Isolated Rat Kidney Research Articles

Interaction of Angiotensin-(1−7) with kinins in the kidney circulation: Role of B1 receptors

Changes in renal hemodynamics impact renal function during physiological and pathological conditions. In this context, renal vascular resistance (RVR) is regulated by components of the Renin-Angiotensin System (RAS) and the Kallikrein-Kinin System (KKS). However, the interaction between these vasoactive peptides on RVR is still poorly understood. Here, we studied the crosstalk between angiotensin-(1−7) and kinins on RVR. The right kidneys of Wistar rats were isolated and perfused in a closed-circuit system. The perfusion pressure and renal perfusate flow were continuously monitored. Ang-(1−7) (1.0–25.0 nM) caused a sustained, dose-dependent reduction of relative RVR (rRVR). This phenomenon was sensitive to 10 nM A-779, a specific Mas receptor (MasR) antagonist. Bradykinin (BK) promoted a sustained and transient reduction in rRVR at 1.25 nM and 125 nM, respectively. The transient effect was abolished by 4 μM des-Arg9-Leu8-bradykinin (DALBK), a specific kinin B1 receptor (B1R) antagonist. Accordingly, des-Arg9-bradykinin (DABK) 1 μM (a B1R agonist) increased rRVR. Interestingly, pre-perfusion of Ang-(1−7) changed the sustained reduction of rRVR triggered by 1.25 nM BK into a transient effect. On the other hand, pre-perfusion of Ang-(1−7) primed and potentiated the DABK response, this mechanism being sensitive to A-779 and DALBK. Binding studies performed with CHO cells stably transfected with MasR, B1R, and kinin B2 receptor (B2R) showed no direct interaction between Ang-(1−7) with B1R or B2R. In conclusion, our findings suggest that Ang-(1−7) differentially modulates kinin's effect on RVR in isolated rat kidneys. These results help to expand the current knowledge regarding the crosstalk between the RAS and KKS complex network in RVR.

Isotopomer analyses with the tricarboxylic acid cycle intermediates and exchanging metabolites from the rat kidney.

Renal metabolism is essential for kidney functions and energy homeostasis in the body. The TCA cycle is the hub of metabolism, but the metabolic activities of the cycle in the kidney have rarely been investigated. This study is to assess metabolic processes at the level of the TCA cycle in the kidney based on isotopomer distributions in multiple metabolites. Isolated rat kidneys were perfused with media containing common substrates including lactate and alanine for an hour. One group of kidneys received [U-13 C3 ]lactate instead of natural abundance lactate while the other group received [U-13 C3 ]alanine instead of natural abundance alanine. Perfused kidneys and effluent were prepared for analysis using NMR spectroscopy. 13 C-labeling patterns in glutamate, fumarate, aspartate and succinate from the kidney extracts showed that pyruvate carboxylase and oxidative metabolism through the TCA cycle were comparably very active, but pyruvate cycling and pyruvate dehydrogenase were relatively less active. Isotopomer analyses with fumarate and malate from effluent, however, indicated that pyruvate carboxylase was much more active than the TCA cycle and other metabolic processes. The reverse equilibrium of oxaloacetate with four-carbon intermediates of the cycle was nearly complete (92%), based on the ratio of [2,3,4-13 C3 ]/[1,2,3-13 C3 ] in aspartate or malate. 13 C enrichment in glucose with 13 C-lactate supply was higher than that with 13 C-alanine. Isotopomer analyses with multiple metabolites (i.e., glutamate, fumarate, aspartate, succinate and malate) allowed us to assess relative metabolic processes in the TCA cycle in the kidney supplied with [U-13 C3 ]lactate. Data from the analytes were generally consistent, indicating highly active pyruvate carboxylase and oxidative metabolism through the TCA cycle. Different 13 C-labeling patterns in analytes from the kidney extracts versus effluent suggested metabolic compartmentalization.

Validation of Freshly Isolated Rat Renal Cells as a Tool for Preclinical Assessment of Radiolabeled Receptor-Specific Peptide Uptake in the Kidney.

The synthetic analogs of regulatory peptides radiolabeled with adequate radionuclides are perspective tools in nuclear medicine. However, undesirable uptake and retention in the kidney limit their application. Specific in vitro methods are used to evaluate undesirable renal accumulation. Therefore, we investigated the usefulness of freshly isolated rat renal cells for evaluating renal cellular uptake of receptor-specific peptide analogs. Special attention was given to megalin as this transport system is an important contributor to the active renal uptake of the peptides. Freshly isolated renal cells were obtained from native rat kidneys by the collagenase method. Compounds with known accumulation in renal cells were used to verify the viability of cellular transport systems. Megalin expressions in isolated rat renal cells were compared to two other potential renal cell models by Western blotting. Specific tubular cell markers were used to confirm the presence of proximal tubular cells expressing megalin in isolated rat renal cell preparations by immunohistochemistry. Colocalization experiments on isolated rat kidney cells confirmed the presence of proximal tubular cells bearing megalin in preparations. The applicability of the method was tested by an accumulation study with several analogs of somatostatin and gastrin labeled with indium-111 or lutetium-177. Therefore, isolated rat renal cells may be an effective screening tool for in vitro analyses of renal uptake and comparative renal accumulation studies of radiolabeled peptides or other radiolabeled compounds with potential nephrotoxicity.

Cover Image, Volume 36, Issue 3

The cover image is based on the Research Article Oxidation of hyperpolarized [1-13C]pyruvate in isolated rat kidneys by Gaurav Sharma et al., https://doi.org/10.1002/nbm.4857.

Oxidation of hyperpolarized [1-13 C]pyruvate in isolated rat kidneys.

Kidneys play a central role in numerous disorders but current imaging methods have limited utility to probe renal metabolism. Hyperpolarized (HP) 13 C magnetic resonance imaging is uniquely suited to provide metabolite-specific information about key biochemical pathways and it offers the further advantage that renal imaging is practical in humans. This study evaluated the feasibility of hyperpolarization examinations in a widely used model for analysis of renal physiology, the isolated kidney, which enables isolation of renal metabolism from the effects of other organs and validation of HP results by independent measurements. Isolated rat kidneys were supplied with either HP [1-13 C]pyruvate only or HP [1-13 C]pyruvate plus octanoate. Metabolic activity in both groups was confirmed by stable renal oxygen consumption. HP [1-13 C]pyruvate was readily metabolized to [13 C]bicarbonate, [1-13 C]lactate, and [1-13 C]alanine, detectable seconds after HP [1-13 C]pyruvate was injected. Octanoate suppressed but did not eliminate the production of HP [13 C]bicarbonate from [1-13 C]pyruvate. Steady-state flux analyses using non-HP 13 C substrates validated the utilization of HP [1-13 C]pyruvate, as observed by HP 13 C NMR. In the presence of octanoate, lactate is generated from a tricarboxylic acid cycle intermediate, oxaloacetate. The isolated rat kidney may serve as an excellent model for investigating and establishing new HP 13 C metabolic probes for future kidney imaging applications.

Peroxiredoxin 6 Prevents Reperfusion Injury to Isolated Rat Kidney

Peroxiredoxin 6 Prevents Reperfusion Injury to Isolated Rat Kidney

MO940: Effects of Erythrocytes and Albumin in The Perfusate of an In Situ Perfusion Model of Isolated Rat Kidney

Abstract BACKGROUND AND AIMS The conventional perfusion model of the isolated rat kidney circulates perfusate to the isolated kidney removed from the body. On the other hand, an in situ perfusion model, within the body, of an isolated kidney is expected to allow research on drug metabolism and physiological functioning of the kidney under physiological conditions and also allow local organ therapy, such as selective perfusion of the organ with therapeutic agents such as anticancer agents. The aims of the present study were to establish an in situ perfusion model of an isolated rat kidney and to clarify the composition of the perfusate necessary to obtain physiological perfusion of the isolated kidney model. METHOD Male Sprague Dawley rats (300–350 g) were used for the experiments. Cannulations of the renal vein, renal artery and ureter were performed, in that order, for the left kidney, and the perfusion experiment was performed for 2 h. The flow rate of the perfusate during the organ perfusion was adjusted (0.3–2.0 mL/min) to maintain the renal artery pressure at 100–120 mm Hg. The circulation conditions were adjusted to maintain the renal artery pressure in the aforementioned range and urine production until the end of the perfusion experiment. The perfusate was oxidized with a 95% oxygen and 5% carbon dioxide gas mixture during the experiment and had the following composition: Na+, 140 mEq/L; K+, 5 mEq/L; Ca2+, 1.9 mEq/L; Mg2+, 2.5 mEq/L; Cl−, 126 Eq/L; and HCO3−, 25 mEq/L. The effects of erythrocytes (Ht: 25%) in the perfusate on the renal arterial pressure, urine production rate and oxygen-carrying capacity were examined, as also those of albumin (4 g/dL) in the erythrocyte-containing perfusate on the blood flow resistance of the kidney and urine production rate during the circulation experiment. RESULTS When a perfusate not containing erythrocytes was used, urine production from the isolated kidney decreased with time, and the renal artery pressure failed to be maintained within the target range. In contrast, when a perfusate containing erythrocytes was used, urine was constantly produced, and the renal artery pressure was maintained within the target range throughout the 2-hour experimental period. When a perfusate containing erythrocytes and albumin was used, the urine production rate of the kidney was close to the physiological urine production rate in rats and significantly lower than that observed when the perfusate did not contain albumin. Urine production was maintained from the isolated rat kidney until the end of the perfusion experiment when the perfusate contained erythrocytes, suggesting that a perfusate with adequate viscosity and sufficient oxygen-carrying capacity is important to maintain the renal artery pressure and renal functions. The addition of albumin to the perfusate containing erythrocytes resulted in appropriate physiological urine production, probably due to the higher colloid osmotic pressure of the perfusate. CONCLUSION We established an in situ perfusion model of an isolated rat kidney that exhibited almost physiological functioning when the perfusate used contained erythrocytes to supply sufficient oxygen and albumin to maintain the colloid osmotic pressure.

The effect of exogenous peroxiredoxin 6 on morphofunctional state of isolated rat kidney

Objective: to investigate the role of peroxiredoxin 6 (PRX6) in preserving the morphofunctional state of ischemic isolated kidney during perfusion.Materials and methods. The model of an isolated perfused rat kidney was used. Ischemia time was 5 and 20 minutes, perfusion was 50 minutes. To evaluate the effectiveness of PRX6 at different ischemia times, we used the conventional criteria of kidney function and histological methods.Results. During short warm ischemia times, exogenous PRX6 improves the morphofunctional state of an isolated kidney during perfusion. During this period, the main criteria for functioning of the isolated ischemic kidney reach acceptable values, renal parenchyma is without severe damage. By the end of perfusion, there was an increase in urine flow rate, glomerular filtration rate, fractional glucose reabsorption, urine urea concentration and proportion of primary urine from 1.5 to 2 times compared with the control lesion. At 20-minute ischemia, the isolated kidney can be recognized as non-viable according to the functioning criteria; the positive effect of PRX6 is leveled.Conclusion. The use of recombinant peroxiredoxin 6 for preserving the morphofunctional state of isolated kidneys can be an effective approach in preventing ischemia–reperfusion injury.

Bothrops pauloensis snake venom-derived Asp-49 and Lys-49 phospholipases A2 mediates acute kidney injury by oxidative stress and release of inflammatory cytokines

The envenomation caused by the Bothrops pauloensis snake leads to severe local and systemic effects including acute kidney injury. In this study, we investigated the renal effects by phospholipases A2 (PLA2s), divided into two main subgroups, Asp-49 and Lys-49, isolated from the Bothrops pauloensis snake venom (BpV) in isolated rat kidney system. Both PLA2s (3 μg/mL), added alone to the perfusion system and analyzed for 120 min, had significant effects on isolated rat kidney. Asp-49 reduced Glomerular Filtration Rate (GFR) at 60, 90 and 120 min, and the percentage of total tubular sodium transport (%TNa+) and potassium transport (%TK+) at 120 min. Lys-49 increased Perfusion Pressure (PP) at 120 min and reduced GFR, %TNa+ and the percentage of total tubular chloride transport (%TCl−) at 60, 90 and 120 min. Cytokine release in the kidney tissues were increased with Asp-49 PLA2 (IL-10) and Lys-49 PLA2 (TNF-α, IL-1β, IL-10). Both increased MPO activity. Asp-49 PLA2 decreased Glutathione (GSH) and increased nitrite levels, while Lys-49 PLA2 increased Malondialdehyde (MDA), GSH and nitrite levels. Histological analysis of the perfused kidneys revealed the presence of glomerular degeneration and atrophy, deposit of proteinaceous material in Bowman's space and intratubular with both PLA2s. These findings indicated that both PLA2s modified the functional parameters in an isolated perfused kidney model with increased oxidative stress and cytokine release. PLA2s are one of the components at high concentration in BpV and our results provide important knowledge about their involvement with the nephrotoxic mechanism.

Electrolyte handling in the isolated perfused rat kidney: demonstration of vasopressin V2-receptor-dependent calcium reabsorption

Background The most profound effect of vasopressin on the kidney is to increase water reabsorption through V2-receptor (V2R) stimulation, but there are also data suggesting effects on calcium transport. To address this issue, we have established an isolated perfused kidney model with accurate pressure control, to directly study the effects of V2R stimulation on kidney function, isolated from systemic effects. Methods The role of V2R in renal calcium handling was studied in isolated rat kidneys using a new pressure control system that uses a calibration curve to compensate for the internal pressure drop up to the tip of the perfusion cannula. Results Kidneys subjected to V2R stimulation using desmopressin (DDAVP) displayed stable osmolality and calcium reabsorption throughout the experiment, whereas kidneys not administered DDAVP exhibited a simultaneous fall in urine osmolality and calcium reabsorption. Epithelial sodium channel (ENaC) inhibition using amiloride resulted in a marked increase in potassium reabsorption along with decreased sodium reabsorption. Conclusions A stable isolated perfused kidney model with computer-controlled pressure regulation was developed, which retained key physiological functions. The preparation responds to pharmacological inhibition of ENaC channels and activation of V2R. Using the model, the dynamic effects of V2R stimulation on calcium handling and urine osmolality could be visualised. The study thereby provides evidence for a stimulatory role of V2R in renal calcium reabsorption.

The Effect of Exogenous Peroxiredoxin 6 on the Functional Parameters of the Isolated Rat Kidney

Abstract—The aim of this study was to investigate the protective effect of exogenous peroxiredoxins with ischemia–reperfusion of an isolated kidney. The study was carried out using a model of isolated rat kidney perfusion ex vivo. A recombinant peroxiredoxin 6 was injected directly in the perfusion buffer. The high-molecular-weight dye Blue Dextran 2000 and urea were added to the perfusion buffer to determine the functionality of an isolated kidney. It was demonstrated that exogenous peroxiredoxin 6 in the cortical layer of an isolated kidney was localized in the vessels of renal glomeruli; in the medulla it was found in microvessels surrounding the thin tubules. The use of peroxiredoxin 6 decreases the degree of damage of nephron structures by two times compared with the damage in the control, which provides the preservation of ultrafiltration processes. A decrease in glomerular damage leads to a decrease in the content of Blue Dextran by two times compared with the damage in the control at the end of the perfusion period. A decrease in the damage of tubular structures indicates the active urea transport during perfusion. Thus, the inclusion of peroxiredoxin 6 in the perfusion buffer mediated a decrease in the damage of nephron structures and maintenance of the multifunctional state of renal glomeruli and tubules.

Renal Alterations Induced by the Venom of Colombian Scorpion Centruroides Margaritatus.

Scorpion venom causes renal injury and affects vascular ion-channels function. Centruroides margaritatus scorpion is found in Colombia and is frequently the cause of envenomation accidents; however, its renal impact has never been investigated. To evaluate the effects of C. margaritatus venom (CmV) on renal parameters using isolated rat kidney and renal cell culture models. Wistar rats (n = 5, weighing 240-300 g) were first perfused with Krebs-Henseleit solution containing 6 g 100 mL-1 bovine serum albumin. After 30 minutes, the kidneys were perfused with CmV to a final concentration of 10 μgmL-1; evaluation was performed by measuring Perfusion Pressure (PP), Renal Vascular Resistance (RVR), Urinary Flow (UF), Glomerular Filtration Rate (GFR), and percentage of electrolyte tubular transport. Moreover, kidney histological analyses and cell cytotoxicity in renal tubule epithelial cells (MDCK) and proximal tubular cells (LLC-MK2) were assessed. CmV increased PP and RVR 60 min after perfusion. On the other hand, UF, GFR, and the percentages of sodium, potassium and chloride tubular transport decreased after experimental envenomation. UF dropped after 120 min, while GFR and percentage of electrolyte tubular transport diminished after 60, 90 and 120 min. CmV was not toxic to MDCK cell line but reduced the viability of LLC-MK2 cells at concentrations ranging from 6.25 to 200 μgmL-1. Histological analyses disclosed hydropic degeneration, edema, and protein deposits. Flow cytometry disclosed that cell death occurred predominantly by necrosis. Our results suggest that C. margaritatus venom can trigger renal impairment, mainly in the proximal kidney tubule.

Nephrotoxicity induced by the venom of Hypnale hypnale from Sri Lanka: Studies on isolated perfused rat kidney and renal tubular cell lines

The hump-nosed pit viper Hypnale hypnale is responsible for a high number of snakebite cases in southwestern India and Sri Lanka. Although most patients only develop local signs and symptoms of envenoming, there is a growing body of evidence indicating that these envenomings may be associated with systemic alterations, including acute kidney injury. In this study we evaluated the renal toxicity of H. hypnale venom by using a perfused isolated rat kidney system and by assessing cytotoxicity in two different renal tubular cell lines in culture. The venom caused alterations in several renal functional parameters, such as reduction on perfusion pressure, renal vascular resistance, and sodium and chloride tubular transport, whereas glomerular filtration rate and urinary flow initially decreased and then increased after venom perfusion. In addition, this venom was cytotoxic to proximal and distal renal tubular cells in culture, with predominance of necrosis over apoptosis. Moreover, the venom affected the mitochondrial membrane potential and induced an increment in reactive oxygen species in these cells. Taken together, our results demonstrate a nephrotoxic activity of H. hypnale venom in these experimental models, in agreement with clinical observations.

Renal Effects Induced by Two Phospholipase A2 (Asp‐49 and Lys‐49) Isolated from Bothropoides pauloensis Snake Venom

Acute renal failure (ARF) wich is one of the most serious complications of Bothrops snakebites, is one of the main causes of death and consequences for victims. However, its pathogenesis remains unclear. Considering that isolated toxins are relevant tools for understanding the actions of the whole venom, we studied properties of phospholipases A2 (Asp‐49 and Lys‐49) isolated from Bothrops pauloensis (from State of São Paulo, Southeast region of Brazil), on isolated perfused kidney of rat. The control group perfused with modified Krebs–Henseleit solution alone. The PLA2s (3μg/mL) caused significant effects in isolated rat kidney, Asp‐49 reducing perfusion pressure(PP), the Renal Vascular Resistence (RVR), the Urinary Flow (UF), the Glomerular Filtration Rate (GFR) and the percentage of tubular sodium (%TNa+), chloride (%TCl−) and potassium (%TK+). The Lys‐49 increased PP and UF and decreased the RFG and the %TNa+, %TK+ and %TCl‐. Oxidative stress in kidney tissue was evaluated by TBARS (thiobarbituric acid reactive substances), Nitrite and reduced Glutathione (GSH) level measurement. A significant increase was observed in TBARS (Lys‐49) and Nitrito (Asp‐49 and Lys‐49) and a decrease in GSH levels (Asp‐49) and increase (Lys‐49). The levels of inflammatory cytokines were measured. We analized the inflammatory cytokines, pro‐inflammatory interleukin 1 (IL‐1), tumor necrosis factor‐α (TNF‐α) and anti‐inflammatory interleukin 10 (IL‐10). The results showed high levels of both cytokines (by Lys‐49) and incresed TNF‐α and IL‐10 by Asp‐49. Increased TBARS, Nitrito level and decreased of GSH activity might be implicated in the presence of oxidative stress in patients with snake bite envenomation. These findings demonstrated that PLA2s caused nephrotoxicity in isolated kidney. The characterization of the effects in the isolated kidney gives strong evidences that the acute renal failure induced by these PLA2s is a result of the direct nephrotoxicity which may involve oxidative and inflammatory mechanisms.Support or Funding InformationCAPES, CNPq, UFC.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A new protocol for isoheart rat kydney blood perfusion

Introduction and the purpose of the work. The perfusion of isolated laboratory animal organs is important for estimation of the effectiveness of methods elaborated for donor organ preservation. Materials and methods. Hemoperfusion of isolated rat kidney was performed after long-term hypothermal ischemia in order to reproduce reperfusion for the subsequent estimation of morphological changes in the cloth of nephritic parenchyma. Results. This method proved to be effective and technically simpler than different modifications of the classical procedure of kidney transplantation in small laboratory animals. Conclusions. The proposed procedure is an alternative to the classical kidney transplantation if reperfusion is required in an acute experiment for several hours.

High Salt Intake Promotes Different Responses to Urodilatin and Uroguanylin in the Isolated Rat Kidney.

Urodilatin (UD) and uroguanylin (UGN) have been implicated in the regulation of salt and water homeostasis, particularly in the balance handling of salt intake. In this sense, the aim of the present work was to study the main effects of these peptides in kidneys from animals subjected to high NaCl (2%) intake, during 10 days in metabolic cages. The control group received only normal water, whereas the treated group drank 2% solution of NaCl (NaCl 2%). In addition, we studied effect of subthreshold UD (0.14 nM) and UGN (0.06 μM) doses in NaCl 2% after a 30-min control period. Kidney perfusion was performed with Krebs-Henseleit containing 6 g% bovine albumin previously dialyzed. The effects of UD (0.14 nM) promoted reduction of PP, RVR, and UF in the NaCl 2% group. We also observed an increase in %TNa+ and %TCl-. The main effects of UGN in NaCl 2% were increase in PP, UF, and GFR, followed by a reduction in %TNa+ and %TCl-. After an increased intake of salt, physiological pathways are activated and regulated in order to eliminate excess sodium. In this study, we observed that in a subthreshold dose, UD does not promotes natriuresis and diuresis, suggesting that UGN is an important hormone in inducing salt excretion in a chronic salt overload. Therefore, the effects herein described may play a contributory role in the regulation of kidney function after ingestion of salty meals.

Perfusion of isolated rat kidney with Mesenchymal Stromal Cells/Extracellular Vesicles prevents ischaemic injury.

Kidney donation after circulatory death (DCD) is a less than ideal option to meet organ shortages. Hypothermic machine perfusion (HMP) with Belzer solution (BS) improves the viability of DCD kidneys, although the graft clinical course remains critical. Mesenchymal stromal cells (MSC) promote tissue repair by releasing extracellular vesicles (EV). We evaluated whether delivering MSC‐/MSC‐derived EV during HMP protects rat DCD kidneys from ischaemic injury and investigated the underlying pathogenic mechanisms. Warm ischaemic isolated kidneys were cold‐perfused (4 hrs) with BS, BS supplemented with MSC or EV. Renal damage was evaluated by histology and renal gene expression by microarray analysis, RT‐PCR. Malondialdehyde, lactate, LDH, glucose and pyruvate were measured in the effluent fluid. MSC‐/EV‐treated kidneys showed significantly less global ischaemic damage. In the MSC/EV groups, there was up‐regulation of three genes encoding enzymes known to improve cell energy metabolism and three genes encoding proteins involved in ion membrane transport. In the effluent fluid, lactate, LDH, MDA and glucose were significantly lower and pyruvate higher in MSC/EV kidneys as compared with BS, suggesting the larger use of energy substrates by MSC/EV kidneys. The addition of MSC/EV to BS during HMP protects the kidney from ischaemic injury by preserving the enzymatic machinery essential for cell viability and protects the kidney from reperfusion damage.

Polyphenolic compositions and in vitro angiotensin-I-converting enzyme inhibitory properties of common green leafy vegetables: A comparative study.

This study compared the phenolic compositions of common green leafy vegetable extracts from Vernonia amygdalina (VA), Telfairia occidentalis (TO), Talinium triangulare (TT), and Amaranthus hybridus (AH) and their effects on the angiotensin-I-converting enzyme (ACE) and cisplatin-induced malonylaldehyde (MDA) production in an isolated rat kidney homogenate. HPLC confirmed the presence of phenolic compounds in the extracts. Furthermore, all extracts inhibited ACE activity dosedependently; however, the extract from VA exhibited the highest ACE activity while TT exhibited the least. Incubation of the kidney homogenate with 1mM cisplatin caused an increase in MDA production; however, all the extracts inhibited the level of MDA produced. Nevertheless, VA extract exhibited the highest inhibition. These activities of the vegetable extracts could be attributed to their phenolic compositions and may suggest some possible mechanism of the actions. However, VA appeared to be the most potent among the vegetables tested.

Effects of Tityus stigmurus (Thorell 1876) (Scorpiones: Buthidae) venom in isolated perfused rat kidneys.

Scorpions belonging to the Tityus genus are of medical interest in Brazil. Among them, Tityus stigmurus is the main scorpion responsible for stings in the Northeast region. After a sting, the scorpion venom distributes rapidly to the organs, reaching the kidneys quickly. However, there are few studies concerning the renal pathophysiology of scorpion poisoning. In this study, we evaluated the effects of T. stigmurus venom (TsV) on renal parameters in isolated rat kidneys. Wistar rats (n = 6), weighing 250-300 g, were perfused with Krebs-Henseleit solution containing 6 g/100 mL bovine serum albumin. TsV at 0.3 and 1.0 μg/mL was tested, and the effects on perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR), and electrolyte excretion were analyzed. Effects were observed only at TsV concentration of 1.0 μg/mL, which increased PP (controlPP40' = 92.7 ± 1.95; TsVPP40' = 182.0 ± 4.70* mmHg, *p < 0.05), RVR (controlRVR40' = 3.28 ± 0.23 mmHg; TstRVR40' = 6.76 ± 0.45* mmHg, *p < 0.05), UF (controlUF50' = 0.16 ± 0.04; TstUF50' = 0.60 ± 0.10* mL/g/min,*p < 0.05), GFR and electrolyte excretion, with histological changes that indicate renal tubular injury. In conclusion, T. stigmurus venom induces a transient increase in PP with tubular injury, both of which lead to an augmented electrolyte excretion.

Diglycolic acid, the toxic metabolite of diethylene glycol, chelates calcium and produces renal mitochondrial dysfunction in vitro

Context: Diethylene glycol (DEG) has caused many cases of acute kidney injury and deaths worldwide. Diglycolic acid (DGA) is the metabolite responsible for the renal toxicity, but its toxic mechanism remains unclear. Objective: To characterize the mitochondrial dysfunction produced from DGA by examining several mitochondrial processes potentially contributing to renal cell toxicity. Materials and methods: The effect of DGA on mitochondrial membrane potential was examined in normal human proximal tubule (HPT) cells. Isolated rat kidney mitochondria were used to assess the effects of DGA on mitochondrial function, including respiratory parameters (States 3 and 4), electron transport chain complex activities and calcium-induced opening of the mitochondrial permeability transition pore. DGA was compared with ethylene glycol tetraacetic acid (EGTA) to determine calcium chelating ability. DGA cytotoxicity was assessed using lactate dehydrogenase leakage from cultured proximal tubule cells. Results: DGA decreased the mitochondrial membrane potential in HPT cells. In rat kidney mitochondria, DGA decreased State 3 respiration, but did not affect State 4 respiration or the ADP/O ratio. DGA reduced glutamate/malate respiration at lower DGA concentrations (0.5 mmol/L) than succinate respiration (100 mmol/L). DGA inhibited Complex II activity without altering Complex I, III or IV activities. DGA blocked calcium-induced mitochondrial swelling, indicating inhibition of the calcium-dependent mitochondrial permeability transition. DGA and EGTA reduced the free calcium concentration in solution in an equimolar manner. DGA toxicity and mitochondrial dysfunction occurred as similar concentrations. Discussion: DGA inhibited mitochondrial respiration, but without uncoupling oxidative phosphorylation. The more potent effect of DGA on glutamate/malate respiration and the inhibition of mitochondrial swelling was likely due to its chelation of calcium. Conclusion: These results indicate that DGA produces mitochondrial dysfunction by chelating calcium to decrease the availability of substrates and of reducing equivalents to access Complex I and by inhibiting Complex II activity at higher concentrations.