Original articleChronic impairment of mitochondrial bioenergetics and β-oxidation promotes experimental AKI-to-CKD transition induced by folic acid
Graphical abstract
Introduction
The term chronic kidney disease (CKD) describes a group of pathologies characterized by the progressive loss of renal function, resulting in a progressive decrement in the glomerular filtration rate (GFR), with or without an increment in the conventional clinical renal damage markers, like creatinine and blood urea nitrogen (BUN) [1,2]. In recent years, the number of patients with CKD has increased dramatically [[3], [4], [5], [6], [7]], representing a growing worldwide public health problem [8]. As a result of the lack of understanding of the complex pathological mechanisms involved in CKD development [9], the current clinical strategies and treatments do not significantly improve kidney function or prevent illness progression [10], thus highlighting the urgent need to investigate and understand the mechanisms involved in the generation and progression of CKD [11,12].
Furthermore, dysfunction in mitochondrial bioenergetics and redox state have recently emerged as key components of the sudden deterioration of renal functions in a relatively short time-interval, known as acute kidney injury (AKI). In fact, mitochondrial dysfunction in AKI is involved in tubular dysfunction, cell death, oxidative stress and inflammation [[13], [14], [15], [16], [17], [18], [19]]. Likewise, in both animal models and patients, a severe episode of AKI, or a series of them, results in CKD development [2,[20], [21], [22]]. The growing evidence suggests that renal mitochondrial alterations [9,23,24] favor hemodynamic alterations, Ca2+ deregulation, cell death, inflammation, fibrotic processes and epithelial-to-mesenchymal transition (EMT), which participate in the progression of several types of CKD in both non-diabetic and diabetic contexts [9,[22], [23], [24], [25], [26], [27]]. However, currently there is no information about the mitochondrial bioenergetics and redox state alterations over a time-course. Also, the possible effects of their attenuation in the progression of renal damage are not clear.
In this context, folic acid (FA)-induced kidney damage is a widely recurred to experimental model for AKI induction [28,29]. This model also induces chronic processes such as the persistence of cell death, EMT, cytokines release, inflammation and fibrosis, which lead to CKD transition usually in less than 28 days [28,30,31]. Furthermore, it recreates the AKI pathology reported in the clinic [32] and is highly reproducible [28,29]. Together, these make the FA model good for the exploration of the mechanisms involved in AKI-to-CKD transition [28,30,31]. In the present work, we refer to “the acute stage” as the period that begins with the administration of FA and ends 72 h later, bringing about the AKI-to-CKD transition or “the chronic stage”, which encompasses the time period between day 4 until day 28. It is important to highlight that although high doses of FA have pathological effects on kidney function [28,30,31], nephroprotective effects and the reduction of cardiovascular risk have also been reported by lower FA doses in CKD patients with elevated homocysteine levels, but with baseline vitamin B12 levels [33,34]. In fact, vitamin B12 and the metabolite of FA, 5-methyltetrahydrofolate (5-THF), act as cofactors of the enzyme methionine synthetase, that catalyzes the transfer to the methyl group from 5-THF to homocysteine, producing methionine [33]. Therefore, the protection in this context may be related to reduced homocysteine levels mediated by FA and vitamin B12 [[35], [36], [37]]. Additionally, it must be taken into account that the FA dose used in these papers (<10 mg/day) is much lower compared to the dose used in the FA-induced renal damage models (>250 mg/day) and it is well documented that FA at higher levels triggers prooxidant and proinflammatory states in kidneys [38,39]. So, a protective or prooxidant effect of FA could depend on the dose, the administration route and the preexistent kidney damage conditions.
In the case of renal damage induced by high levels of FA, the redox imbalance generated by FA metabolism is one of the main mechanisms involved in the genesis of the pathology [13,38,40], because FA metabolism requires higher levels of NADPH to reduce folate to THF [41,42], decreasing the antioxidant defense [13,38]. This redox imbalance is particularly harmful for kidney's mitochondria, due to the high internalization rates of folate in kidney [38,43,44] and because mitochondria store approximately 40% of folates [43,45]. Therefore, mitochondria are especially vulnerable to high doses of FA [13,46]. Additionally, the mitochondrial redox imbalance can be directly related to mitochondrial bioenergetics alterations [47]. In fact, we recently demonstrated that at 24 h after its administration FA induced in kidney mitochondria the decrease in glutathione (GSH) levels, the increase in glutathione disulfide (GSSG) and the decrease in total glutathione content (GSH + GSSG), that together with higher S-glutathionylation removal activity of mitochondrial glutaredoxin (Grx), leads to complex I (CI) dysfunction [13]. Furthermore, CI dysfunction induced by FA triggers the decrease in mitochondrial oxidative phosphorylation (OXPHOS) capacity, mitochondrial decoupling, loss of mitochondrial membrane potential (ΔΨm), increased oxidative stress and mitochondrial hydrogen peroxide (H2O2) production. Such effects were associated with AKI development [13]. Additionally, we also demonstrated that the pretreatment with N-acetyl-cysteine (NAC), a donor of sulfhydryl groups and a precursor of GSH [48,49] prevented the mitochondrial bioenergetics and redox state impairment, which prevented the development of AKI 24 h after FA administration [13].
The progressive nature of the renal disease induced by FA was further evidenced by the reduction in mRNA levels of mitochondrial electron transport system (ETS) components in the days 2–14 after FA administration [39,46], the persistence of oxidative stress in kidneys after FA administration [38,50,51] and the inflammatory and fibrotic processes [[52], [53], [54], [55]]. Interestingly, inflammatory and fibrotic processes have been related to mitochondrial impairment [[56], [57], [58]], suggesting that mitochondrial bioenergetics and redox state impairment is persistent in AKI-to-CKD transition in this model. However, such hypothesis has not yet been evaluated.
Therefore, in the present study, we characterize for the first time, the time course of mitochondrial bioenergetics and redox state alterations and their possible association to changes in mitochondrial mass and biogenesis in the AKI-to-CKD transition induced by FA. We also assess the effects of NAC pre-administration in the temporal evolution of mitochondrial bioenergetics and redox state alterations. Lastly, we evaluate if the prevention of mitochondrial dysfunction by NAC during AKI prevents transition to CKD in the FA model.
Section snippets
Reagents
Adenosine 5′-diphosphate sodium salt (ADP), amplex red, antimycin A, l-arginine, fat free bovine serum albumin (BSA), bromophenol blue, β-mercaptoethanol, carbonyl cyanide m-chlorophenylhydrazone (CCCP), cytochrome c from equine heart, D-(+)-glucose, D-mannitol, decylubiquinone (DUB), 2,6-dichlorophenolindophenol sodium salt hydrate (DCPIP), 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB), ethylene glycol-bis(2-aminoethylether)-N,N,N′,N'-tetraacetic acid (EGTA), glycerol, GSH, glucose-6-phosphate
FA induces renal damage progression
Our first goal was to characterize the kidney damage progression induced by FA. Therefore, we evaluated the levels of the classic renal damage markers BUN and creatinine in plasma. As shown in Fig. 1A–C, at day 2 after its administration, FA induced a remarkable increase in BUN and creatinine plasma levels, as well as in the kidney weight/rat weight ratio compared to the control group. These alterations were prevented at day 2 by the pre-administration of NAC (Fig. 1A–C). Furthermore,
Discussion
The administration of high levels of FA leads to an increase of its metabolized product (tetrahydrofolate) in blood [42,43], which is then transported inside the cell by the reduced folate carrier exchanger and by the high-affinity folate receptor [42,43,45,68]. In the case of the kidney, folate metabolites accumulate in higher concentrations than in other tissues due to the expression of the high-affinity folate receptor in this organ, especially in PT cells [43]. The folate metabolites are
Conclusion
FA induces an acute stage of renal damage alterations in mitochondrial β-oxidation and CI-linked respiration. Although in a chronic stage of renal damage there is partial recovery in CI-linked respiration parameters, the mitochondrial β-oxidation alterations persist over time, triggering a permanent reduction in ATP production and mitochondrial decoupling, favoring the progression to CKD.
Finally, the renal mitochondrial preservation that results from NAC pre-treatment in FA-induced AKI may be a
Declaration of competing interestCOI
The authors report no conflict of interests.
Acknowledgements
Research conducted for this publication was supported by grants from “Consejo Nacional de Ciencia y Tecnología” (CONACyT, A1-S-7495 and 281967), Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT, #IN202219) of Universidad Nacional Autónoma de México (UNAM), Programa de Apoyo a la Investigación y el Posgrado (PAIP, 5000-9105), and Fondos del Gasto Directo autorizado a la Subdirección de Investigación Básica from Instituto Nacional de Cardiología Ignacio Chávez to ET
References (97)
- et al.
Chronic kidney disease
Lancet
(2012) - et al.
Chronic kidney disease: definition, epidemiology, cost, and outcomes
- et al.
The growth of acute kidney injury: a rising tide or just closer attention to detail?
Kidney Int.
(2015) - et al.
Chronic kidney disease, gender, and access to care: a global perspective
Semin. Nephrol.
(2017) - et al.
Chronic kidney disease
Lancet
(2017) - et al.
The management of CKD: a look into the future
Kidney Int.
(2007) - et al.
Acute kidney injury: global health alert, Hong Kong
J. Nephrol.
(2013) - et al.
Impact of acute kidney injury on distant organ function: recent findings and potential therapeutic targets
Kidney Int.
(2016) - et al.
Protective effects of N-acetyl-cysteine in mitochondria bioenergetics, oxidative stress, dynamics and S-glutathionylation alterations in acute kidney damage induced by folic acid
Free Radic. Biol. Med.
(2019) - et al.
Curcumin prevents potassium dichromate (K2Cr2O7)-induced renal hypoxia
Food Chem. Toxicol.
(2018)