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Intracellular traficking and acute kidney injury: Rab GTPase as markers of tubular lesion.
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Investigating the role of the mercapturate pathway in kideney function for the identification of an early indicator tubular dysfunction
Publication . Dias, Clara G.; Pereira, Sofia Azeredo; Rios, Miriam Karina Soto
The general objective of this project was to respond to a pressing need in clinical practice, which is the diagnosis of tubular dysfunction. This knowledge would not only allow the prediction of the early onset and progression of tubular dysfunction, but also the management of strategies to stop the progression of renal disease, which at present are non-existent. On the other hand, it would be of significant importance in the development of strategies to minimize tubular toxicity induced by drugs and substances used in diagnostic methods that pose a major challenge in clinical practice. It would also have added value in the preclinical tests in drug development.
There is a good correlation between the degree of damage to the tubulo-interstitial compartment and the deterioration of renal function. Moreover, the primary role of kidney as a detoxification organ renders it vulnerable to develop various forms of injury. Motivated by the relevant detoxification activity and its main expression in kidney proximal tubular cells, the mercapturate pathway was chosen as a starting point of this mechanistic-based investigation for a reliable biomarker reflective of tubular dysfunction. We were particularly interested in N-acetyltransferase 8 (NAT8), the last enzyme of the mercapturate pathway, that acetylates cysteine-S-conjugates (Cys-S-conjugates), rendering their urinary elimination. Cys-S-conjugates can be more toxic than the parent compounds. The formation of Cys-S-conjugates have been reported in inflammatory diseases and a hypothesis-based review of their contribution and the contribution of NAT8 as a balancer of their availability was built up exemplified by a tubular view of diabetic nephropathy.
As the kidney is highly susceptible to oxidative conditions and is exposed to relatively high concentrations of cysteine (Cys), which becomes easily oxidized, promoting the accumulation of its disulfides (in both the free and protein forms), we though that the measurement of Cys-S-conjugates which are disulfides could be a starting point for the identification of new markers of tubular dysfunction.
With the general goal of non-invasively evaluate kidney tubular dysfunction, it was first needed to develop a method that allowed to phenotype patients as higher or lower Cys-Sconjugates detoxifiers inspired on the activity of NAT8, which has been recognized as a nephroprotective factor mainly expressed in proximal tubular cells. This method quantifies the mercapturates of Cys-S-conjugates that are disulfides (CysSSX). We measured the N-acetylcysteine moiety of these mercapturates, that we named uNAC, as a surrogate of detoxification of CysSSX. In Chapter I, the rationale for the mercapturates of CysSSX as indicators of tubular dysfunction and for method development and validation is presented. The developed method was validated and applied to urine from individuals with healthy kidney and kidney disease as well as to urine from mice and rats. We have shown that CysSSX are acetylated and eliminated in urine as mercapturates. Rats and mice showed higher levels of uNAC than man. No intra-individual variability was found for uNAC levels in urine samples from rats and mice. In addition, lower levels of uNAC were found in urine of four patients that progressed to chronic kidney disease (CKD) after an episode of acute kidney injury (AKI) in comparison with volunteers.
In order to evaluate the potential of uNAC as an early indicator of AKI, we measured uNAC concentrations in two animal models of drug-induced AKI (Chapter II). Cisplatin (CISP) was chosen because is a tubular toxicant that undergoes the mercapturate pathway and gentamicin (G) also promotes tubular dysfunction, but it does not undergo this metabolic circuitry. For this, male Wistar rats were allocated in individual metabolic cages and randomly divided into three groups: (1) control group (CTL), receiving daily vehicle intraperitoneal for 6 days; (2) cisplatin group (CISP), receiving a single intraperitoneal dose of cisplatin (5 mg/kg); and (3) gentamicin group (G), receiving gentamicin intraperitoneal (150 mg/kg) for 6 days. The levels of uNAC decreased prior to the increase in pCr, particularly in CISP model (p < 0.001). This tendency was also observed for rats treated with G, despite only reaching significance belatedly than CISP (p = 0.044).
Next, we were interested to know the inter-individual variability of uNAC in man and factors that could influence it. For that, uNAC was measured in a population of humanimmunodeficiency virus (HIV)-infected patients as a proof-of-principle (Chapter III). The levels of uNAC varied highly (59%) among 242 HIV-infected patients. A multivariable analysis showed that the levels of uNAC were positively associated with estimated glomerular filtration rate (eGFR) (p = 0.020) and negatively associated with lamivudine use (3TC) (p = 0.019) and Black race (p < 0.001). Notably, a prospective analysis of uNAC in 118 HIV-infected patients allowed to discriminate patients on kidney disease progression from those that maintained a stable renal function, that might imply changes in cysteine disulfides availability and dynamics in kidney disease progression.
Thus, the next step was to evaluate the temporal changes of cysteine disulfides dynamics and uNAC in models of chronic diseases that affect the kidney, aiming to evaluate if they are prior to kidney dysfunction and also its relation to disease development (e.g. hypertension, insulin resistance). For that, we selected animal models associated with metabolic inflammation, namely two pre-diabetic mice models induced by high fructose (HFruct) or hypercaloric (HFat) diet for 15 weeks (Chapter IV); and a rat model of hypertension and insulin resistance induced by chronic intermittent hypoxia (CIH) to a maximum of 60 days (Chapter V). In the three models the disease progression and severity diseases are mild, being appropriated to study temporal changes of kidney tubular dysfunction and its relation to the development of hypertension and insulin resistance. HFat animals presented lower uNAC right after 1 week of exposure to the diet while the HFruct group presented a mild increase. Nevertheless, the increment on the chronicity of exposure to the diets decreased uNAC until the end of the study, that was particularly evident for HFat model (45% lower than Chow group, p = 0.005). Moreover, Cys availability and protein cysteinylation (CysSSP) increased in HFruct (p = 0.046, p = 0.045 and p = 0.017, respectively for total, free total and CysSSP); while HFat diet induced an overall decrease (p = 0.028, p = 0.065 and p = 0.049, respectively for total, free total and CysSSP fractions). Although no clear renal pathology was observed in both pre-diabetic groups at the end of the study, changes in urinary albumin-to-creatinine ratio (uACR) were earlier and more evident in the HFat, comparing with the HFruct group. HFat mice also presented higher renal expression of NAT8 (p = 0.001), fibrotic markers and enzymes of glucose and lipid metabolism. Both diets induced increases in the renal expression of inflammatory, epithelial to mesenchymal transition and endoplasmic reticulum stress markers.
In the first day of exposure to CIH, there was an acute increase of almost 100% in uNAC in comparison with normoxic (Nx) animals. Afterwards, its levels decreased overtime, reaching a difference of -80% in CIH versus Nx group (p < 0.001). Kidney NAT8 gene expression initially decreased at days 1 (p = 0.006 for renal cortex – RC; p = 0.041 for renal medulla – RM) and 7 (p = 0.021 for RC and p = 0.024 for RM) of CIH exposure, further increasing at 21 days when hypertension is established but prior to the establishment of insulin resistance, and decreased at 60 days (p = 0.037 for RC and p = 0.024 for RM), coincident with the previously reported increase in renal fibronectin expression. At 7 days of CIH exposure, an increase in renal total Cys (p = 0.003 for RC and p = 0.001 for RM) and CysSSP (p = 0.041 for RC and p = 0.028 for RM) was observed, further decreasing with the chronicity of exposure to CIH (p < 0.001). An increase was also observed in the ratio of free Cys by CysSSX at day 7 (p = 0.008 for RC and p = 0.017 for RM), indicative of a more reduced state of Cys, decreasing from day 14 to 21 (p < 0.001) and returning to values closer to control conditions by the end of the experiment. Nevertheless, at the end of the study, no histological alterations were observed neither changes in uACR, despite there was a tendency to increase in CIH animals.
Herein, we provided evidence in order to respond to a lack of tubular dysfunction markers, a necessity that is mostly driven due to the need to better understand the pathophysiological mechanisms underlying it. Secondly, a method was developed to evaluate tubular dysfunction in a non-invasive manner. Lastly and although still very preliminary, new knowledge was herein provided based on a hypothesis that, as far as the author knowledge, is an innovative and never addressed issue that could pave the way for the identification of new mechanisms of inflammatory diseases through a tubulocentric perspective.
The preliminary data herein reported supports uNAC as an indicator of tubular dysfunction progression. Our approach shows that Cys-S-conjugate’s dynamics changes throughout disease progression. The results support that this dynamic might be associated not only to adaptive mechanism, with increases in CysSSP and uNAC, but also to mechanisms of disease progression with the chronicity of injury, characterized by opposite changes on this dynamic. The translation of these results together with the deepening of this study might lead to the identification of new therapeutic targets to slow down or stop disease progression and to establish preventive strategies achieved through early diagnosis. Furthermore, the identification of a new marker could also help to evaluate the success of preventive/management strategies.
The mercapturomic profile of health and non-communicable diseases
Publication . Gonçalves-Dias, Clara; Morello, Judit; Semedo, Valdir; Correia, M. João; Coelho, Nuno R.; Monteiro, Emilia C.; Monteiro, E.C.; Antunes, Alexandra M.M.; Pereira, Sofia A.; SA, Pereira; NOVA Medical School|Faculdade de Ciências Médicas (NMS|FCM); Centro de Estudos de Doenças Crónicas (CEDOC); MDPI - Multidisciplinary Digital Publishing Institute
The mercapturate pathway is a unique metabolic circuitry that detoxifies electrophiles upon adducts formation with glutathione. Since its discovery over a century ago, most of the knowledge on the mercapturate pathway has been provided from biomonitoring studies on environmental exposure to toxicants. However, the mercapturate pathway-related metabolites that is formed in humans—the mercapturomic profile—in health and disease is yet to be established. In this paper, we put forward the hypothesis that these metabolites are key pathophysiologic factors behind the onset and development of non-communicable chronic inflammatory diseases. This review goes from the evidence in the formation of endogenous metabolites undergoing the mercapturate pathway to the methodologies for their assessment and their association with cancer and respiratory, neurologic and cardiometabolic diseases.
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Fundação para a Ciência e a Tecnologia
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PD/BD/105892/2014