Temporal Alterations in Mitochondrial ß-Oxidation and Oxidative Stress Aggravate Chronic Kidney Disease Development in 5/6 Nephrectomy Induced Renal Damage.

Five-sixths nephrectomy (5/6Nx) model is widely used for studying the mechanisms involved in chronic kidney disease (CKD) progression, a kidney pathology that has increased dramatically in recent years. Mitochondrial impairment is a key mechanism that aggravates CKD progression; however, the information on mitochondrial bioenergetics and redox alterations along a time course in a 5/6Nx model is still limited and in some cases contradictory. Therefore, we performed for the first time a time-course study of mitochondrial alterations by high-resolution respirometry in the 5/6Nx model. Our results show a decrease in mitochondrial ß-oxidation at early times, as well as a permanent impairment in adenosine triphosphate (ATP) production in CI-linked respiration, a permanent oxidative state in mitochondria and decoupling of these organelles. These pathological alterations are linked to the early decrease in complex I and ATP synthase activities and to the further decrease in complex III activity. Therefore, our results may suggest that mitochondrial bioenergetics impairment is an early event in renal damage, whose persistence in time aggravates CKD development in the 5/6Nx model.

Role of Autophagy on Heavy Metal-Induced Renal Damage and the Protective Effects of Curcumin in Autophagy and Kidney Preservation.

Curcumin is a hydrophobic polyphenol compound extracted from the rhizome of turmeric. The protective effect of curcumin on kidney damage in multiple experimental models has been widely described. Its protective effect is mainly associated with its antioxidant and anti-inflammatory properties, as well as with mitochondrial function maintenance. On the other hand, occupational or environmental exposure to heavy metals is a serious public health problem. For a long time, heavy metals-induced nephrotoxicity was mainly associated with reactive oxygen species overproduction and loss of endogenous antioxidant activity. However, recent studies have shown that in addition to oxidative stress, heavy metals also suppress the autophagy flux, enhancing cell damage. Thus, natural compounds with the ability to modulate and restore autophagy flux represent a promising new therapeutic strategy. Furthermore, it has been reported in other renal damage models that curcumin's nephroprotective effects are related to its ability to regulate autophagic flow. The data indicate that curcumin modulates autophagy by classic signaling pathways (suppression of protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and/or by stimulation of adenosine monophosphate-activated protein kinase (AMPK) and extracellular signal-dependent kinase (ERK) pathways). Moreover, it allows lysosomal function preservation, which is crucial for the later stage of autophagy. However, future studies of autophagy modulation by curcumin in heavy metals-induced autophagy flux impairment are still needed.

Inflammation- and cancer-related microRNAs in rat renal cortex after subchronic exposure to fluoride.

The proximal tubule is a target of subchronic exposure to fluoride (F) in the kidney. Early markers are used to classify kidney damage, stage, and prognosis. MicroRNAs (miRNAs) are small sequences of non-coding single-stranded RNA that regulate gene expression and play an essential role in developing many pathologies, including renal diseases. This study aimed to evaluate the expression of Cytokine-Chemokine molecules (IL-1α/1ß/4/6/10, INF-γ, MIP-1α, MCP-1, RANTES, and TGF ß1/2/3) and inflammation-related miRNAs to evidence the possible renal mechanisms involved in subchronic exposure to F. Total protein and miRNAs were obtained from the renal cortex of male Wistar rats exposed to 0, 15 and 50 mg NaF/L through drinking water during 40 and 80 days. In addition, cytokines-chemokines were analyzed by multiplexing assay, and a panel of 77 sequences of inflammatory-related miRNAs was analyzed by qPCR. The results show that cytokines-chemokines expression was concentration- and time-dependent with F, where the 50 mg NaF/L were the main altered groups. The miRNAs expression resulted in statistically significant differences in thirty-four miRNAs in the 50 mg NaF/L groups at 40 and 80 days. Furthermore, a molecular interaction network analysis was performed. The relevant pathways modified by subchronic exposure to fluoride were related to extracellular matrix-receptor interaction, Mucin type O-glycan biosynthesis, Gap junction, and miRNAs involved with renal cell carcinoma. Thus, F-induced cytokines-chemokines suggest subchronic inflammation; detecting miRNAs related to cancer and proliferation indicates a transition from renal epithelium to pathologic tissue after fluoride exposure.

Effects of fluoride exposure on mitochondrial function: Energy metabolism, dynamics, biogenesis and mitophagy.

Fluoride is ubiquitous in the environment. Furthermore, drinking water represents the main source of exposure to fluoride for humans. Interestingly, low fluoride concentrations have beneficial effects on bone and teeth development; however, chronic fluoride exposure has harmful effects on human health. Besides, preclinical studies associate fluoride toxicity with oxidative stress, inflammation, and apoptosis. On the other hand, it is well-known that mitochondria play a key role in reactive oxygen species production. By contrast, fluoride's effect on processes such as mitochondrial dynamics, biogenesis and mitophagy are little known. These processes modulate the size, content, and distribution of mitochondria and their depuration help to counter the reactive oxygen species production and cytochrome c release, thereby allowing cell survival. However, a maladaptive response could enhance fluoride-induced toxicity. The present review gives a brief account of fluoride-induced mitochondrial alterations on soft and hard tissues, including liver, reproductive organs, heart, brain, lung, kidney, bone, and tooth.

Natural antioxidants' effects on endoplasmic reticulum stress-related diseases.

Endoplasmic reticulum (ER) stress is a normal molecular process induced by the over-accumulation of misfolded or unfolded proteins. ER stress induces the unfolded protein response (UPR), which reduces global protein synthesis, increases ER capacity and protein degradation, to restart ER homeostasis, allowing cell survival. However, the over-induction of UPR can also trigger inflammatory processes, tissue damage and cell death. ER stress is involved in several pathologies, like endothelial dysfunction, diabetes and heart, liver, kidney or neurological diseases. Although the progression of these diseases is the result of several pathological mechanisms, oxidative stress has been widely related to these pathologies. Moreover, ER stress can establish a progressive pathological cycle with oxidative stress. Therefore, the use of natural antioxidants, able to modulate both oxidative and ER stress, can be a new strategy to mitigate these diseases. This review is focused on the effects of natural antioxidant compounds on ER stress in endothelial dysfunction, diabetes and heart, liver, kidney or neurological diseases.

Chronic impairment of mitochondrial bioenergetics and ß-oxidation promotes experimental AKI-to-CKD transition induced by folic acid.

Recent studies suggest that mitochondrial bioenergetics and oxidative stress alterations may be common mechanisms involved in the progression of renal damage. However, the evolution of the mitochondrial alterations over time and the possible effects that their prevention could have in the progression of renal damage are not clear. Folic acid (FA)-induced kidney damage is a widely used experimental model to induce acute kidney injury (AKI), which can evolve to chronic kidney disease (CKD). Therefore, it has been extensively applied to study the mechanisms involved in AKI-to-CKD transition. We previously demonstrated that one day after FA administration, N-acetyl-cysteine (NAC) pre-administration prevented the development of AKI induced by FA. Such therapeutic effect was related to mitochondrial preservation. In the present study, we characterized the temporal course of mitochondrial bioenergetics and redox state alterations along the progression of renal damage induced by FA. Mitochondrial function was studied at different time points and showed a sustained impairment in oxidative phosphorylation capacity and a decrease in ß-oxidation, decoupling, mitochondrial membrane potential depolarization and a pro-oxidative state, attributed to the reduction in activity of complexes I and III and mitochondrial cristae effacement, thus favoring the transition from AKI to CKD. Furthermore, the mitochondrial protection by NAC administration before AKI prevented not only the long-term deterioration of mitochondrial function at the chronic stage, but also CKD development. Taken together, our results support the idea that the prevention of mitochondrial dysfunction during an AKI event can be a useful strategy to prevent the transition to CKD.

Alterations in mitochondrial homeostasis in a potassium dichromate model of acute kidney injury and their mitigation by curcumin.

Curcumin has protective effects in several acute kidney injury models, including that induced by potassium dichromate (K2Cr2O7). The protective effect of curcumin in this experimental model has been associated to the preservation of mitochondrial bioenergetics. This study is aimed at evaluating whether or not curcumin's protective effect in mitochondrial bioenergetics is related to the modulation of mitochondrial dynamics and biogenesis. Wistar rats were treated with a single subcutaneous dose of K2Cr2O7 (12.5 mg/kg) or received curcumin (400 mg/kg/day) by oral gavage 10 days before and one day after the K2Cr2O7 injection. K2Cr2O7 induced kidney dysfunction and increased mitochondrial hydrogen peroxide production, while decreasing the respiration directly attributable to oxidative phosphorylation and mitochondrial membrane potential. In mitochondria, K2Cr2O7 increased fission and reduced fusion. Structural analysis of mitochondria in the proximal tubular cells corroborated their fragmentation and loss of crests' integrity. Regarding mitochondrial biogenesis, K2Cr2O7 decreased peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) levels. Conversely, curcumin treatment mitigated the aforementioned alterations and increased the expression of the mitochondrial transcription factor A (TFAM). Taken together, our results suggest that curcumin can protect against renal injury by modulating mitochondrial homeostasis, mitigating alterations in bioenergetics and dynamics, possibly by stimulating mitochondrial biogenesis.

The Protective Effect of Alpha-Mangostin against Cisplatin-Induced Cell Death in LLC-PK1 Cells is Associated to Mitochondrial Function Preservation.

Cis-dichlorodiammineplatinum II (CDDP) is a chemotherapeutic agent that induces nephrotoxicity by different mechanisms, including oxidative stress, mitochondrial dysfunction, autophagy, and endoplasmic reticulum stress. This study aimed to evaluate if the protective effects of the antioxidant alpha-mangostin (αM) in CDDP-induced damage in proximal tubule Lilly laboratory culture porcine kidney (LLC-PK1) cells, are related to mitochondrial function preservation. It was found that αM co-incubation prevented CDDP-induced cell death. Furthermore, αM prevented the CDDP-induced decrease in cell respiratory states, in the maximum capacity of the electron transfer system (E) and in the respiration associated to oxidative phosphorylation (OXPHOS). CDDP also decreased the protein levels of voltage dependence anion channel (VDAC) and mitochondrial complex subunits, which together with the reduction in E, the mitofusin 2 decrease and the mitochondrial network fragmentation observed by MitoTracker Green, suggest the mitochondrial morphology alteration and the decrease in mitochondrial mass induced by CDDP. CDDP also induced the reduction in mitochondrial biogenesis observed by transcription factor A, mitochondria (TFAM) decreased protein-level and the increase in mitophagy. All these changes were prevented by αM. Taken together, our results imply that αM's protective effects in CDDP-induced toxicity in LLC-PK1 cells are associated to mitochondrial function preservation.

Sulforaphane prevents maleic acid-induced nephropathy by modulating renal hemodynamics, mitochondrial bioenergetics and oxidative stress.

Maleic acid (MA)-induced nephropathy that is characterized by proteinuria, glycosuria, phosphaturia and a deficient urinary acidification and concentration. Sulforaphane (SF) is an indirect antioxidant that shows nephroprotective effects. The aim of the present work was to test the pre-treatment with SF against the MA-induced nephropathy. Wistar rats (230-260 g) were separated in the following groups: control, MA (which received 400 mg/kg of MA), SF + MA (which received MA and 1 mg/kg of SF each day for four days) and SF (which only received SF). MA induced proteinuria, an increase in urinary excretion of N-acetyl-ß-d-glucosaminidase, and a decrease in plasma glutathione peroxidase activity, renal blood flow, and oxygenation and perfusion of renal cortex. All these impairments correlated with higher levels of oxidative damage markers and exacerbated superoxide anion production on renal cortex. Moreover, MA impaired mitochondrial bioenergetics associated to complex I, mitochondrial membrane potential and respiratory control index and increased the mitochondrial production of hydrogen peroxide. Further it disrupted mitochondrial morphology. SF prevented all the above-described alterations. In conclusion, the protective effect of SF against MA-induced nephropathy is associated with preservation of mitochondrial bioenergetics, amelioration of oxidative stress and improvement of renal hemodynamics and renal cortex oxygenation.

Formalin injection produces long-lasting hypersensitivity with characteristics of neuropathic pain.

The purpose of this study was to investigate whether 1%, 2% or 5% formalin injection produce hypersensitivity with characteristics of the neuropathic pain induced by spinal nerve injury. Formalin injection (1%, 2% and 5%) produced concentration-dependent long-lasting (at least 14 days) mechanical allodynia and hyperalgesia in both paws. Likewise, L5/L6 spinal nerve ligation induced allodynia and hyperalgesia in both paws. The intensity of hypersensitivity was greater in the ipsilateral than in the contralateral paw in all models. Systemic gabapentin or morphine completely reduced 1% formalin-induced hypersensitivity. In contrast, both drugs were not able to fully diminish 2-5% formalin- and nerve injury-induced hypersensitivity. Indomethacin produced a significant effect in the chronic 1% formalin test. Conversely, this drug did not modify 2 or 5% formalin- and nerve injury-induced hypersensitivity. Spinal nerve injury and 2-5%, but not 1%, formalin injection enhanced ATF3 protein expression and immunofluorescence in dorsal root ganglia (DRG) in a time-dependent manner. Furthermore, 2-5%, but not 1%, formalin injection or spinal nerve injury also enhanced α2δ-1 subunit protein levels in DRG. Our results suggest that 5% and, at lesser extent, 2% formalin injection produces long-lasting hypersensitivity with a pharmacological and molecular pattern that resembles neuropathic pain induced by spinal nerve ligation.

Role of spinal 5-HT5A, and 5-HT1A/1B/1D, receptors in neuropathic pain induced by spinal nerve ligation in rats.

Serotonin (5-HT) participates in pain modulation by interacting with different 5-HT receptors. The role of 5-HT5A receptor in neuropathic pain has not previously studied. The purpose of this study was to investigate: A) the role of 5-HT5A receptors in rats subjected to spinal nerve injury; B) the expression of 5-HT5A receptors in dorsal spinal cord and dorsal root ganglia (DRG). Neuropathic pain was induced by L5/L6 spinal nerve ligation. Tactile allodynia in neuropathic rats was assessed with von Frey filaments. Western blot methodology was used to determine 5-HT5A receptor protein expression. Intrathecal administration (on day 14th) of 5-HT (10-100 nmol) or 5-carboxamidotryptamine (5-CT, 0.03-0.3 nmol) reversed nerve injury-induced tactile allodynia. Intrathecal non-selective (methiothepin, 0.1-0.8 nmol) and selective (SB-699551, 1-10 nmol) 5-HT5A receptor antagonists reduced, by ~60% and ~25%, respectively, the antiallodynic effect of 5-HT (100 nmol) or 5-CT (0.3 nmol). Moreover, both selective 5-HT1A and 5-HT1B/1D receptor antagonists, WAY-100635 (0.3-1 nmol) and GR-127935 (0.3-1 nmol), respectively, partially diminished the antiallodynic effect of 5-HT or 5-CT by about 30%. Injection of antagonists, by themselves, did not affect allodynia. 5-HT5A receptors were expressed in the ipsilateral dorsal lumbar spinal cord and DRG and L5/L6 spinal nerve ligation did not modify 5-HT5A receptor protein expression in those sites. Results suggest that 5-HT5A receptors reduce pain processing in the spinal cord and that 5-HT and 5-CT reduce neuropathic pain through activation of 5-HT5A and 5-HT1A/1B/1D receptors. These receptors could be an important part of the descending pain inhibitory system.