical. Xanthine is further catabolized to uric acid by xanthine oxidase and more O2 ˉ is also generated, which causes excessive oxidative stress on cells5 . IRI can be reduced if certain reagents can suppress free radical generation or scavenge them. Allopurinol, an inhibitor of xanthine oxidase, which was used mainly for clinical treatment of gout may also have beneficial effects on ischemia-reperfused kidneys5, 6 . However, Bussmann et al. reported that Allopurinol did not exert protective effects on the kidneys of rats subjected to ischemia-reperfusion injury7 , Therefore, there is yet to be a universally successful result of using allopurinol to treat IRI. Superoxide dismutase (SOD), however, could be more effective in treating renal IRI, as it is an O2ˉ scavenger. In 1993, Pollak et al. found that post-operative renal function did not change when human recombinant SOD was administered immediately prior to renal allograft and 1 hour after it in a randomized double-blind trial8 . In 2001, Yin et al reported that Sod gene transduction minimized ischemia-reperfusion–induced acute renal failure9 . Therefore, SOD plays an inclusive role in renal IRI, as almost no findings have been published regarding the topic since. This also indicates the urgency of finding AKI interventions, as hundreds of thousands of patients experience this life-threatening illness every year. Vitamin B12 (B12, cobalamin) has been shown to exhibit a SOD mimetic activity which scavenges superoxide free radicals10. In a reverse direction, B12 deficiency has been found to result in lower SOD activity in livers of C57BL/6 mice11. In addition to its antioxidant effect, B12 has anti-inflammation and autophagy properties, as it is involved in the production of S-Adenosyl Methionine (SAM) 12, the universal donor to over 100 DNA, protein, and lipid methylation reactions13. More specifically, B12 is a cofactor for the conversion of homocysteine to methionine, which is then converted to SAM. Therefore, the addition of B12 reverses impaired autophagy caused by high concentration of homocysteine resulting from B12 deficiency in mouse astrocytes15 . SAM additionally inhibits the expression of tumor necrosis factor-alpha (TNFα), an inflammatory protein induced by lipopolysaccharides (LPS) in human leukocytes. Based on these previous findings, the first objective of my study was to test whether B12 has a beneficial effect on hypoxia/reperfusion injury and the mechanism(s) involved in the effect of this molecule in vitro."
