Pub Date : 2024-07-01Epub Date: 2024-05-23DOI: 10.1152/ajprenal.00268.2023
Sai Sindhu Thangaraj, Christina S Oxlund, Henrik Andersen, Per Svenningsen, Jane Stubbe, Yaseelan Palarasah, Micaella Pereira Da Fonseca, Daniel F J Ketelhuth, Camilla Enggaard, Maria Høj Hansen, Jan Erik Henriksen, Ib Abildgaard Jacobsen, Boye L Jensen
Interleukin (IL)-17A contributes to hypertension in preclinical models. T helper 17 and dendritic cells are activated by NaCl, which could involve the epithelial Na+ channel (ENaC). We hypothesized that the ENaC blocker amiloride reduces plasma IL-17A and related cytokines in patients with hypertension. Concentrations of IL-17A, IFN-γ, TNF, IL-6, IL-1β, and IL-10 were determined by immunoassays in plasma from two patient cohorts before and after amiloride treatment: 1) patients with type 2 diabetes mellitus (T2DM) and treatment-resistant hypertension (n = 69, amiloride 5-10 mg/day for 8 wk) and 2) patients with hypertension and type 1 diabetes mellitus (T1DM) (n = 29) on standardized salt intake (amiloride 20-40 mg/day, 2 days). Plasma and tissue from ANG II-hypertensive mice with T1DM treated with amiloride (2 mg/kg/day, 4 days) were analyzed. The effect of amiloride and benzamil on macrophage cytokines was determined in vitro. Plasma cytokines showed higher concentrations (IL-17A ∼40-fold) in patients with T2DM compared with T1DM. In patients with T2DM, amiloride had no effect on IL-17A but lowered TNF and IL-6. In patients with T1DM, amiloride had no effect on IL-17A but increased TNF. In both cohorts, blood pressure decline and plasma K+ increase did not relate to plasma cytokine changes. In mice, amiloride exerted no effect on IL-17A in the plasma, kidney, aorta, or left cardiac ventricle but increased TNF in cardiac and kidney tissues. In lipopolysaccharide-stimulated human THP-1 macrophages, amiloride and benzamil (from 1 nmol/L) decreased TNF, IL-6, IL-10, and IL-1β. In conclusion, inhibition of ENaC by amiloride reduces proinflammatory cytokines TNF and IL-6 but not IL-17A in patients with T2DM, potentially by a direct action on macrophages.NEW & NOTEWORTHY ENaC activity may contribute to macrophage-derived cytokine release, since amiloride exerts anti-inflammatory effects by suppression of TNF and IL-6 cytokines in patients with resistant hypertension and type 2 diabetes and in THP-1-derived macrophages in vitro.
{"title":"Amiloride lowers plasma TNF and interleukin-6 but not interleukin-17A in patients with hypertension and type 2 diabetes.","authors":"Sai Sindhu Thangaraj, Christina S Oxlund, Henrik Andersen, Per Svenningsen, Jane Stubbe, Yaseelan Palarasah, Micaella Pereira Da Fonseca, Daniel F J Ketelhuth, Camilla Enggaard, Maria Høj Hansen, Jan Erik Henriksen, Ib Abildgaard Jacobsen, Boye L Jensen","doi":"10.1152/ajprenal.00268.2023","DOIUrl":"10.1152/ajprenal.00268.2023","url":null,"abstract":"<p><p>Interleukin (IL)-17A contributes to hypertension in preclinical models. T helper 17 and dendritic cells are activated by NaCl, which could involve the epithelial Na<sup>+</sup> channel (ENaC). We hypothesized that the ENaC blocker amiloride reduces plasma IL-17A and related cytokines in patients with hypertension. Concentrations of IL-17A, IFN-γ, TNF, IL-6, IL-1β, and IL-10 were determined by immunoassays in plasma from two patient cohorts before and after amiloride treatment: <i>1</i>) patients with type 2 diabetes mellitus (T2DM) and treatment-resistant hypertension (<i>n</i> = 69, amiloride 5-10 mg/day for 8 wk) and <i>2</i>) patients with hypertension and type 1 diabetes mellitus (T1DM) (<i>n</i> = 29) on standardized salt intake (amiloride 20-40 mg/day, 2 days). Plasma and tissue from ANG II-hypertensive mice with T1DM treated with amiloride (2 mg/kg/day, 4 days) were analyzed. The effect of amiloride and benzamil on macrophage cytokines was determined in vitro. Plasma cytokines showed higher concentrations (IL-17A ∼40-fold) in patients with T2DM compared with T1DM. In patients with T2DM, amiloride had no effect on IL-17A but lowered TNF and IL-6. In patients with T1DM, amiloride had no effect on IL-17A but increased TNF. In both cohorts, blood pressure decline and plasma K<sup>+</sup> increase did not relate to plasma cytokine changes. In mice, amiloride exerted no effect on IL-17A in the plasma, kidney, aorta, or left cardiac ventricle but increased TNF in cardiac and kidney tissues. In lipopolysaccharide-stimulated human THP-1 macrophages, amiloride and benzamil (from 1 nmol/L) decreased TNF, IL-6, IL-10, and IL-1β. In conclusion, inhibition of ENaC by amiloride reduces proinflammatory cytokines TNF and IL-6 but not IL-17A in patients with T2DM, potentially by a direct action on macrophages.<b>NEW & NOTEWORTHY</b> ENaC activity may contribute to macrophage-derived cytokine release, since amiloride exerts anti-inflammatory effects by suppression of TNF and IL-6 cytokines in patients with resistant hypertension and type 2 diabetes and in THP-1-derived macrophages in vitro.</p>","PeriodicalId":93867,"journal":{"name":"American journal of physiology. Renal physiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141081867","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-01Epub Date: 2024-05-23DOI: 10.1152/ajprenal.00029.2024
Cole S Hudson, Anirban Roy, Qingtian Li, Aniket S Joshi, Taijun Yin, Ashok Kumar, David Sheikh-Hamad, Vincent H Tam
Polymyxins are a last-resort treatment option for multidrug-resistant gram-negative bacterial infections, but they are associated with nephrotoxicity. Gelofusine was previously shown to reduce polymyxin-associated kidney injury in an animal model. However, the mechanism(s) of renal protection has not been fully elucidated. Here, we report the use of a cell culture model to provide insights into the mechanisms of renal protection. Murine epithelial proximal tubular cells were exposed to polymyxin B. Cell viability, lactate dehydrogenase (LDH) release, polymyxin B uptake, mitochondrial superoxide production, nuclear morphology, and apoptosis activation were evaluated with or without concomitant gelofusine. A megalin knockout cell line was used as an uptake inhibition control. Methionine was included in selected experiments as an antioxidant control. A polymyxin B concentration-dependent reduction in cell viability was observed. Increased viability was observed in megalin knockout cells following comparable polymyxin B exposures. Compared with polymyxin B exposure alone, concomitant gelofusine significantly increased cell viability as well as reduced LDH release, polymyxin B uptake, mitochondrial superoxide, and apoptosis. Gelofusine and methionine were more effective at reducing renal cell injury in combination than either agent alone. In conclusion, the mechanisms of renal protection by gelofusine involve decreasing cellular drug uptake, reducing subsequent oxidative stress and apoptosis activation. These findings would be valuable for translational research into clinical strategies to attenuate drug-associated acute kidney injury.NEW & NOTEWORTHY Gelofusine is a gelatinous saline solution with the potential to attenuate polymyxin-associated nephrotoxicity. We demonstrated that the mechanisms of gelofusine renal protection involve reducing polymyxin B uptake by proximal tubule cells, limiting subsequent oxidative stress and apoptosis activation. In addition, gelofusine was more effective at reducing cellular injury than a known antioxidant control, methionine, and a megalin knockout cell line, indicating that gelofusine likely has additional pharmacological properties besides only megalin inhibition.
导言:多粘菌素是治疗耐多药革兰氏阴性菌感染的最后选择,但它与肾毒性有关。之前有研究表明,在动物模型中,Gelofusine 可减轻多粘菌素相关的肾损伤。然而,肾脏保护机制尚未完全阐明。在此,我们报告了利用细胞培养模型深入了解肾脏保护机制的情况:细胞存活率、多粘菌素 B 吸收、线粒体超氧化物产生、核形态和细胞凋亡活化均在同时使用或不使用凝胶磷脂的情况下进行评估。用巨球蛋白剔除细胞系作为摄取抑制对照。在选定的实验中加入蛋氨酸作为抗氧化剂对照:结果:观察到多粘菌素 B 浓度依赖性降低细胞活力。在暴露于类似的多粘菌素 B 后,观察到megalin 基因剔除细胞的活力增加。与单独暴露于多粘菌素 B 相比,同时暴露于凝胶磷脂和蛋氨酸可显著提高细胞活力,减少线粒体超氧化物的产生,并改善细胞核形态。凝胶磷脂(而非蛋氨酸)能明显减少多粘菌素 B 的吸收和 Bax/Bcl-2 蛋白的比率(内在凋亡的生物标志物)。与单独使用两种药物相比,明胶和蛋氨酸联合使用能更有效地减轻肾细胞损伤:结论:格列福辛对肾脏的保护机制包括降低细胞对药物的吸收、减少随后的氧化应激和细胞凋亡激活。这些发现对于将研究成果转化为减轻药物相关急性肾损伤的临床策略很有价值。
{"title":"Mechanisms of gelofusine protection in an in vitro model of polymyxin B-associated renal injury.","authors":"Cole S Hudson, Anirban Roy, Qingtian Li, Aniket S Joshi, Taijun Yin, Ashok Kumar, David Sheikh-Hamad, Vincent H Tam","doi":"10.1152/ajprenal.00029.2024","DOIUrl":"10.1152/ajprenal.00029.2024","url":null,"abstract":"<p><p>Polymyxins are a last-resort treatment option for multidrug-resistant gram-negative bacterial infections, but they are associated with nephrotoxicity. Gelofusine was previously shown to reduce polymyxin-associated kidney injury in an animal model. However, the mechanism(s) of renal protection has not been fully elucidated. Here, we report the use of a cell culture model to provide insights into the mechanisms of renal protection. Murine epithelial proximal tubular cells were exposed to polymyxin B. Cell viability, lactate dehydrogenase (LDH) release, polymyxin B uptake, mitochondrial superoxide production, nuclear morphology, and apoptosis activation were evaluated with or without concomitant gelofusine. A megalin knockout cell line was used as an uptake inhibition control. Methionine was included in selected experiments as an antioxidant control. A polymyxin B concentration-dependent reduction in cell viability was observed. Increased viability was observed in megalin knockout cells following comparable polymyxin B exposures. Compared with polymyxin B exposure alone, concomitant gelofusine significantly increased cell viability as well as reduced LDH release, polymyxin B uptake, mitochondrial superoxide, and apoptosis. Gelofusine and methionine were more effective at reducing renal cell injury in combination than either agent alone. In conclusion, the mechanisms of renal protection by gelofusine involve decreasing cellular drug uptake, reducing subsequent oxidative stress and apoptosis activation. These findings would be valuable for translational research into clinical strategies to attenuate drug-associated acute kidney injury.<b>NEW & NOTEWORTHY</b> Gelofusine is a gelatinous saline solution with the potential to attenuate polymyxin-associated nephrotoxicity. We demonstrated that the mechanisms of gelofusine renal protection involve reducing polymyxin B uptake by proximal tubule cells, limiting subsequent oxidative stress and apoptosis activation. In addition, gelofusine was more effective at reducing cellular injury than a known antioxidant control, methionine, and a megalin knockout cell line, indicating that gelofusine likely has additional pharmacological properties besides only megalin inhibition.</p>","PeriodicalId":93867,"journal":{"name":"American journal of physiology. Renal physiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141082172","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-01Epub Date: 2024-05-23DOI: 10.1152/ajprenal.00067.2024
Mikhail Burmakin, Peter S Gilmour, Magnus Gram, Nelli Shushakova, Ruben M Sandoval, Bruce A Molitoris, Tobias E Larsson
α-1-Microglobulin (A1M) is a circulating glycoprotein with antioxidant, heme-binding, and mitochondrial protection properties. The investigational drug RMC-035, a modified therapeutic A1M protein, was assessed for biodistribution and pharmacological activity in a broad set of in vitro and in vivo experiments, supporting its clinical development. Efficacy and treatment posology were assessed in various models of kidney ischemia and reperfusion injury (IRI). Real-time glomerular filtration rate (GFR), functional renal biomarkers, tubular injury biomarkers (NGAL and KIM-1), and histopathology were evaluated. Fluorescently labeled RMC-035 was used to assess biodistribution. RMC-035 demonstrated consistent and reproducible kidney protection in rat IRI models as well as in a model of IRI imposed on renal impairment and in a mouse IRI model, where it reduced mortality. Its pharmacological activity was most pronounced with combined dosing pre- and post-ischemia and weaker with either pre- or post-ischemia dosing alone. RMC-035 rapidly distributed to the kidneys via glomerular filtration and selective luminal uptake by proximal tubular cells. IRI-induced expression of kidney heme oxygenase-1 was inhibited by RMC-035, consistent with its antioxidative properties. RMC-035 also dampened IRI-associated inflammation and improved mitochondrial function, as shown by tubular autofluorescence. Taken together, the efficacy of RMC-035 is congruent with its targeted mechanism(s) and biodistribution profile, supporting its further clinical evaluation as a novel kidney-protective therapy.NEW & NOTEWORTHY A therapeutic A1M protein variant (RMC-035) is currently in phase 2 clinical development for renal protection in patients undergoing open-chest cardiac surgery. It targets several key pathways underlying kidney injury in this patient group, including oxidative stress, heme toxicity, and mitochondrial dysfunction. RMC-035 is rapidly eliminated from plasma, distributing to kidney proximal tubules, and demonstrates dose-dependent efficacy in numerous models of ischemia-reperfusion injury, particularly when administered before ischemia. These results support its continued clinical evaluation.
α-1-微球蛋白(A1M)是一种循环糖蛋白,具有抗氧化、血红素结合和线粒体保护特性。研究药物 RMC-035 是一种改良的治疗用 A1M 蛋白,在一系列广泛的体外和体内实验中对其生物分布和药理活性进行了评估,为其临床开发提供了支持。在各种肾缺血和再灌注损伤(IRI)模型中评估了药效和治疗效果。对实时肾小球滤过率、肾功能生物标志物、肾小管损伤生物标志物(NGAL 和 KIM-1)和组织病理学进行了评估。荧光标记的 RMC-035 用于评估生物分布。RMC-035 在大鼠 IRI 模型、强加于肾功能损伤的 IRI 模型和小鼠 IRI 模型中均表现出一致且可重复的肾脏保护作用,并降低了死亡率。缺血前后联合用药的药理活性最明显,而缺血前后单独用药的药理活性较弱。RMC-035 通过肾小球滤过和近端肾小管细胞的选择性管腔摄取迅速分布到肾脏。RMC-035 可抑制 IRI 诱导的肾脏血红素加氧酶-1 的表达,这与其抗氧化特性是一致的。RMC-035 还能抑制 IRI 引起的炎症,并通过肾小管自发荧光改善线粒体功能。总之,RMC-035 的疗效与其靶向机制和生物分布特征相一致,支持将其作为一种新型肾脏保护疗法进行进一步的临床评估。
{"title":"Therapeutic α-1-microglobulin ameliorates kidney ischemia-reperfusion injury.","authors":"Mikhail Burmakin, Peter S Gilmour, Magnus Gram, Nelli Shushakova, Ruben M Sandoval, Bruce A Molitoris, Tobias E Larsson","doi":"10.1152/ajprenal.00067.2024","DOIUrl":"10.1152/ajprenal.00067.2024","url":null,"abstract":"<p><p>α-1-Microglobulin (A1M) is a circulating glycoprotein with antioxidant, heme-binding, and mitochondrial protection properties. The investigational drug RMC-035, a modified therapeutic A1M protein, was assessed for biodistribution and pharmacological activity in a broad set of in vitro and in vivo experiments, supporting its clinical development. Efficacy and treatment posology were assessed in various models of kidney ischemia and reperfusion injury (IRI). Real-time glomerular filtration rate (GFR), functional renal biomarkers, tubular injury biomarkers (NGAL and KIM-1), and histopathology were evaluated. Fluorescently labeled RMC-035 was used to assess biodistribution. RMC-035 demonstrated consistent and reproducible kidney protection in rat IRI models as well as in a model of IRI imposed on renal impairment and in a mouse IRI model, where it reduced mortality. Its pharmacological activity was most pronounced with combined dosing pre- and post-ischemia and weaker with either pre- or post-ischemia dosing alone. RMC-035 rapidly distributed to the kidneys via glomerular filtration and selective luminal uptake by proximal tubular cells. IRI-induced expression of kidney heme oxygenase-1 was inhibited by RMC-035, consistent with its antioxidative properties. RMC-035 also dampened IRI-associated inflammation and improved mitochondrial function, as shown by tubular autofluorescence. Taken together, the efficacy of RMC-035 is congruent with its targeted mechanism(s) and biodistribution profile, supporting its further clinical evaluation as a novel kidney-protective therapy.<b>NEW & NOTEWORTHY</b> A therapeutic A1M protein variant (RMC-035) is currently in phase 2 clinical development for renal protection in patients undergoing open-chest cardiac surgery. It targets several key pathways underlying kidney injury in this patient group, including oxidative stress, heme toxicity, and mitochondrial dysfunction. RMC-035 is rapidly eliminated from plasma, distributing to kidney proximal tubules, and demonstrates dose-dependent efficacy in numerous models of ischemia-reperfusion injury, particularly when administered before ischemia. These results support its continued clinical evaluation.</p>","PeriodicalId":93867,"journal":{"name":"American journal of physiology. Renal physiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141082188","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Diabetes is closely associated with K+ disturbances during disease progression and treatment. However, it remains unclear whether K+ imbalance occurs in diabetes with normal kidney function. In this study, we examined the effects of dietary K+ intake on systemic K+ balance and renal K+ handling in streptozotocin (STZ)-induced diabetic mice. The control and STZ mice were fed low or high K+ diet for 7 days to investigate the role of dietary K+ intake in renal K+ excretion and K+ homeostasis and to explore the underlying mechanism by evaluating K+ secretion-related transport proteins in distal nephrons. K+-deficient diet caused excessive urinary K+ loss, decreased daily K+ balance, and led to severe hypokalemia in STZ mice compared with control mice. In contrast, STZ mice showed an increased daily K+ balance and elevated plasma K+ level under K+-loading conditions. Dysregulation of the NaCl cotransporter (NCC), epithelial Na+ channel (ENaC), and renal outer medullary K+ channel (ROMK) was observed in diabetic mice fed either low or high K+ diet. Moreover, amiloride treatment reduced urinary K+ excretion and corrected hypokalemia in K+-restricted STZ mice. On the other hand, inhibition of SGLT2 by dapagliflozin promoted urinary K+ excretion and normalized plasma K+ levels in K+-supplemented STZ mice, at least partly by increasing ENaC activity. We conclude that STZ mice exhibited abnormal K+ balance and impaired renal K+ handling under either low or high K+ diet, which could be primarily attributed to the dysfunction of ENaC-dependent renal K+ excretion pathway, despite the possible role of NCC.NEW & NOTEWORTHY Neither low dietary K+ intake nor high dietary K+ intake effectively modulates renal K+ excretion and K+ homeostasis in STZ mice, which is closely related to the abnormality of ENaC expression and activity. SGLT2 inhibitor increases urinary K+ excretion and reduces plasma K+ level in STZ mice under high dietary K+ intake, an effect that may be partly due to the upregulation of ENaC activity.
{"title":"Impaired distal renal potassium handling in streptozotocin-induced diabetic mice.","authors":"Peng Wu, Shu-Ting Li, Ting-Ting Shu, Zi-Hui Mao, Wen-Jia Fu, Yuan-Yuan Yang, Shao-Kang Pan, Dong-Wei Liu, Zhang-Suo Liu, Zhong-Xiuzi Gao","doi":"10.1152/ajprenal.00240.2023","DOIUrl":"10.1152/ajprenal.00240.2023","url":null,"abstract":"<p><p>Diabetes is closely associated with K<sup>+</sup> disturbances during disease progression and treatment. However, it remains unclear whether K<sup>+</sup> imbalance occurs in diabetes with normal kidney function. In this study, we examined the effects of dietary K<sup>+</sup> intake on systemic K<sup>+</sup> balance and renal K<sup>+</sup> handling in streptozotocin (STZ)-induced diabetic mice. The control and STZ mice were fed low or high K<sup>+</sup> diet for 7 days to investigate the role of dietary K<sup>+</sup> intake in renal K<sup>+</sup> excretion and K<sup>+</sup> homeostasis and to explore the underlying mechanism by evaluating K<sup>+</sup> secretion-related transport proteins in distal nephrons. K<sup>+</sup>-deficient diet caused excessive urinary K<sup>+</sup> loss, decreased daily K<sup>+</sup> balance, and led to severe hypokalemia in STZ mice compared with control mice. In contrast, STZ mice showed an increased daily K<sup>+</sup> balance and elevated plasma K<sup>+</sup> level under K<sup>+</sup>-loading conditions. Dysregulation of the NaCl cotransporter (NCC), epithelial Na<sup>+</sup> channel (ENaC), and renal outer medullary K<sup>+</sup> channel (ROMK) was observed in diabetic mice fed either low or high K<sup>+</sup> diet. Moreover, amiloride treatment reduced urinary K<sup>+</sup> excretion and corrected hypokalemia in K<sup>+</sup>-restricted STZ mice. On the other hand, inhibition of SGLT2 by dapagliflozin promoted urinary K<sup>+</sup> excretion and normalized plasma K<sup>+</sup> levels in K<sup>+</sup>-supplemented STZ mice, at least partly by increasing ENaC activity. We conclude that STZ mice exhibited abnormal K<sup>+</sup> balance and impaired renal K<sup>+</sup> handling under either low or high K<sup>+</sup> diet, which could be primarily attributed to the dysfunction of ENaC-dependent renal K<sup>+</sup> excretion pathway, despite the possible role of NCC.<b>NEW & NOTEWORTHY</b> Neither low dietary K<sup>+</sup> intake nor high dietary K<sup>+</sup> intake effectively modulates renal K<sup>+</sup> excretion and K<sup>+</sup> homeostasis in STZ mice, which is closely related to the abnormality of ENaC expression and activity. SGLT2 inhibitor increases urinary K<sup>+</sup> excretion and reduces plasma K<sup>+</sup> level in STZ mice under high dietary K<sup>+</sup> intake, an effect that may be partly due to the upregulation of ENaC activity.</p>","PeriodicalId":93867,"journal":{"name":"American journal of physiology. Renal physiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141081905","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-01Epub Date: 2024-05-23DOI: 10.1152/ajprenal.00043.2024
James D Odum, Juheb Akhter, Vivek Verma, Giacynta Vollmer, Ahmad Davidson, Kelly A Hyndman, Subhashini Bolisetty
Sepsis-associated acute kidney injury (SA-AKI) is a key contributor to the life-threatening sequelae attributed to sepsis. Mechanistically, SA-AKI is a consequence of unabated myeloid cell activation and oxidative stress that induces tubular injury. Iron mediates inflammatory pathways directly and through regulating the expression of myeloid-derived ferritin, an iron storage protein comprising ferritin light (FtL) and ferritin heavy chain (FtH) subunits. Previous work revealed that myeloid FtH deletion leads to a compensatory increase in intracellular and circulating FtL and is associated with amelioration of SA-AKI. We designed this study to test the hypothesis that loss of myeloid FtL and subsequently, circulating FtL will exacerbate the sepsis-induced inflammatory response and worsen SA-AKI. We generated a novel myeloid-specific FtL knockout mouse (FtLLysM-/-) and induced sepsis via cecal ligation and puncture or lipopolysaccharide endotoxemia. As expected, serum ferritin levels were significantly lower in the knockout mice, suggesting that myeloid cells dominantly contribute to circulating ferritin. Interestingly, although sepsis induction led to a marked production of pro- and anti-inflammatory cytokines, there was no statistical difference between the genotypes. There was a similar loss of kidney function, as evidenced by a rise in serum creatinine and cystatin C and renal injury identified by expression of kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin. Finally, RNA sequencing revealed upregulation of pathways for cell cycle arrest and autophagy postsepsis, but no significant differences were observed between genotypes, including in key genes associated with ferroptosis, an iron-mediated form of cell death. The loss of FtL did not impact sepsis-mediated activation of NF-κB or HIF-1a signaling, key inflammatory pathways associated with dysregulated host response. Taken together, while FtL overexpression was shown to be protective against sepsis, the loss of FtL did not influence sepsis pathogenesis.NEW & NOTEWORTHY Hyperferritinemia in sepsis is often associated with a proinflammatory phenotype and poor prognosis. We previously showed the myeloid deletion of FtH results in a compensatory increase in FtL and is associated with reduced circulating cytokines and decreased rates of SA-AKI in animal sepsis models. Here, we show that myeloid deletion of FtL does not impact the severity of SA-AKI following CLP or LPS, suggesting that FtH plays the predominant role in propagating myeloid-induced proinflammatory pathways.
{"title":"Myeloid-specific ferritin light chain deletion does not exacerbate sepsis-associated AKI.","authors":"James D Odum, Juheb Akhter, Vivek Verma, Giacynta Vollmer, Ahmad Davidson, Kelly A Hyndman, Subhashini Bolisetty","doi":"10.1152/ajprenal.00043.2024","DOIUrl":"10.1152/ajprenal.00043.2024","url":null,"abstract":"<p><p>Sepsis-associated acute kidney injury (SA-AKI) is a key contributor to the life-threatening sequelae attributed to sepsis. Mechanistically, SA-AKI is a consequence of unabated myeloid cell activation and oxidative stress that induces tubular injury. Iron mediates inflammatory pathways directly and through regulating the expression of myeloid-derived ferritin, an iron storage protein comprising ferritin light (FtL) and ferritin heavy chain (FtH) subunits. Previous work revealed that myeloid FtH deletion leads to a compensatory increase in intracellular and circulating FtL and is associated with amelioration of SA-AKI. We designed this study to test the hypothesis that loss of myeloid FtL and subsequently, circulating FtL will exacerbate the sepsis-induced inflammatory response and worsen SA-AKI. We generated a novel myeloid-specific FtL knockout mouse (FtL<sup>LysM-/-</sup>) and induced sepsis via cecal ligation and puncture or lipopolysaccharide endotoxemia. As expected, serum ferritin levels were significantly lower in the knockout mice, suggesting that myeloid cells dominantly contribute to circulating ferritin. Interestingly, although sepsis induction led to a marked production of pro- and anti-inflammatory cytokines, there was no statistical difference between the genotypes. There was a similar loss of kidney function, as evidenced by a rise in serum creatinine and cystatin C and renal injury identified by expression of kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin. Finally, RNA sequencing revealed upregulation of pathways for cell cycle arrest and autophagy postsepsis, but no significant differences were observed between genotypes, including in key genes associated with ferroptosis, an iron-mediated form of cell death. The loss of FtL did not impact sepsis-mediated activation of NF-κB or HIF-1a signaling, key inflammatory pathways associated with dysregulated host response. Taken together, while FtL overexpression was shown to be protective against sepsis, the loss of FtL did not influence sepsis pathogenesis.<b>NEW & NOTEWORTHY</b> Hyperferritinemia in sepsis is often associated with a proinflammatory phenotype and poor prognosis. We previously showed the myeloid deletion of FtH results in a compensatory increase in FtL and is associated with reduced circulating cytokines and decreased rates of SA-AKI in animal sepsis models. Here, we show that myeloid deletion of FtL does not impact the severity of SA-AKI following CLP or LPS, suggesting that FtH plays the predominant role in propagating myeloid-induced proinflammatory pathways.</p>","PeriodicalId":93867,"journal":{"name":"American journal of physiology. Renal physiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141082186","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-01Epub Date: 2024-05-09DOI: 10.1152/ajprenal.00001.2024
Shervin Hakimi, Pritha Dutta, Anita T Layton
Pregnancy is associated with elevated demand of most nutrients, with many trace elements and minerals critical for the development of fetus. In particular, calcium (Ca2+) and magnesium (Mg2+) are essential for cellular function, and their deficiency can lead to impaired fetal growth. A key contributor to the homeostasis of these ions is the kidney, which in a pregnant rat undergoes major changes in morphology, hemodynamics, and molecular structure. The goal of this study is to unravel the functional implications of these pregnancy-induced changes in renal handling of Ca2+ and Mg2+, two cations that are essential in a healthy pregnancy. To achieve that goal, we developed computational models of electrolyte and water transport along the nephrons of a rat in mid and late pregnancy. Model simulations reveal a substantial increase in the reabsorption of Mg2+ along the proximal tubules and thick ascending limbs. In contrast, the reabsorption of Ca2+ is increased in the proximal tubules but decreased in the thick ascending limbs, due to the lower transepithelial concentration gradient of Ca2+ along the latter. Despite the enhanced transport capacity, the marked increase in glomerular filtration rate results in elevated urinary excretions of Ca2+ and Mg2+ in pregnancy. Furthermore, we conducted simulations of hypocalcemia and hypomagnesemia. We found that hypocalcemia lowers Ca2+ excretion substantially more than Mg2+ excretion, with this effect being more pronounced in virgin rats than in pregnant ones. Conversely, hypomagnesemia reduces the excretion of Mg2+ and Ca2+ to more similar degrees. These differences can be explained by the greater sensitivity of the calcium-sensing receptor (CaSR) to Ca2+ compared with Mg2+.NEW & NOTEWORTHY A growing fetus' demands of minerals, notably calcium and magnesium, necessitate adaptations in pregnancy. In particular, the kidney undergoes major changes in morphology, hemodynamics, and molecular structure. This computational modeling study provides insights into how these pregnancy-induced renal adaptation impact calcium and magnesium transport along different nephron segments. Model simulations indicate that, despite the enhanced transport capacity, the marked increase in glomerular filtration rate results in elevated urinary excretions of calcium and magnesium in pregnancy.
{"title":"Renal calcium and magnesium handling during pregnancy: modeling and analysis.","authors":"Shervin Hakimi, Pritha Dutta, Anita T Layton","doi":"10.1152/ajprenal.00001.2024","DOIUrl":"10.1152/ajprenal.00001.2024","url":null,"abstract":"<p><p>Pregnancy is associated with elevated demand of most nutrients, with many trace elements and minerals critical for the development of fetus. In particular, calcium (Ca<sup>2+</sup>) and magnesium (Mg<sup>2+</sup>) are essential for cellular function, and their deficiency can lead to impaired fetal growth. A key contributor to the homeostasis of these ions is the kidney, which in a pregnant rat undergoes major changes in morphology, hemodynamics, and molecular structure. The goal of this study is to unravel the functional implications of these pregnancy-induced changes in renal handling of Ca<sup>2+</sup> and Mg<sup>2+</sup>, two cations that are essential in a healthy pregnancy. To achieve that goal, we developed computational models of electrolyte and water transport along the nephrons of a rat in mid and late pregnancy. Model simulations reveal a substantial increase in the reabsorption of Mg<sup>2+</sup> along the proximal tubules and thick ascending limbs. In contrast, the reabsorption of Ca<sup>2+</sup> is increased in the proximal tubules but decreased in the thick ascending limbs, due to the lower transepithelial concentration gradient of Ca<sup>2+</sup> along the latter. Despite the enhanced transport capacity, the marked increase in glomerular filtration rate results in elevated urinary excretions of Ca<sup>2+</sup> and Mg<sup>2+</sup> in pregnancy. Furthermore, we conducted simulations of hypocalcemia and hypomagnesemia. We found that hypocalcemia lowers Ca<sup>2+</sup> excretion substantially more than Mg<sup>2+</sup> excretion, with this effect being more pronounced in virgin rats than in pregnant ones. Conversely, hypomagnesemia reduces the excretion of Mg<sup>2+</sup> and Ca<sup>2+</sup> to more similar degrees. These differences can be explained by the greater sensitivity of the calcium-sensing receptor (CaSR) to Ca<sup>2+</sup> compared with Mg<sup>2+</sup>.<b>NEW & NOTEWORTHY</b> A growing fetus' demands of minerals, notably calcium and magnesium, necessitate adaptations in pregnancy. In particular, the kidney undergoes major changes in morphology, hemodynamics, and molecular structure. This computational modeling study provides insights into how these pregnancy-induced renal adaptation impact calcium and magnesium transport along different nephron segments. Model simulations indicate that, despite the enhanced transport capacity, the marked increase in glomerular filtration rate results in elevated urinary excretions of calcium and magnesium in pregnancy.</p>","PeriodicalId":93867,"journal":{"name":"American journal of physiology. Renal physiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140891524","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-01Epub Date: 2024-05-09DOI: 10.1152/ajprenal.00426.2023
Maya Brewer, Lukasz G Migas, Kelly A Clouthier, Jamie L Allen, David M Anderson, Ellie Pingry, Melissa Farrow, Ellen M Quardokus, Jeffrey M Spraggins, Raf Van de Plas, Mark P de Caestecker
The lack of standardization in antibody validation remains a major contributor to irreproducibility of human research. To address this, we have applied a standardized approach to validate a panel of antibodies to identify 18 major cell types and 5 extracellular matrix compartments in the human kidney by immunofluorescence (IF) microscopy. We have used these to generate an organ mapping antibody panel for two-dimensional (2-D) and three-dimensional (3-D) cyclical IF (CyCIF) to provide a more detailed method for evaluating tissue segmentation and volumes using a larger panel of markers than would normally be possible using standard fluorescence microscopy. CyCIF also makes it possible to perform multiplexed IF microscopy of whole slide images, which is a distinct advantage over other multiplexed imaging technologies that are applicable to limited fields of view. This enables a broader view of cell distributions across larger anatomical regions, allowing a better chance to capture localized regions of dysfunction in diseased tissues. These methods are broadly accessible to any laboratory with a fluorescence microscope, enabling spatial cellular phenotyping in normal and disease states. We also provide a detailed solution for image alignment between CyCIF cycles that can be used by investigators to perform these studies without programming experience using open-sourced software. This ability to perform multiplexed imaging without specialized instrumentation or computational skills opens the door to integration with more highly dimensional molecular imaging modalities such as spatial transcriptomics and imaging mass spectrometry, enabling the discovery of molecular markers of specific cell types, and how these are altered in disease.NEW & NOTEWORTHY We describe here validation criteria used to define on organ mapping panel of antibodies that can be used to define 18 cell types and five extracellular matrix compartments using cyclical immunofluorescence (CyCIF) microscopy. As CyCIF does not require specialized instrumentation, and image registration required to assemble CyCIF images can be performed by any laboratory without specialized computational skills, this technology is accessible to any laboratory with access to a fluorescence microscope and digital scanner.
{"title":"Validation of an organ mapping antibody panel for cyclical immunofluorescence microscopy on normal human kidneys.","authors":"Maya Brewer, Lukasz G Migas, Kelly A Clouthier, Jamie L Allen, David M Anderson, Ellie Pingry, Melissa Farrow, Ellen M Quardokus, Jeffrey M Spraggins, Raf Van de Plas, Mark P de Caestecker","doi":"10.1152/ajprenal.00426.2023","DOIUrl":"10.1152/ajprenal.00426.2023","url":null,"abstract":"<p><p>The lack of standardization in antibody validation remains a major contributor to irreproducibility of human research. To address this, we have applied a standardized approach to validate a panel of antibodies to identify 18 major cell types and 5 extracellular matrix compartments in the human kidney by immunofluorescence (IF) microscopy. We have used these to generate an organ mapping antibody panel for two-dimensional (2-D) and three-dimensional (3-D) cyclical IF (CyCIF) to provide a more detailed method for evaluating tissue segmentation and volumes using a larger panel of markers than would normally be possible using standard fluorescence microscopy. CyCIF also makes it possible to perform multiplexed IF microscopy of whole slide images, which is a distinct advantage over other multiplexed imaging technologies that are applicable to limited fields of view. This enables a broader view of cell distributions across larger anatomical regions, allowing a better chance to capture localized regions of dysfunction in diseased tissues. These methods are broadly accessible to any laboratory with a fluorescence microscope, enabling spatial cellular phenotyping in normal and disease states. We also provide a detailed solution for image alignment between CyCIF cycles that can be used by investigators to perform these studies without programming experience using open-sourced software. This ability to perform multiplexed imaging without specialized instrumentation or computational skills opens the door to integration with more highly dimensional molecular imaging modalities such as spatial transcriptomics and imaging mass spectrometry, enabling the discovery of molecular markers of specific cell types, and how these are altered in disease.<b>NEW & NOTEWORTHY</b> We describe here validation criteria used to define on organ mapping panel of antibodies that can be used to define 18 cell types and five extracellular matrix compartments using cyclical immunofluorescence (CyCIF) microscopy. As CyCIF does not require specialized instrumentation, and image registration required to assemble CyCIF images can be performed by any laboratory without specialized computational skills, this technology is accessible to any laboratory with access to a fluorescence microscope and digital scanner.</p>","PeriodicalId":93867,"journal":{"name":"American journal of physiology. Renal physiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11390132/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140891759","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-01Epub Date: 2024-03-28DOI: 10.1152/ajprenal.00014.2024
Tjessa Bondue, Laleh Khodaparast, Ladan Khodaparast, Sara Cairoli, Bianca Maria Goffredo, Rik Gijsbers, Lambertus van den Heuvel, Elena Levtchenko
Cystinosis is an autosomal recessive lysosomal storage disorder, caused by mutations in the CTNS gene, resulting in an absent or altered cystinosin (CTNS) protein. Cystinosin exports cystine out of the lysosome, with a malfunction resulting in cystine accumulation and a defect in other cystinosin-mediated pathways. Cystinosis is a systemic disease, but the kidneys are the first and most severely affected organs. In the kidney, the disease initially manifests as a generalized dysfunction in the proximal tubules (also called renal Fanconi syndrome). MFSD12 is a lysosomal cysteine importer that directly affects the cystine levels in melanoma cells, HEK293T cells, and cystinosis patient-derived fibroblasts. In this study, we aimed to evaluate MFSD12 mRNA levels in cystinosis patient-derived proximal tubular epithelial cells (ciPTECs) and to study the effect of MFSD12 knockout on cystine levels. We showed similar MFSD12 mRNA expression in patient-derived ciPTECs in comparison with the control cells. CRISPR MFSD12 knockout in a patient-derived ciPTEC (CTNSΔ57kb) resulted in significantly reduced cystine levels. Furthermore, we evaluated proximal tubular reabsorption after injection of mfsd12a translation-blocking morpholino (TB MO) in a ctns-/- zebrafish model. This resulted in decreased cystine levels but caused a concentration-dependent increase in embryo dysmorphism. Furthermore, the mfsd12a TB MO injection did not improve proximal tubular reabsorption or megalin expression. In conclusion, MFSD12 mRNA depletion reduced cystine levels in both tested models without improvement of the proximal tubular function in the ctns-/- zebrafish embryo. In addition, the apparent toxicity of higher mfsd12a TB MO concentrations on the zebrafish development warrants further evaluation.NEW & NOTEWORTHY In this study, we show that MFSD12 depletion with either CRISPR/Cas9-mediated gene editing or a translation-blocking morpholino significantly reduced cystine levels in cystinosis ciPTECs and ctns-/- zebrafish embryos, respectively. However, we observed no improvement in the proximal tubular reabsorption of dextran in the ctns-/- zebrafish embryos injected with mfsd12a translation-blocking morpholino. Furthermore, a negative effect of the mfsd12a morpholino on the zebrafish development warrants further investigation.
{"title":"<i>MFSD12</i> depletion reduces cystine accumulation without improvement in proximal tubular function in experimental models for cystinosis.","authors":"Tjessa Bondue, Laleh Khodaparast, Ladan Khodaparast, Sara Cairoli, Bianca Maria Goffredo, Rik Gijsbers, Lambertus van den Heuvel, Elena Levtchenko","doi":"10.1152/ajprenal.00014.2024","DOIUrl":"10.1152/ajprenal.00014.2024","url":null,"abstract":"<p><p>Cystinosis is an autosomal recessive lysosomal storage disorder, caused by mutations in the <i>CTNS</i> gene, resulting in an absent or altered cystinosin (CTNS) protein. Cystinosin exports cystine out of the lysosome, with a malfunction resulting in cystine accumulation and a defect in other cystinosin-mediated pathways. Cystinosis is a systemic disease, but the kidneys are the first and most severely affected organs. In the kidney, the disease initially manifests as a generalized dysfunction in the proximal tubules (also called renal Fanconi syndrome). MFSD12 is a lysosomal cysteine importer that directly affects the cystine levels in melanoma cells, HEK293T cells, and cystinosis patient-derived fibroblasts. In this study, we aimed to evaluate <i>MFSD12</i> mRNA levels in cystinosis patient-derived proximal tubular epithelial cells (ciPTECs) and to study the effect of <i>MFSD12</i> knockout on cystine levels. We showed similar <i>MFSD12</i> mRNA expression in patient-derived ciPTECs in comparison with the control cells. CRISPR <i>MFSD12</i> knockout in a patient-derived ciPTEC (<i>CTNS<sup>Δ57kb</sup></i>) resulted in significantly reduced cystine levels. Furthermore, we evaluated proximal tubular reabsorption after injection of <i>mfsd12a</i> translation-blocking morpholino (TB MO) in a <i>ctns<sup>-/-</sup></i> zebrafish model. This resulted in decreased cystine levels but caused a concentration-dependent increase in embryo dysmorphism. Furthermore, the <i>mfsd12a</i> TB MO injection did not improve proximal tubular reabsorption or megalin expression. In conclusion, <i>MFSD12</i> mRNA depletion reduced cystine levels in both tested models without improvement of the proximal tubular function in the <i>ctns<sup>-/-</sup></i> zebrafish embryo. In addition, the apparent toxicity of higher <i>mfsd12a</i> TB MO concentrations on the zebrafish development warrants further evaluation.<b>NEW & NOTEWORTHY</b> In this study, we show that <i>MFSD12</i> depletion with either CRISPR/Cas9-mediated gene editing or a translation-blocking morpholino significantly reduced cystine levels in cystinosis ciPTECs and <i>ctns<sup>-/-</sup></i> zebrafish embryos, respectively. However, we observed no improvement in the proximal tubular reabsorption of dextran in the <i>ctns<sup>-/-</sup></i> zebrafish embryos injected with <i>mfsd12a</i> translation-blocking morpholino. Furthermore, a negative effect of the <i>mfsd12a</i> morpholino on the zebrafish development warrants further investigation.</p>","PeriodicalId":93867,"journal":{"name":"American journal of physiology. Renal physiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140308232","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}