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Carfilzomib increased collecting duct transporters gene expression and activated mineralocorticoid receptor signaling, independently of renin-angiotensin-aldosterone axis. Graphs of (A) plasma angiotensin-converting enzyme (ACE) activity (nmol/min, n = 10 per group), (B) serum angiotensin II (AngII, n = 5 per group), (C) Plasma aldosterone (pg/mL, n = 5-6 per group) and (D) urine aldosterone excretion (pg/24 h, n = 5-6 per group). (E) Graph of relative mRNA levels expressed as fold change of controls of Sglt1. Nhe1, Nkcc2, Aqp2, β-Enac, Uta1, Na-K-atpase in the kidneys (n = 5-6 per group). (F) Representative Western blot images and (G-J) relative densitometry analysis of mineralocorticoid receptor (MR)-SGK-1 related proteins in the cytosolic and nuclear fractions of the kidneys (n = 5-6 per group). Data are presented as mean ± SEM. All phosphorylated proteins are normalized to their respective total proteins and total proteins are normalized to GAPDH or α-actinin (loading controls) Student's T-Test, unpaired, 2-tailed. *P < 0.05. Aqp2 = aquaporin 2; β-ENaC = epithelial sodium channel ENaC beta subunit; MR = mineralocorticoid receptor; NEDD4 = neural precursor cell expressed developmentally down-regulated protein 4; Nhe1 = sodium-hydrogen antiporter 1; Nkcc2 = Na-K-Cl cotransporter; Sglt1 = sodium-glucose cotransporter 1; SGK-1 = serum and glucocorticoid-regulated kinase 1; Uta1 = urea transporter A1.
Source publication
Carfilzomib is an irreversible proteasome inhibitor indicated for relapsed/refractory multiple myeloma. Carfilzomib toxicity includes renal adverse effects (RAEs) of obscure pathobiology. Therefore, we investigated the mechanisms of nephrotoxicity developed by Carfilzomib. In a first experimental series, we used our previously established in vivo m...
Contexts in source publication
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... We found that 4 doses of Cfz did not activate RAAS, as shown by the unchanged levels of angiotensin-converting enzyme (ACE) activity and angiotensin II (AngII) levels in the sera. Moreover, aldosterone levels were found to be decreased in the serum and without any significant changes in 24 hours aldosterone excretion as assessed in the urine (Fig. 4A-D). The latter might indicate a negative feedback loop on Aldosterone biosynthesis that was herein not investigated. Taking under consideration that AMPKα plays a key transcriptional role in the kidneys, by regulating numerous transporters' gene expression in the proximal tubules, Henle loop, and collecting duct 20 and since we already ...
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... exchanger 1; Nhe1) and Henle loop (Na + -K + -Cl -cotransporter 2; Nkcc2) remained unchanged, whereas gene expression of transporters in the collecting duct (epithelial sodium channel beta; β-Enac, urea transporter-1 Uta1) and the ubiquitously expressed Sodium-Potassium ATPase (Na + -K + -ATPase) were significantly increased in the Cfz group (Fig. 4E). Since collecting duct epithelium and especially β-ENaC are key mediators of Na + reabsorption, 21 while Na + reabsorption and Na + gradient are involved with Figure 2. Metabolic, protein repair, and molecular transport pathways are differentially regulated by Carfilzomib in the kidneys. Emerging role of AMPKα. (A) Three-dimensional ...
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... of activated B cells; Raptor = regulatory-associated protein of mTOR; VEGF-A = vascular endothelial growth factor A; VEGFR2 = vascular endothelial growth factor receptor 2. mineralocorticoid receptor (MR) activation, we focused our interest on the MR signaling. We found an upregulation of β-ENaC expression in whole kidney lysates (Fig. 4F, G), while in the subcellular fractionation experiments, we found an upregulation of MR in the nucleus, an upregulation of neural precursor cell expressed developmentally down-regulated protein 4 (NEDD-4) and an increased phosphorylation and expression of Serum/Glucocorticoid-Regulated Kinase 1 (SGK-1) in the cytosol (Fig. 4F, H, J). ...
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... whole kidney lysates (Fig. 4F, G), while in the subcellular fractionation experiments, we found an upregulation of MR in the nucleus, an upregulation of neural precursor cell expressed developmentally down-regulated protein 4 (NEDD-4) and an increased phosphorylation and expression of Serum/Glucocorticoid-Regulated Kinase 1 (SGK-1) in the cytosol (Fig. 4F, H, J). Moreover, the MR cytosolic/nuclear expression ratio was found to be decreased in the Cfz group, indicating a translocation of the receptor to the nucleus and thus increased MR activity (Fig. 4F, I). 22 Collectively our data suggest that Cfz increased collecting duct transporters mRNA expression and activated MR/ SGK-1 signaling in the ...
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... protein 4 (NEDD-4) and an increased phosphorylation and expression of Serum/Glucocorticoid-Regulated Kinase 1 (SGK-1) in the cytosol (Fig. 4F, H, J). Moreover, the MR cytosolic/nuclear expression ratio was found to be decreased in the Cfz group, indicating a translocation of the receptor to the nucleus and thus increased MR activity (Fig. 4F, I). 22 Collectively our data suggest that Cfz increased collecting duct transporters mRNA expression and activated MR/ SGK-1 signaling in the kidneys, independently of RAAS activation. Since MR activation is implicated with Na + reabsorption and K + sparing from the urine, 23 we further confirmed our finding by measuring plasma and urine ...
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... To investigate the effect of MR/SGK-1 activation on blood pressure in vivo, we assessed systolic blood pressure (SBP) and heart rate (HR) in the 2-and 4-dose protocols. We found that SBP was increased only in the 4-dose protocol, in which renal MR/SGK-1 axis is shown to be activated in line with the presence of nephrotoxicity, whereas SBP remained unchanged in the 2-dose protocol ( Figure S4A-D). Thus, the increased blood pressure in the 4-dose protocol complements our findings on MR/SGK-1 derived phenotype in vivo. ...
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... concerning MR-induced Na + retention and K + sparing, Eplerenone abrogated Na + and K + imbalance in the urine in the co-administration group, an effect which confirmed MR/SGK-1 inhibition (Table S3). Additionally, eplerenone prevented the Cfz-induced increase in SBP in vivo without altering HR (Figure S4E-F). Regarding the kidney, urine, and plasma metabolic phenotype, Cfz exhibited a distinct metabolic profile, as shown by Partial Least-Squares Discriminant Figure 7. Eplerenone prevented Carfilzomib-induced SGK-1/MR activation in the kidneys and restored the metabolic profile in the kidneys, urine, and plasma in vivo. ...
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... We found that 4 doses of Cfz did not activate RAAS, as shown by the unchanged levels of angiotensin-converting enzyme (ACE) activity and angiotensin II (AngII) levels in the sera. Moreover, aldosterone levels were found to be decreased in the serum and without any significant changes in 24 hours aldosterone excretion as assessed in the urine (Fig. 4A-D). The latter might indicate a negative feedback loop on Aldosterone biosynthesis that was herein not investigated. Taking under consideration that AMPKα plays a key transcriptional role in the kidneys, by regulating numerous transporters' gene expression in the proximal tubules, Henle loop, and collecting duct 20 and since we already ...
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... exchanger 1; Nhe1) and Henle loop (Na + -K + -Cl -cotransporter 2; Nkcc2) remained unchanged, whereas gene expression of transporters in the collecting duct (epithelial sodium channel beta; β-Enac, urea transporter-1 Uta1) and the ubiquitously expressed Sodium-Potassium ATPase (Na + -K + -ATPase) were significantly increased in the Cfz group (Fig. 4E). Since collecting duct epithelium and especially β-ENaC are key mediators of Na + reabsorption, 21 while Na + reabsorption and Na + gradient are involved with Figure 2. Metabolic, protein repair, and molecular transport pathways are differentially regulated by Carfilzomib in the kidneys. Emerging role of AMPKα. (A) Three-dimensional ...
Context 10
... of activated B cells; Raptor = regulatory-associated protein of mTOR; VEGF-A = vascular endothelial growth factor A; VEGFR2 = vascular endothelial growth factor receptor 2. mineralocorticoid receptor (MR) activation, we focused our interest on the MR signaling. We found an upregulation of β-ENaC expression in whole kidney lysates (Fig. 4F, G), while in the subcellular fractionation experiments, we found an upregulation of MR in the nucleus, an upregulation of neural precursor cell expressed developmentally down-regulated protein 4 (NEDD-4) and an increased phosphorylation and expression of Serum/Glucocorticoid-Regulated Kinase 1 (SGK-1) in the cytosol (Fig. 4F, H, J). ...
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... whole kidney lysates (Fig. 4F, G), while in the subcellular fractionation experiments, we found an upregulation of MR in the nucleus, an upregulation of neural precursor cell expressed developmentally down-regulated protein 4 (NEDD-4) and an increased phosphorylation and expression of Serum/Glucocorticoid-Regulated Kinase 1 (SGK-1) in the cytosol (Fig. 4F, H, J). Moreover, the MR cytosolic/nuclear expression ratio was found to be decreased in the Cfz group, indicating a translocation of the receptor to the nucleus and thus increased MR activity (Fig. 4F, I). 22 Collectively our data suggest that Cfz increased collecting duct transporters mRNA expression and activated MR/ SGK-1 signaling in the ...
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... protein 4 (NEDD-4) and an increased phosphorylation and expression of Serum/Glucocorticoid-Regulated Kinase 1 (SGK-1) in the cytosol (Fig. 4F, H, J). Moreover, the MR cytosolic/nuclear expression ratio was found to be decreased in the Cfz group, indicating a translocation of the receptor to the nucleus and thus increased MR activity (Fig. 4F, I). 22 Collectively our data suggest that Cfz increased collecting duct transporters mRNA expression and activated MR/ SGK-1 signaling in the kidneys, independently of RAAS activation. Since MR activation is implicated with Na + reabsorption and K + sparing from the urine, 23 we further confirmed our finding by measuring plasma and urine ...
Context 13
... To investigate the effect of MR/SGK-1 activation on blood pressure in vivo, we assessed systolic blood pressure (SBP) and heart rate (HR) in the 2-and 4-dose protocols. We found that SBP was increased only in the 4-dose protocol, in which renal MR/SGK-1 axis is shown to be activated in line with the presence of nephrotoxicity, whereas SBP remained unchanged in the 2-dose protocol ( Figure S4A-D). Thus, the increased blood pressure in the 4-dose protocol complements our findings on MR/SGK-1 derived phenotype in vivo. ...
Context 14
... concerning MR-induced Na + retention and K + sparing, Eplerenone abrogated Na + and K + imbalance in the urine in the co-administration group, an effect which confirmed MR/SGK-1 inhibition (Table S3). Additionally, eplerenone prevented the Cfz-induced increase in SBP in vivo without altering HR (Figure S4E-F). Regarding the kidney, urine, and plasma metabolic phenotype, Cfz exhibited a distinct metabolic profile, as shown by Partial Least-Squares Discriminant Figure 7. Eplerenone prevented Carfilzomib-induced SGK-1/MR activation in the kidneys and restored the metabolic profile in the kidneys, urine, and plasma in vivo. ...
Citations
... Carfilzomib (Cfz) is an antineoplastic agent employed for the cure of relapse/refractory multiple myeloma; however, its action is compromised by high percentages of cardiovascular and renal side effects. Previous studies have shown that Cfz nephrotoxicity is caused by the activation of the mineralocorticoid receptor (MR) through the dysregulation of water/ion transport and the urine electrolyte balance [1]. The existing metabolomics studies show that Cfz leads to metabolic dysregulations that are mainly located in the kidneys and urinary system [1,2]. ...
... Previous studies have shown that Cfz nephrotoxicity is caused by the activation of the mineralocorticoid receptor (MR) through the dysregulation of water/ion transport and the urine electrolyte balance [1]. The existing metabolomics studies show that Cfz leads to metabolic dysregulations that are mainly located in the kidneys and urinary system [1,2]. Furthermore, the found metabolites prove the correlation between the drug administration with conditions of inflammation, kidney injury, and oxidative stress [2]. ...
... This study aimed to enrich the existing knowledge on Cfz-induced nephrotoxicity by the metabolomics prospective, performing RP chromatographic analysis, as a complementary step to Barla et al.'s published workflow [1]. Metabolomics deals with thousands of compounds, therefore, the combination of different separation methodologies offers more opportunities for 'good performance' for a larger number of metabolites. ...
(1) Carfilzomib (Cfz) is an antineoplastic agent indicated for the treatment of multiple myeloma. However, its beneficial action is attenuated by the occurrence of cardiotoxicity and nephrotoxicity as the most common adverse effects. Presently, there is well-established knowledge on the pathomechanisms related to these side effects; however, the research on the metabolic alterations provoked by the drug is limited. (2) An in vivo simulation of Cfz-induced toxicity was developed in (i) Cfz-treated and (ii) control mice. An RP-HRMS-based protocol and an advanced statistical treatment were used to investigate the impact of Cfz on the non-polar metabolome. (3) The differential analysis classified the Cfz-treated and control mice and resulted in a significant number of identified biomarkers with AUC > 0.9. The drug impaired the biosynthesis and degradation of aromatic amino acids (AAA) and led to alterations of uremic toxins in the renal and urine levels. Furthermore, the renal degradation of tryptophan was affected, inducing its degradation via the kynurenine pathway. (4) The renal levels of metabolites showed impaired excretion and degradation of AAAs. Cfz was, finally, correlated with the biosynthesis of renal dopamine, explaining the biochemical causes of water and ion retention and the increase in systolic pressure.
... As a result, an increase in plasma creatinine and BUN was identified as an indication of renal failure. Similarly, Efentakis et al.'s 2022 investigation found that CFZ treatment at 4 and 8 mg/kg was linked with significant acute renal damage in C57BI/6J mice, indicated biochemically by an increase in blood creatinine and lactate dehydrogenase (LDH) [10]. Moreover, our study revealed that 4 mg/kg of CFZ for 16 days resulted in a significant rise in the levels of plasma creatinine, blood urea nitrogen (BUN), and uric acid compared to the vehicletreated group in rats. ...
Citation: Qadri, M.M.; Alam, M.F.; Khired, Z.A.; Alaqi, R.O.; Khardali, A.A.; Alasmari, M.M.; Alrashah, A.S.S.; Muzafar, H.M.A.; Qahl, A.M. Thymoquinone Ameliorates Carfilzomib-Induced Renal Impairment by Modulating Oxidative Stress Markers, Inflammatory/Apoptotic Mediators, and Augmenting Nrf2 in Rats. Int. J. Mol. Sci. 2023, 24, 10621. https:// Abstract: Chemotherapy-induced kidney damage is an emerging problem that restricts cancer treatment effectiveness. The proteasome inhibitor carfilzomib (CFZ) is primarily used to treat multiple myeloma and has been associated with severe renal injury in humans. CFZ-induced nephrotoxicity remains an unmet medical need, and there is an urgent need to find and develop a nephroprotective and antioxidant therapy for this condition. Thymoquinone (TQ) is a bioactive compound that has been isolated from Nigella sativa seeds. It has a wide range of pharmacological properties. Therefore, this experimental design aimed to study the effectiveness of TQ against CFZ-induced renal toxicity in rats. The first group of rats was a normal control (CNT); the second group received CFZ (4 mg/kg b.w.); the third and fourth groups received TQ (10 and 20 mg/kg b.w.) 2 h before receiving CFZ; the fifth group received only TQ (20 mg/kg b.w.). This experiment was conducted for 16 days, and at the end of the experiment, blood samples and kidney tissue were collected for biochemical assays. The results indicated that administration of CFZ significantly enhanced serum marker levels such as BUN, creatinine, and uric acid in the CFZ group. Similarly, it was also noticed that CFZ administration induced oxidative stress by reducing antioxidants (GSH) and antioxidant enzymes (CAT and SOD) and increasing lipid peroxidation. CFZ treatment also enhanced the expression of IL-1β, IL-6, and TNF-α production. Moreover, CFZ increased caspase-3 concentrations and reduced Nrf2 expression in the CFZ-administered group. However, treatment with 10 and 20 mg/kg TQ significantly decreased serum markers and increased antioxidant enzymes. TQ treatment considerably reduced IL-1β, IL-6, TNF-α, and caspase-3 concentrations. Overall, this biochemical estimation was also supported by histopathological outcomes. This study revealed that TQ administration significantly mitigated the negative effects of CFZ treatment on Nrf2 expression. Thus, it indicates that TQ may have utility as a potential drug to prevent CFZ-induced nephrotoxicity in the future.
Carfilzomib (CFZ) is a proteasome inhibitor approved for relapsed/refractory multiple myeloma (MM) but its clinical use is limited by cardiovascular toxicity. The mechanisms of CFZ-induced cardiovascular toxicity are not fully understood but endothelial dysfunction may be a common denominator. Here, we first characterized the direct toxic effects of CFZ on endothelial cells (HUVECs and EA.hy926 cells) and tested whether SGLT2 inhibitors, known to have cardioprotective effects, can protect against CFZ-induced toxicity. To determine the chemotherapeutic effect of CFZ in the presence of SGLT2 inhibitors, MM and lymphoma cells were treated with CFZ with or without canagliflozin. CFZ decreased cell viability and induced apoptotic cell death in endothelial cells in a concentration-dependent manner. CFZ also upregulated ICAM-1 and VCAM-1 and downregulated VEGFR-2. These effects were associated with the activation of Akt and MAPK pathways, inhibition of p70s6k, and downregulation of AMPK. Canagliflozin, but not empagliflozin or dapagliflozin, protected endothelial cells from CFZ-induced apoptosis. Mechanistically, canagliflozin abrogated CFZ-induced JNK activation and AMPK inhibition. AICAR (an AMPK activator) protected from CFZ-induced apoptosis, and compound C (an AMPK inhibitor) abrogated the protective effect of canagliflozin, strongly suggesting that AMPK mediates these effects. Canagliflozin did not interfere with the anticancer effect of CFZ in cancer cells. In conclusion, our findings demonstrate for the first time the direct toxic effects of CFZ in endothelial cells and the associated signaling changes. Canagliflozin abrogated the apoptotic effects of CFZ in endothelial cells in an AMPK-dependent mechanism, without interfering with its cytotoxicity in cancer cells.
Background:
Carfilzomib (Cfz) is an anti-cancer drug related to cardiorenal adverse events, with cardiovascular and renal complications limiting its clinical use. Despite the important progress concerning the discovery of the underlying causes of Cfz-induced nephrotoxicity, the molecular/biochemical background is still not well clarified. Furthermore, the number of metabolomics-based studies concerning Cfz-induced nephrotoxicity is limited.
Methods:
A metabolomics UPLC-HRMS-DIA methodology was applied to three bio-sample types i.e., plasma, kidney, and urine, obtained from two groups of mice, namely (i) Cfz (8 mg Cfz/ kg) and (ii) Control (0.9% NaCl) (n = 6 per group). Statistical analysis, involving univariate and multivariate tools, was applied for biomarker detection. Furthermore, a sub-study was developed, aiming to estimate metabolites' correlation among bio-samples, and to enlighten potential mechanisms.
Results:
Cfz mostly affects the kidneys and urine metabolome. Fifty-four statistically important metabolites were discovered, and some of them have already been related to renal diseases. Furthermore, the correlations between bio-samples revealed patterns of metabolome alterations due to Cfz.
Conclusions:
Cfz causes metabolite retention in kidney and dysregulates (up and down) several metabolites associated with the occurrence of inflammation and oxidative stress.
