Renal Mitochondrial Respiration Research Articles

The effect of chronic unpredictable stress on renal mitochondrial function in female mice

Background: Chronic stress can induce high blood pressure and lead to decreased renal function by mechanisms that remain unclear. The exposure of rodents to chronic unpredictable stress (CUS) is a translational model used for investigating the physiological impact of stress. The CUS protocol consists of unpredictable and random stressors that mimic real life exposure to stress. While stress is known to impact mitochondrial function, little is understood about the direct impact of chronic stress on renal mitochondrial respiration. Moreover, while studies have shown a link between estrogen and mitochondria through estrogen receptors, the relation between stress induced renal mitochondrial dysfunction and estrous cycle is unknown. Hypothesis: Exposing female mice to CUS will increase blood pressure and cause decreased renal mitochondrial respiration. Methods: We exposed adult female C57BL/6 mice to CUS for 28 days. Before and after stress, all control (CON, n=8) and CUS (n=10) mice were placed in metabolic cages for overnight urine collection. Urine albumin was measured by dipstick assay. On day 28, mice were assessed for blood pressure measurement via tail cuff. 24 hours later, mice were euthanized, and vaginal lavage was performed. The left kidney was removed, transected into thirds, and the poles were placed in relaxing buffer. Kidney mitochondrial oxygen consumption was assessed using high-resolution respirometry. Results: Data were stratified by phases of the estrus cycle. Neither blood pressure nor urinary albumin was significantly altered by CUS in stressed females. Females in estrus and proestrus exposed to CUS (n=5) exhibited a decrease of 20.8% ± 5.1 (n=5, p=0.03) in renal mitochondrial maximal uncoupled respiration compared to non-stressed CON. Renal mitochondrial respiration in diestrus was similar between CUS and CON. Conclusion: Results from this study reveal a link between the female estrus cycle, stress, and mitochondrial function. This work was supported by NIH grants P20GM109091 (F.H.), R01HL130972-01A1 (F.G.S.), R01HL5949 (F.G.S.), RO1DK132948 (C.W. & F.P.) and R01 MH129798 (SKW); VA grants: VISN7 RDA (F.H.), Merit awards: BX000168-10A1 (F.G.S.), BX005320 (F.G.S.), and BX002604 (M.J.R.). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Diurnal Differences in Mitochondrial Bioenergetics is Lost in Bmal1 Knockout Rats

In the kidney, 95% of O2 consumed through mitochondrial respiration is used for Na+ transport. The circadian clock regulates Na+ transport with a necessary diurnal variation for optimal health. The circadian clock transcription factor BMAL1 is proposed to regulate mitochondrial bioenergetic function; however, this has not been extensively investigated in the kidney. Therefore, we tested the hypothesis that renal mitochondrial respiration may be impaired or altered in a novel Bmal1 clock gene knock‐out rat model and is associated with loss of diurnal control of Na+ excretion. Male and female global Bmal1+/+ and Bmal1‐/‐ rats at 12‐14 weeks of age maintained in a regular 12‐hour light/dark cycle was used to measure renal mitochondrial O2consumption. Outer renal medullary tissue was dissected and prepared for respiration measurements at either day (ZT2‐4) or night (ZT14‐16) periods to correspond with minimum and maximum whole‐body energy consumption as well as peak and trough BMAL1 protein expression, respectively (n = 10, Bmal1+/+; n = 22, Bmal1‐/‐). Respiration was assessed using permeabilized tissue in the Oroboros Oxygraph and analyzed using Data Lab 2 software (O2K, Oroboros Instruments GmbH). Mitochondrial gene expression analysis was assessed using digital drop PCR. Data from both sexes were combined for each genotype at the two time points and analyzed by two‐way ANOVA. Mitochondrial state 3 O2 consumption was significantly higher (main effect of genotype p = 0.0310) in Bmal1‐/‐ (118 ± 156 pmol/s*mg) compared to BMAL1+/+ rats (75 ± 15 pmol/s*mg; Tukey post‐hoc p = 0.0303). We also observed a significant increase in complex IV activity in Bmal1‐/‐ rats during the dark vs. light period (164 ± 40 vs. 306 ± 40 pmol/s*mg; p=0.0031, t‐test). Mitochondrial‐associated genes, optic atrophy 1 (Opal1) and mitofusion 1 (Mfn1) show as significant day‐night difference in mRNA expression in kidneys from BMAL1+/+ control rats that is lost in Bmal1‐/‐ rats consistent with disrupted mitochondrial fusion and fission processes. Our lab previously reported that male Bmal1‐/‐ rats do not have the typical night‐day difference in Na+ excretion, which is consistent with our new data showing an alteration in mitochondrial function in kidneys from Bmal1‐/‐ rats. Therefore, our findings demonstrate that BMAL1 may play a role in maintaining the coupling of mitochondrial respiration and ATP generation in the kidney and support the hypothesis that diurnal Na+handling is regulated in part by BMAL1‐dependent mitochondrial function.

P0536HYPOTHERMIA DURING RENAL ISCHEMIA-REPERFUSION IN MICE: A PROTECTIVE EFFECT ON RENAL AND MITOCHONDRIAL FUNCTIONS

Abstract Background and Aims Renal ischemia reperfusion (RIR) can induce mitochondrial stress triggering cell death and eventually leading to acute kidney injury (AKI). It has been suggested that mild hypothermia could be protective in RIR without clear underlying mechanisms. We aimed to show that mild hypothermia (34°C) during RIR protects renal mitochondrial function and prevents AKI. Method Male C57BL6 mice were assigned to 4 groups: normothermic ischemic (RIR-37°C) group (n=14) and hypothermic ischemic (RIR-34°C) group (n=14) with body temperature maintained at respectively 37°C or 34°C during 20 minutes of renal ischemia by bilateral renal clamping under general anesthesia; normothermic sham (Sham-37°C) group (n=10) and hypothermic sham (Sham-34°C) group (n=10) with only anesthesia and laparotomy at 37°C or 34°C respectively. Renal function (serum urea concentration) and isolated renal mitochondria function (capacity of mitochondria to retain calcium i.e. calcic retention capacity (CRC), and oxidative phosphorylation capacity of electron transport chain complexes (complex I, II and IV)) were assessed 2 hours and 24 hours after reperfusion. All animal procedures were approved by local Ethics Committee. Data are presented as median with IQR. Results All the parameters monitored were not modified by the temperature in the sham groups, and there was no mortality in those 2 groups. Mortality was 33% in the RIR-37°C group and 11% in the RIR-34°C group 24 hours after reperfusion (p=0.58). Renal ischemia was responsible for a significant increase of serum urea level 2 hours after reperfusion at 37°C [18.7 (17.3–19.0) mmol/L] compared to sham groups (p=0.02), whereas no significant increase was observed in the RIR-34°C group. After 24 hours of reperfusion serum urea level was improved in the RIR-34°C group [22.7 (11.5–42.0) mmol/L] compared to RIR-37°C [60.8 (58.0–69.7) mmol/L, p=0.001]. CRC was not modified by RIR after 2 hours of reperfusion in both groups. CRC was preserved 24 hours after reperfusion in the RIR-34°C group [260 (210–320) nmol Ca2+/mg protein] with no difference compared to Sham-37°C [320 (280–360) nmol Ca2+/mg protein p=0.18] whereas CRC was significantly decreased in the RIR-37°C group compared to Sham-37°C [120 (0–130) vs 320 (280–360) nmol Ca2+/mg protein p=0.004). Complexes I, II and IV were lowered after 2 hours of reperfusion in the RIR-37°C group (p<0.05), and complexes II and IV activities remained altered 24 hours after reperfusion, compared to Sham-37°C (p=0.009 and p=0.02 respectively). In the RIR-34°C group, complexes I, II and IV activities were preserved 2 hours after reperfusion but complex I activity decreased 24 hours after reperfusion. We found significant difference between complexes II and IV activities between IRI-34°C and RIR-37°C. Conclusion Mild hypothermia (34°C) during RIR significantly protected renal mitochondrial respiration and mitochondrial stress, associated with a preserved renal function after 2 hours of reperfusion and an improved renal function 24 hours after reperfusion compared to normothermic mice (37°C).

Blockade of endogenous angiotensin II type I receptor agonistic autoantibody activity improves mitochondrial reactive oxygen species and hypertension in a rat model of preeclampsia

Preeclampsia (PE) is characterized by new-onset hypertension that usually occurs in the third trimester of pregnancy and is associated with oxidative stress and angiotensin II type 1 receptor agonistic autoantibodies (AT1-AAs). Inhibition of the AT1-AAs in the reduced uterine perfusion pressure (RUPP) rat, a model of PE, attenuates hypertension and many other characteristics of PE. We have previously shown that mitochondrial oxidative stress (mtROS) is a newly described PE characteristic exhibited by the RUPP rat that contributes to hypertension. However, the factors that cause mtROS in PE or RUPP are unknown. Thus, the objective of the current study is to use pharmacologic inhibition of AT1-AAs to examine their role in mtROS in the RUPP rat model of PE. AT1-AA inhibition in RUPP rats was achieved by administration of an epitope-binding peptide ('n7AAc'). Female Sprague-Dawley rats were divided into the following two groups: RUPP and RUPP + AT1-AA inhibition (RUPP + 'n7AAc'). On day 14 of gestation (GD), RUPP surgery was performed; 'n7AAc' peptide (2 µg/μL) was administered by miniosmotic pumps in a subset of RUPP rats; and on GD19, sera, placentas, and kidneys were collected. mitochondrial respiration and mtROS were measured in isolated mitochondria using the Oxygraph 2K and fluorescent microplate reader, respectively. Placental and renal mitochondrial respiration and mtROS were improved in RUPP + 'n7AAc' rats compared with RUPP controls. Moreover, endothelial cells (human umbilical vein endothelial cells) treated with RUPP + 'n7AAc' sera exhibited less mtROS compared with those treated with RUPP sera. Overall, our findings suggest that AT1-AA signaling is one stimulus of mtROS during PE.

Hyperglycemia induced damage to mitochondrial respiration in renal mesangial and tubular cells: Implications for diabetic nephropathy

Damage to renal tubular and mesangial cells is central to the development of diabetic nephropathy (DN), a complication of diabetes which can lead to renal failure. Mitochondria are the site of cellular respiration and produce energy in the form of ATP via oxidative phosphorylation, and mitochondrial dysfunction has been implicated in DN. Since the kidney is an organ with high bioenergetic needs, we postulated that hyperglycemia causes damage to renal mitochondria resulting in bioenergetic deficit. The bioenergetic profiles and the effect of hyperglycemia on cellular respiration of human primary mesangial (HMCs) and proximal tubular cells (HK-2) were compared in normoglycemic and hyperglycemic conditions using the seahorse bio-analyzer. In normoglycemia, HK-2 had significantly lower basal, ATP-linked and maximal respiration rates, and lower reserve capacity compared to HMCs. Hyperglycemia caused a down-regulation of all respiratory parameters within 4 days in HK-2 but not in HMCs. After 8 days of hyperglycemia, down-regulation of respiratory parameters persisted in tubular cells with compensatory up-regulated glycolysis. HMCs had reduced maximal respiration and reserve capacity at 8 days, and by 12 days had compromised mitochondrial respiration despite which they did not enhance glycolysis. These data suggest that diabetes is likely to lead to a cellular deficit in ATP production in both cell types, although with different sensitivities, and this mechanism could significantly contribute to the cellular damage seen in the diabetic kidney. Prevention of diabetes induced damage to renal mitochondrial respiration may be a novel therapeutic approach for the prevention/treatment of DN.

Grape Powder Improves Age-Related Decline in Mitochondrial and Kidney Functions in Fischer 344 Rats.

We examined the effects and mechanism of grape powder- (GP-) mediated improvement, if any, on aging kidney function. Adult (3-month) and aged (21-month) Fischer 344 rats were treated without (controls) and with GP (1.5% in drinking water) and kidney parameters were measured. Control aged rats showed higher levels of proteinuria and urinary kidney injury molecule-1 (KIM-1), which decreased with GP treatment in these rats. Renal protein carbonyls (protein oxidation) and gp91phox-NADPH oxidase levels were high in control aged rats, suggesting oxidative stress burden in these rats. GP treatment in aged rats restored these parameters to the levels of adult rats. Moreover, glomerular filtration rate and sodium excretion were low in control aged rats suggesting compromised kidney function, which improved with GP treatment in aged rats. Interestingly, low renal mitochondrial respiration and ATP levels in control aged rats were associated with reduced levels of mitochondrial biogenesis marker MtTFA. Also, Nrf2 proteins levels were reduced in control aged rats. GP treatment increased levels of MtTFA and Nrf2 in aged rats. These results suggest that GP by potentially regulating Nrf2 improves aging mitochondrial and kidney functions.

Mitochonic Acid 5 Binds Mitochondria and Ameliorates Renal Tubular and Cardiac Myocyte Damage.

Mitochondrial dysfunction causes increased oxidative stress and depletion of ATP, which are involved in the etiology of a variety of renal diseases, such as CKD, AKI, and steroid-resistant nephrotic syndrome. Antioxidant therapies are being investigated, but clinical outcomes have yet to be determined. Recently, we reported that a newly synthesized indole derivative, mitochonic acid 5 (MA-5), increases cellular ATP level and survival of fibroblasts from patients with mitochondrial disease. MA-5 modulates mitochondrial ATP synthesis independently of oxidative phosphorylation and the electron transport chain. Here, we further investigated the mechanism of action for MA-5. Administration of MA-5 to an ischemia-reperfusion injury model and a cisplatin-induced nephropathy model improved renal function. In in vitro bioenergetic studies, MA-5 facilitated ATP production and reduced the level of mitochondrial reactive oxygen species (ROS) without affecting activity of mitochondrial complexes I-IV. Additional assays revealed that MA-5 targets the mitochondrial protein mitofilin at the crista junction of the inner membrane. In Hep3B cells, overexpression of mitofilin increased the basal ATP level, and treatment with MA-5 amplified this effect. In a unique mitochondrial disease model (Mitomice with mitochondrial DNA deletion that mimics typical human mitochondrial disease phenotype), MA-5 improved the reduced cardiac and renal mitochondrial respiration and seemed to prolong survival, although statistical analysis of survival times could not be conducted. These results suggest that MA-5 functions in a manner differing from that of antioxidant therapy and could be a novel therapeutic drug for the treatment of cardiac and renal diseases associated with mitochondrial dysfunction.

Alterations in renal mitochondrial respiration in response to the reactive oxoaldehyde methylglyoxal

Chronic hyperglycemia has been linked to alterations in mitochondrial function, suggesting an important role in the pathophysiology of the complications of diabetes mellitus. In the diabetic kidney, ultrastructural changes in mitochondria are associated with impaired tubular function. The goal of this study was to determine if methylglyoxal (MGO), a dicarbonyl compound reaching high levels in hyperglycemic conditions, has direct toxicity for renal mitochondria. Intact mitochondria isolated from the renal cortex of rats were incubated with MGO to determine 1) its effect on mitochondrial respiration, 2) the conditions under which MGO exerts these effects, and 3) the potential mitochondrial targets of MGO influence. This study demonstrates that MGO has an inhibitory effect on both the tricarboxylic acid cycle and the electron respiratory chain. The modifications appear to be specific to certain mitochondrial proteins. Alterations of these proteins lead to disturbances in mitochondria that may play an important role in renal cellular toxicity and in the development of diabetic nephropathy.

Mitochondrial dysfunction at the early stage of cisplatin-induced acute renal failure in rats

The present study was undertaken to clarify the pathogenesis of cisplatin-induced acute renal failure at the early stage. Male Sprague-Dawley rats were given an intravenous administration of 10 mg/kg cisplatin. 0.9% saline was infused into them at a rate of 2 ml/h for 3 h, starting with a 2-ml bolus injection before cisplatin administration. 3 h following cisplatin administration, no evident morphological abnormalities were found by both light and electron microscopy; there were also no significant changes in GFR. Thirty min after cisplatin injection, urine sodium and potassium excretion increased by 56% and 260% those of the control animals, respectively. Apparent renal mitochondrial respiration dysfunction was observed in cisplatintreated rats 3 h later; the state 4 respiration increased by 100% and state 3 respiration, respiratory control ratio and carbonyl cyanide p-trifluoromethoxyphenyl hydrazone-uncoupled respiration decreased by 46%, 74% and 47% of the controls, respectively. The present data suggest that mitochondrial dysfunction may be a very early event in cisplatin-induced acute renal failure in rats.

MITOCHONDRIAL DYSFUNCTION AT THE EARLY STAGE OF CISPLATIN-INDUCED ACUTE RENAL FAILURE IN RATS

The present study was undertaken to clarify the pathogenesis of cisplatin induced acute renal failure at the early stage. Male Sprague Dawley rats were given an intravenous administration of 10 mg/kg cisplatin. 0.9% saline was infused into them at a rate of 2 ml/h for 3 h, starting with a 2 ml bolus injection before cisplatin administration. 3 h following cisplatin administration, no evident morphological abnormalities were found by both light and electron microscopy; there were also no significant changes in GFR. Thirty min after cisplatin injection, urine sodium and potassium excretion increased by 56% and 260% those of the control animals, respectively. Apparent renal mitochondrial respiration dysfunction was observed in cisplatin treated rats 3 h later; the state 4 respiration increased by 100% and state 3 respiration, respiratory control ratio and carbonyl cyanide p trifluoromethoxyphenyl hydrazone uncoupled respiration decreased by 46%, 74% and 47% of the controls, respectively. The present data suggest that mitochondrial dysfunction may be a very early event in cisplatin induced acute renal failure in rats.

Experimental and clinical studies on renal disturbance in obstructive jaundice.

Renal functions and the clinical courses of 14 patients with renal disturbances associated with obstructive jaundice were studied. All patients had high concentrations of serum bilirubin, long durations of jaundice and episodes of shock due to massive hemorrhage or severe inflammation. In an experimental study on jaundiced rats, effects of hypotension or bilateral renal artery occlusion on renal function and renal cortical mitochondrial respiration at 1 week, 3 or 6-weeks after biliary obstruction were investigated. In jaundiced rats, there was no remarkable difference in renal function, but the renal mitochondrial respiration indices decreased with prolongation of the biliary obstruction. In hypotensive rats with prolonged biliary obstruction, the mitochondrial function was impaired, and in the rats with renal artery occlusion, the mitochondrial impairment was more severe. Based on these clinical and experimental data, it is tentatively suggested that patients with prolonged jaundice should be considered in a prodromal state of renal failure and that any minute circulatory failure may induce acute renal failure.

Chronic low-level lead toxicity in the rat: III. An integrated assessment of long-term toxicity with special reference to the kidney

Male and female rats whose mothers had been exposed to Pb before and during pregnancy and lactation at exposure levels of 0, 0.5, 5, 25, 50, and 250 ppm Pb as Pb-acetate in drinking water were continued on the respective regimens for 6 or 9 months. Body weights of males and females were not significantly different from controls at 6 months of age; however, female body weights were significantly decreased at 250 ppm at 9 months of age. In males at 9 months of age, spleen weights were significantly increased at 250 ppm Pb and kidney weights were increased at 0.5 ppm Pb and above; in females the liver, pituitary, and heart weights were affected at 250 ppm Pb. No significant Pb effects were found in sperm counts or sperm morphology, hematology profiles, or serum chemistries. Blood, brain, femur, and kidney Pb levels as well as urinary ALA excretion were all significantly dose related. Histopathological lesions were noted in the spleen (250 ppm) and in the kidney as evidenced by cytomegaly/karyomegaly (beginning at 5 ppm in males; 25 ppm in females), nuclear inclusion body formation and increased numbers of iron-positive granules within renal proximal tubule cells. These effects were more marked after 9 months exposure. Ultrastructural studies revealed mitochondrial swelling and the presence of increased numbers of lysosomes within renal proximal tubule cells. Energy-dispersive X-ray microanalysis, performed on adjacent sections, showed the highest intracellular Pb concentrations in nuclear inclusion bodies within renal proximal tubule cells. Inhibition of renal mitochondrial respiration for both succinate and NAD-linked substrates was found in 50- and 250-ppm Pb exposure groups at 9 months but not at 6 months. Mitochondrial δ-aminolevulinic acid synthetase and ferrochelatase, but not δ-aminolevulinic dehydratase, were also found to be inhibited at these Pb levels at 9 months. The lowest exposure level resulting in a detectable effect of Pb (cytomegaly/karyomegaly in renal proximal tubule cells) was 5 ppm associated with a median blood Pb concentration of 11 μg/dl.