Rat Kidney Cortex Research Articles

Abnormal activation of the mineralocorticoid receptor in the aldosterone-sensitive distal nephron contributes to fructose-induced salt-sensitive hypertension.

Fructose high-salt (FHS) diets increase blood pressure (BP) in an angiotensin II (Ang II)-dependent manner. Ang II stimulates aldosterone release, which, by acting on the mineralocorticoid receptor (MR), regulates Na + reabsorption by the aldosterone-sensitive distal nephron (ASDN). The MR can be transactivated by glucocorticoids, including those locally produced by 11β-HSD1. The epithelial sodium channel (ENaC) is a key transporter regulated by MRs. We hypothesized that fructose-induced salt-sensitive hypertension depends in part on abnormal activation of MRs in the ASDN with consequent increases in ENaC expression. We found that aldosterone-upregulated genes in mice ASDN, significantly overlapped with 74 genes upregulated by FHS in the rat kidney cortex (13/74; p≤1x10 -8 ), and that these 74 genes are prominently expressed in rat ASDN cells. Additionally, the average z-score expression of mice-aldosterone-upregulated genes is highly correlated with FHS compared to glucose high-salt (GHS) in the rat kidney cortex (Pearson correlation; r=0.66; p≤0.005). There were no significant differences in plasma aldosterone concentrations between the FHS and GHS. However, 11β-HSD1 transcripts were upregulated by FHS (log 2 FC=0.26, p≤0.02). FHS increased BP by 23±6 mmHg compared to GHS, and blocking MRs with eplerenone prevented this increase. Additionally, inhibiting ENaC with amiloride significantly reduced BP in FHS from 148±6 to 134±5 mmHg (p≤0.019). Compared to GHS, FHS increased total and cleaved αENaC protein by 89±14 % (p≤0.03) and 47±16 % (p≤0.01) respectively. FHS did not change β- or γ-subunit expression. These results suggest that fructose-induced salt-sensitive hypertension depends, in part, on abnormal Na + retention by ENaC, resulting from the activation of MRs by glucocorticoids.

Effect of SGLT2 and dual SGLT1/2 Inhibition on the Development of Salt-induced Hypertension

In recent years, sodium-glucose co-transporter inhibitors (SGLT2i) have become the new mainstay in treating diabetes mellitus and, lately, cardiovascular diseases. Large clinical trials have shown remarkable benefits of SGLT2i on renal and cardiovascular outcomes. SGLT2i have been reported to slow the progression of chronic kidney disease by reducing hyperfiltration and glomerular capillary pressure, kidney hypertrophy, and albuminuria. Recently, the dual SGLT1/2 inhibitor sotagliflozin (Sota) was approved by the FDA for the treatment of heart failure independent of ejection fraction. However, despite these promising outcomes, the effects of SGLTi, particularly in salt-sensitive hypertension, requires further investigation. The main goal of this study was to define the effects of SGLT inhibition on the development of salt-induced hypertension and characterize potential contributing mechanisms. In our recent studies, we showed that SGLTi dapagliflozin (Dapa) caused an increase of diuresis in 8-weeks old male and female Dahl SS rats when fed a high salt (HS, 4% NaCl for 3 weeks) diet. Additionally, Dapa increased glucose and Na+ excretion. Chronic Dapa treatment also resulted in inhibition of the development of hypertension. Interestingly, male rats had no changes in kidney damage, whereas in females Dapa attenuated kidney fibrosis. To define potential molecular mechanisms involved in the beneficial effects of SGLT2i, we performed additional analyses of tissues collected from SS rats on a HS diet treated with either vehicle or Dapa. Transcriptomics analyses revealed that in the kidney cortex, top biological functions affected by SGLT2 inhibition were related to lipid and amino acid metabolism, molecular transport, as well as cell function. Preliminary studies were performed to test lipid mediators in the kidney cortex of male Dahl SS rats fed 3 weeks with HS diet and treated with Dapa. We found that several tested bioactive lipids, specifically pro-inflammatory mediators, were elevated in Dahl SS rats following HS diet, and these increased levels were completely blocked in Dapa-treated rats. Dual SGLT1/2 inhibition by Sota (30 mg/kg/day in food, n=6 rats/group) resulted in rapid attenuation of salt-induced hypertension. Starting from day 5 of HS challenge, MAP of vehicle-treated Dahl SS male rats was significantly higher compared to Sota-treated rats (~33 mmHg MAP difference on day 21), whichout differences in heart rate. In response to Sota treatment, SS rats had ~1.6-fold greater diuresis (at day 21: vehicle 37±2 vs. treated 61±6 ml/day) and glucosuria (values?). Further studies are ongoing to define the effects on GFR, kidney, and heart damage, and the potential resulting mechanisms. Our preliminary data indicate that dual SGLT1/2 inhibition with Sota had a significant impact on salt-induced hypertension in the Dahl SS rats. Further research will provide a more comprehensive understanding of the mechanisms. This research was supported by National Institutes of Health grant R35 HL135749 (to AS). This is the full abstract presented at the American Physiology Summit 2024 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.

Effect of Fasting During Time Restricted Feeding on Mitochondrial Beta-Oxidation Enzyme Expression in the Kidney Cortex of Adult Male Salt-Sensitive Dahl Rats

The Dahl salt-sensitive (SS) rat is a well-characterized model of SS hypertension and renal disease that exhibits enhanced Na+ reabsorption in the kidney. Prior research has shown that SS rats experience a reduction in fatty acid beta oxidation (FAO) in the kidney cortex when placed on a high salt (HS) diet. Time restricted feeding (TRF), a type of intermittent fasting in which food consumption is restricted to <10 hrs/day, has been shown to activate FAO. Since the proximal tubule is responsible for the highest amount of Na+ reabsorption, we are interested in understanding if intermittent salt intake via TRF can mitigate dysfunctional cortical metabolism and slow progression of renal damage. We hypothesized that fasting during TRF would activate FAO throughout the body, as well as in the kidney cortex. To study this, 10-week-old male SS rats were provided ad libitum access to 4.0% NaCl (high salt; HS) or 0.4% (low salt; LS) diet for one week. Rats were then placed into a Promethion Core system (Sable Systems International) for metabolic phenotyping, where some continued ad libitum feeding, and others began time restricted feeding (TRF) for an additional week (n=7-9/group). During TRF, food was provided for 8 hours during the active cycle (2200hrs-0600hrs). Rats were euthanized in the last two hours of the final fasting period. We found that during the last hour of the fasting period, when compared to ad libitum rats, both the LS TRF (0.824 ± 0.01 vs 0.986 ± 0.008) and HS TRF (0.830 ± 0.01 vs 0.980 ± 0.006) rats had a significant reduction in their respiratory exchange ratio (2-way ANOVA with Tukey multiple comparisons, p<0.05, mean ± SEM). This suggests that the TRF protocol induced a net whole-body shift in fuel utilization from primarily carbohydrates to a mix of fuel sources by the end of the 16-hour fasting period, regardless of diet. To investigate the effect of fasting during TRF on the kidney cortex, we performed western blot analysis of acyl-CoA dehydrogenase long chain (ACADL) and acetyl-CoA acyltransferase 2 (ACAA2), enzymes involved in initial and final steps of mitochondrial beta-oxidation, respectively. We found that HS rats had lower ACADL and ACAA2 expression than the LS rats (2-way ANOVA; main effect of HS, p<0.05), and there was no difference between ad libitum and TRF groups within LS or HS groups (2-way ANOVA; main effect of TRF, p>0.05). This data suggests that, even though whole-body FAO may be stimulated by TRF fasting, HS-induced suppression of mitochondrial FAO proteins ACADL and ACAA2 was not restored by TRF. Future studies will investigate the effects of the shift in fuel utilization on other metabolic pathways in the cortex and proximal tubules. Supported by the Advancing a Healthier Wisconsin Endowment, the Medical College of Wisconsin, and NIH 5T32HL007852-25. This is the full abstract presented at the American Physiology Summit 2024 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.

Sex-Specific Mitochondrial Bioenergetics Differences in Heart and Kidney Cortex

Rationale: Mitochondrial bioenergetics of different tissues have been studied widely, but their sex-specific differences have not been systematically studied. The goal of this study was therefore to investigate the sex-specific differences in mitochondrial respiration (oxygen consumption rate; OCR) and membrane potential (Δψ) in the heart and kidney cortex and to explore underlying mechanisms responsible for any observed differences. Methods: Mitochondria were isolated from adult male and female Sprague-Dawley rat heart and kidney cortex and were used to study their bioenergetics responses. Three different substrate combinations, namely, pyruvate+malate (PM), glutamate+malate (GM), and succinate in the presence of complex I inhibitor rotenone (SR) were used. This was followed by two different ADP addition protocols to determine how different substrates influence mitochondrial OCR and Δψ to ADP perturbations during oxidative phosphorylation (OxPhos) in male and female heart and kidney cortex. In one, a single saturated dose of ADP, while in the other, sequentially increasing doses of ADP were added to mitochondria in the presence of different substrates. Mitochondrial OCR was measured using a dual chamber Oroboros Oxygraph-2k Instrument and Δψ was measured using a spectrofluorometer and rhodamine-123 dye. From these measurements, OCR and Δψ alterations under different respiratory states and as functions of added ADP concentration were determined for different substrates and for both male and female rats. Results: Male and female mitochondria exhibited distinct respiratory patterns in the kidney cortex and heart with different substrates. In the kidney cortex, male mitochondria showed significantly higher OCR than female mitochondria when fueled with PM or SR. However, no significant sex differences in the OCR were observed when GM were used as substrates. In contrast, heart mitochondria exhibited negligible sex differences in the OCR for PM and SR, but significant differences emerged with GM, where female mitochondria displayed higher OCR than their male counterparts. Interestingly, sex-specific variations of Δψ in ADP-stimulated respiration were not apparent for either tissue or substrate when a single saturated dose of ADP was used. Instead, a trend similar to the OCR emerged when ADP was administered in gradually increasing concentrations. Conclusion: Heart and kidney cortex mitochondrial Δψ for males and females did not differ conclusively with the two ADP addition protocols. However, there were significant differences in the OCR of mitochondria from the heart and kidney cortex of males and females, depending on the respiratory substrate utilized. Males were found to have enhanced OCR in the kidney cortex, whereas females exhibited higher OCR in the heart. These observed sex-specific differences in the OCR could be attributed to differential regulation of mitochondrial metabolic pathways by respiratory substrates and sex hormones. NIH R01-HL151587. This is the full abstract presented at the American Physiology Summit 2024 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.

The Nephroprotective Activity of Boesenbergia Rotunda Rhizome by Reducing Creatinine, Urea Nitrogen, Glutamic Pyruvic Transaminase, and Malondialdehyde Levels in the Blood and Attenuating the Expression of Havcr1 (KIM-1), Lcn2 (NGAL), Casp3, and Casp7 Genes in the Kidney Cortex of Cisplatin-Induced Sprague-Dawley Rats.

Cisplatin chemotherapy induces nephrotoxicity by producing reactive oxygen species, hence, discovering add-on nephroprotective drugs for patients with cancer is challenging. Boesenbergia rotunda has been reported for its antioxidant properties. This study aims to explore the nephroprotective mechanism of the ethanol extract of Boesenbergia rotunda rhizome (EEBR) in cisplatin-induced rats. The rats were randomly assigned into 6 groups: the normal control (treated with saline); the negative control (cisplatin-induced without any treatment); the positive control (treated with quercetin 50 mg/kg BW); and 3 treatment EEBR (125 mg/kg BW; 250 mg/kg BW; 500 mg/kg BW) groups for 10 days. The % relative organ weight, kidney histopathology, and nephrotoxicity biomarkers expression were evaluated. EEBR decreased creatinine, urea nitrogen, glutamic pyruvate transaminase, and malondialdehyde levels in the blood of cisplatin-induced rats. An insignificant increase in GOT was observed in rats treated with the highest dose of EEBR. EEBR did not significantly alter the BW and the % kidney relative weight. An abnormal shape of the Bowman capsule is observed in the negative control group. EEBR reduced the expression of Havcr1 (KIM-1), Lcn2 (NGAL), Casp3, and Casp7 genes in rats' kidneys. Boesenbergia rotunda could be considered a potential candidate for add-on therapy in cisplatin-treated patients, but further studies are needed to verify its efficacy and safety.

Intestinal and Renal Adaptations to Changes of Dietary Phosphate Concentrations in Rat.

We have studied the role of the intestine, kidney, and several hormones when adapting to changes in dietary P concentration. Normal and parathyroidectomized (PTX) rats were fed pH-matched diets containing 0.1%, 0.6%, and 1.2% P concentrations. 32Pi uptake was determined in the jejunum and kidney cortex brush border membrane vesicles. Several hormone and ion concentrations were determined in the blood and urine of rats. Both jejunum and kidney cortex Pi transport was regulated with 5 d of chronic feeding of P diets in normal rats. Acute adaptation was determined by switching foods on day 6, which was only clearly observed in the kidney cortex of normal rats, with more statistical variability in the jejunum. However, no paradoxical increase of Pi uptake in the jejunum was reproduced after the acute switch to the 1.2% P diet. Pi uptake in the jejunum was parathyroid hormone (PTH)-independent, but in the kidney, the chronic adaptation was reduced, and no acute dietary adaptations were observed. The NaPi2a protein was more abundant in the PTX than the sham kidneys, but contrary to the modest or absent changes in Pi uptake adaptation, the transporter was similarly regulated by dietary P, as in the sham rats. PTH and fibroblast growth factor 23 (FGF23) were the only hormones regulated by all diet changes, even in fasting animals, which exhibited regulated Pi transport despite similar phosphatemia. Evidence of Pi appetite effects was also observed. In brief, our results show new characteristics of Pi adaptations, including a lack of correlation between Pi transport, NaPi2a expression, and PTH/FGF23 concentrations.

Gadolinium-based Contrast Agent Biodistribution and Speciation in Rats.

Background Gadolinium retention has been observed in organs of patients with normal renal function; however, the biodistribution and speciation of residual gadolinium is not well understood. Purpose To compare the pharmacokinetics, distribution, and speciation of four gadolinium-based contrast agents (GBCAs) in healthy rats using MRI, mass spectrometry, elemental imaging, and electron paramagnetic resonance (EPR) spectroscopy. Materials and Methods In this prospective animal study performed between November 2021 and September 2022, 32 rats received a dose of gadoterate, gadoteridol, gadobutrol, or gadobenate (2.0 mmol/kg) for 10 consecutive days. GBCA-naive rats were used as controls. Three-dimensional T1-weighted ultrashort echo time images and R2* maps of the kidneys were acquired at 3, 17, 34, and 52 days after injection. At 17 and 52 days after injection, gadolinium concentrations in 23 organ, tissue, and fluid specimens were measured with mass spectrometry; gadolinium distribution in the kidneys was evaluated using elemental imaging; and gadolinium speciation in the kidney cortex was assessed using EPR spectroscopy. Data were assessed with analysis of variance, Kruskal-Wallis test, analysis of response profiles, and Pearson correlation analysis. Results For all GBCAs, the kidney cortex exhibited higher gadolinium retention at 17 days after injection than all other specimens tested (mean range, 350-1720 nmol/g vs 0.40-401 nmol/g; P value range, .001-.70), with gadoteridol showing the lowest level of retention. Renal cortex R2* values correlated with gadolinium concentrations measured ex vivo (r = 0.95; P < .001), whereas no associations were found between T1-weighted signal intensity and ex vivo gadolinium concentration (r = 0.38; P = .10). EPR spectroscopy analysis of rat kidney cortex samples showed that all GBCAs were primarily intact at 52 days after injection. Conclusion Compared with other macrocyclic GBCAs, gadoteridol administration led to the lowest level of retention. The highest concentration of gadolinium was retained in the kidney cortex, but T1-weighted MRI was not sensitive for detecting residual gadolinium in this tissue. © RSNA, 2023 Supplemental material is available for this article. See also the editorial by Tweedle in this issue.

Abstract P213: Multi-omic Analysis Of The Effect Of SGLT2 Inhibition On Blood Pressure In Dahl SS Rats

SGLT2 inhibitors (SGLT2i) have demonstrated efficacy in reducing heart failure, lowering blood pressure, and mitigating kidney diseases in both diabetic and non-diabetic conditions. Previously, we demonstrated the preventive effects of the SGLT2i dapagliflozin (Dapa) on salt-sensitive hypertension (SS HTN) in male Dahl SS rats (Dapa vs. vehicle: 136±3 vs. 156±6 mmHg) (Kravtsova et al ., AJP Renal 2022). In the current study, we aimed to assess whether Dapa has similar outcomes in female rats. Our findings reveal that Dapa administration (2 mg/kg/day in drinking water for 3 weeks) resulted in a comparable effect on blood pressure in female rats (Dapa vs. vehicle: 140±5 vs. 160±9 mmHg). We further performed transcriptomic and proteomic analyses of the kidney cortex and medulla in both male and female rats fed a high salt (HS) diet, with or without Dapa. Both analyses revealed that the top biological functions affected by Dapa were related to lipid and amino acid metabolism, molecular transport, and cell function. The enrichment analysis of biological functions highlighted altered genes primarily associated with fatty acid oxidation, lipid transport and oxidation, and ion homeostasis primarily in the medulla. Notably, Plin1 , Plin4 , Ucp1 , Ucp3 and Adrb3 , which are involved in lipid metabolism, were the highest expressed genes in the kidney medulla of both sexes. Based on previous studies suggesting the involvement of oxidized phospholipids and metabolites of linoleic and arachidonic acids in the pathogenesis of SS HTN, we conducted preliminary experiments with males to assess the levels of lipid mediators in the kidneys of Dapa-treated rats. HS diet in Dahl SS rats led to elevated levels of several pro-inflammatory mediators, which were significantly reduced in Dapa-treated rats. These reductions were observed for prostaglandins (PGD2 [fold change (FC)=0.28], PGF2a [FC=0.14], 8-iso-PGF2a [FC=0.14]), 12-HHT [FC=0.31], TXB2 [FC=0.18], and 20-OH-LTB4 (FC=0.10). Our results indicated that SGLT2i altered various genes in the kidneys of Dahl SS rats independent of sex. This information provides novel insight on the pleiotropic effects of SGLT2i. Further studies are necessary to validate the predicted targeted pathways and subsequent biological functions.

Computational Modeling of Substrate-Dependent Mitochondrial Respiration and Bioenergetics in the Heart and Kidney Cortex and Outer Medulla.

Integrated computational modeling provides a mechanistic and quantitative framework to characterize alterations in mitochondrial respiration and bioenergetics in response to different metabolic substrates in-silico. These alterations play critical roles in the pathogenesis of diseases affecting metabolically active organs such as heart and kidney. Therefore, the present study aimed to develop and validate thermodynamically constrained integrated computational models of mitochondrial respiration and bioenergetics in the heart and kidney cortex and outer medulla (OM). The models incorporated the kinetics of major biochemical reactions and transport processes as well as regulatory mechanisms in the mitochondria of these tissues. Intrinsic model parameters such as Michaelis-Menten constants were fixed at previously estimated values, while extrinsic model parameters such as maximal reaction and transport velocities were estimated separately for each tissue. This was achieved by fitting the model solutions to our recently published respirometry data measured in isolated rat heart and kidney cortex and OM mitochondria utilizing various NADH- and FADH2-linked metabolic substrates. The models were validated by predicting additional respirometry and bioenergetics data, which were not used for estimating the extrinsic model parameters. The models were able to predict tissue-specific and substrate-dependent mitochondrial emergent metabolic system properties such as redox states, enzyme and transporter fluxes, metabolite concentrations, membrane potential, and respiratory control index under diverse physiological and pathological conditions. The models were also able to quantitatively characterize differential regulations of NADH- and FADH2-linked metabolic pathways, which contribute differently toward regulations of oxidative phosphorylation and ATP synthesis in the heart and kidney cortex and OM mitochondria.

#6817 ROLE OF THE CGAS-STING PATHWAY IN THE PROGRESSION OF DIABETIC NEPHROPATHY

Abstract Background and Aims Diabetic kidney disease (DKD) is the leading cause of chronic renal pathology. Understanding the molecular underpinnings of DKD is critical to designing tailored therapeutic approaches. We have demonstrated previously that type 2 diabetic nephropathy (T2DN) rats develop renal and physiological abnormalities similar to clinical observations in humans with DKD, indicating these rats are an excellent model for studying the progression of renal injury in type 2 DKD [1]. Furthermore, our recent studies revealed sexual dimorphism in this model, indicating that while both male and female T2DN rats developed non-obese DKD phenotype, females had significant protection from the development of severe forms of glomerular and tubular damage [2]. The potential role of the cyclic GMP-AMP synthase (cGAS) / Stimulator of interferon genes (STING) pathway in renal inflammation and fibrosis was also uncovered [3]. Here we focused on sex differences in DKD and the potential mechanisms leading to the progression of DKD. Method To explore the sexual dimorphisms in the development of DKD, we utilized young (12-week-old) and aged (&amp;gt;48 weeks old) type 2 diabetic nephropathy (T2DN) rats. To delineate transcriptional changes, RNA-Seq analysis was performed in the kidney cortex of T2DN rats of both sexes at a younger and older age. Western blotting, immunohistochemistry, and flow cytometry analyses were further used to identify specific pathways contributing to sexual dimorphism and disease progression in T2DN rats. Results We have revealed that the cGAS/STING signaling pathway is upregulated in T2DN rats compared to non-diabetic Wistar rats and in type 2 diabetic human kidneys. The expression of key proteins in the cGAS-STING pathway, such as cGAS, STING, phospho-IRF, mtTFA, and TREX1, was significantly different between male and female T2DN rats and following the progression of DKD. Proinflammatory genes were also upregulated in male T2DN rats compared to female rats of the same age, and their levels were further elevated in aged rats. RNA-Seq analysis also identified significant changes in genes participating in the cGAS-STING inflammatory pathway. Flow cytometry revealed a significantly greater number of infiltrating leukocytes in the male kidneys compared to their age-matched females. Moreover, the leukocytic count was significantly higher in old males versus young males, while it was almost the same in females of different ages. Approximately 50% of the renal leukocytic population was monocytes/macrophages (CD11b/c+), with fewer CD3+ T cells, both CD3+CD4+ T helper and CD3+CD8+ cytotoxic T cells, and CD45R+ B cells. Conclusion Our study provides critical insights into the sexual dimorphism and progression of DKD and identifies the cGAS-STING pathway as an essential contributor to disease development.

Novel Model of Oxalate Diet-Induced Chronic Kidney Disease in Dahl-Salt-Sensitive Rats.

Diet-induced models of chronic kidney disease (CKD) offer several advantages, including clinical relevance and animal welfare, compared with surgical models. Oxalate is a plant-based, terminal toxic metabolite that is eliminated by the kidneys through glomerular filtration and tubular secretion. An increased load of dietary oxalate leads to supersaturation, calcium oxalate crystal formation, renal tubular obstruction, and eventually CKD. Dahl-Salt-Sensitive (SS) rats are a common strain used to study hypertensive renal disease; however, the characterization of other diet-induced models on this background would allow for comparative studies of CKD within the same strain. In the present study, we hypothesized that SS rats on a low-salt, oxalate rich diet would have increased renal injury and serve as novel, clinically relevant and reproducible CKD rat models. Ten-week-old male SS rats were fed either 0.2% salt normal chow (SS-NC) or a 0.2% salt diet containing 0.67% sodium oxalate (SS-OX) for five weeks.Real-time PCR demonstrated an increased expression of inflammatory marker interleukin-6 (IL-6) (p < 0.0001) and fibrotic marker Timp-1 metalloproteinase (p < 0.0001) in the renal cortex of SS-OX rat kidneys compared with SS-NC. The immunohistochemistry of kidney tissue demonstrated an increase in CD-68 levels, a marker of macrophage infiltration in SS-OX rats (p < 0.001). In addition, SS-OX rats displayed increased 24 h urinary protein excretion (UPE) (p < 0.01) as well as significant elevations in plasma Cystatin C (p < 0.01). Furthermore, the oxalate diet induced hypertension (p < 0.05). A renin-angiotensin-aldosterone system (RAAS) profiling (via liquid chromatography-mass spectrometry; LC-MS) in the SS-OX plasma showed significant (p < 0.05) increases in multiple RAAS metabolites including angiotensin (1-5), angiotensin (1-7), and aldosterone. The oxalate diet induces significant renal inflammation, fibrosis, and renal dysfunction as well as RAAS activation and hypertension in SS rats compared with a normal chow diet. This study introduces a novel diet-induced model to study hypertension and CKD that is more clinically translatable and reproducible than the currently available models.

Mitochondrial dysfunction in kidney cortex and medulla of subtotally nephrectomized rats.

Five-sixths nephrectomy is a widely used experimental model of chronic kidney disease (CKD) that is associated with severe mitochondrial dysfunction of the remnant tissue. In this study, we assessed the effect of CKD on mitochondrial respiration separately in the rat kidney cortex and medulla 10 weeks after induction of CKD by subtotal 5/6 nephrectomy (SNX). Mitochondrial oxygen consumption was evaluated on mechanically permeabilized samples of kidney cortex and medulla using high-resolution respirometry and expressed per mg of tissue wet weight or IU citrate synthase (CS) activity. Mitochondrial respiration in the renal cortex of SNX rats was significantly reduced in all measured respiratory states if expressed per unit wet weight and remained lower if recalculated per IU citrate synthase activity, i.e. per mitochondrial mass. In contrast, the profound decrease in the activity of CS in SNX medulla resulted in significantly elevated respiratory states expressing the OXPHOS capacity when Complexes I and II or II only are provided with electrons, LEAK respiration after oligomycin injection, and Complex IV-linked oxygen consumption per unit CS activity suggesting compensatory hypermetabolic state in remaining functional mitochondria that is not sufficient to fully compensate for respiratory deficit expressed per tissue mass. The results document that CKD induced by 5/6 nephrectomy in the rat is likely to cause not only mitochondrial respiratory dysfunction (in the kidney cortex), but also adaptive changes in the medulla that tend to at least partially compensate for mitochondria loss.

Benincasa hispida extracts positively regulated high salt-induced hypertension in Dahl salt-sensitive rats: Impact on biochemical profile and metabolic patterns.

Salt-induced hypertension is one of the major issues worldwide and one of the main factors involved in heart and kidney failure. The objective of this study was to investigate the potential role of Benincasa hispida extracts on high salt-induced hypertension in Dahl-salt sensitive (D-SS) rats and to find out the metabolic and biochemical pattern involved in the reduction of hypertension. Twenty-six Dahl salt-sensitive (D-SS) rats were selected and divided into four groups. The metabolic strategy was applied to test the extracts on salt-sensitive hypertension in kidney. Gas Chromatography-Mass spectrometry (GC-MS) was used to identify the potent biochemical profile in renal medulla and cortex of rat kidneys. The differential metabolites of cortex and medulla, enrichment analysis and pathway analysis were performed using metabolomics data. The GC-MS data revealed that 24 different antihypertensive metabolites was detected in renal cortex, while 16 were detected in renal medulla between different groups. The significantly metabolic pathways namely citrate cycle, glutathione metabolism, glycine, serine, and threonine metabolism, glyoxylate and dicarboxylate metabolism, glycerolipid metabolism, alanine, aspartate and glutamate metabolism in renal cortex and glycerolipid metabolism, pentose phosphate pathway, citrate cycle, glycolysis, glycerophospholipid metabolism, phenylalanine, tyrosine and tryptophan biosynthesis in renal medulla were involved in the process of Hypertension. The results suggest that the extract mainly alter the metabolic pathways of amino acid in Dahl salt-sensitive rats and its antioxidant potential reduced the hypertension patterns of Salt-sensitive rat. The antihypertensive components malic acid, aspartic acid, and glycine of extract can be used as therapeutic drugs to protect kidneys from salt-induced hypertension. PRACTICAL APPLICATIONS: Hypertension is a multifactorial disease and one of the risk factors for heart and kidney failure. Benincasa hispida is a widely used vegetable in China, which belongs to the Cucurbitaceae family. Benincasa hispida (wax gourd) has been used in traditional Chinese medicine for the treatment of inflammation and hypertension. The Benincasa hispida contains many compounds such as amino acids, carbohydrates, volatile compounds, vitamins, and minerals. The amino acid present in the pulp of Benincasa hispida are ornithine, threonine, aspartate, glutamate, serine, glycine, proline, alanine, valine, cysteine, isoleucine, tyrosine, leucine, lysine, phenylalanine, histidine, arginine, and γ-aminobutyric acid. Our results showed that Benincasa hispida is one of the potent natural antioxidants and can maintain normal blood pressure in Dahl salt-sensitive rats (D-SS). In conclusion, the current results provide good theoretical basis for the development and research using Benincasa hispida as an effective natural antioxidant for hypertension.

Reverse Electron Transfer is a More Dominant Source of Mitochondrial ROS Production in the Heart and Kidney Outer Medulla than in the Kidney Cortex

RationaleReactive oxygen species (ROS; e.g. O2·– and H2O2) play important roles in both physiological and pathophysiological processes. ROS in low concentrations contribute to physiological processes, such as cellular redox signaling and phagocytosis, whereas ROS in high concentrations are toxic to the cell causing tissue injury contributing to the pathogenesis of cardiovascular and chronic renal diseases, including salt‐sensitive hypertension. Mitochondria, which produce ROS as byproducts of aerobic respiration via both forward electron transfer (FET) and reverse electron transfer (RET) are known to be one of the major cellular sources of ROS. Although it is recognized that the RET mechanism in which electrons flow back from complex II to complex I contribute significantly to ROS production in cardiac mitochondria, the mechanisms of ROS production and the role of RET in kidney mitochondria has remained poorly understood.MethodWe evaluated the relative contributions of FET and RET towards overall ROS production in mitochondria isolated from the heart and kidney cortex and outer medulla (OM) of adult Sprague‐Dawley rats. H2O2 emission was measured by a spectrofluorometer in isolated mitochondria in the presence of either Succinate (Suc) simulating RET or succinate+rotenone (Suc+Rot) simulating FET. Furthermore, we measured mitochondrial rates of H2O2 production along with respiration and membrane potential under three respiratory states namely (i) leak state (state 2; after substrate addition), (ii) oxidative phosphorylation (OxPhos) state (state 3; after ADP addition), and (iii) maximum respiratory state (state 5; after the addition of the uncoupler FCCP).ResultsIt was found that mitochondria isolated from the heart and kidney cortex produced the least and the most ROS, respectively. The rate of ROS production in the presence of Suc+Rot compared to Suc alone decreased significantly in the heart and to a lesser extent in OM, indicating significant contribution of RET to overall ROS production in the heart and slightly in the OM. In contrast, there was not significant difference in ROS production rates in the presence of Suc and Suc +Rot in mitochondria from kidney cortex, showing that RET is not predominant in the kidney cortex. Also, we observed significant reduction in the ROS production rate in state 4 compared to state 2 in the heart mitochondria compared to kidney cortex and OM. A possible explanation for these differential results is that oxaloacetate (OAA), produced by the tricarboxylic acid cycle, accumulates resulting in succinate dehydrogenase (SDH) inhibition more rapidly in the heart than in the kidney affecting mitochondrial ROS production, respiration, and bioenergetics.ConclusionRET mechanism contributes to mitochondrial ROS production significantly in the heart and slightly in the kidney OM, but not in the kidney cortex. OAA accumulation contributes to SDH inhibition significantly in the heart than in the kidney.

Computational Modeling of Substrate‐Dependent Differential Regulation of Mitochondrial Bioenergetics in the Heart and Kidney Cortex and Outer Medulla

RationaleIt is recognized that the kinetics and efficiency of mitochondrial O2 consumption for ATP production depend on the choice of respiratory substrates. Various substrates differentially generate the reducing equivalents NADH and FADH2 via the tricarboxylic acid (TCA) cycle that feed electrons to the electron transport chain (ETC) driving oxidative phosphorylation (OxPhos). However, the contributions of these substrates and their combinations have not been systematically characterized in either the heart or the kidney, the two major energy consuming organs in the body.MethodBioenergetic responses (respiration and membrane potential) of mitochondria isolated from the heart and kidney cortex and outer medulla (OM) of adult Sprague‐Drawly rats were characterized with different substrate combinations and different ADP perturbations. To better understand these distinct substrate and tissue‐specific mitochondrial bioenergetics, thermodynamically‐constrained mechanistic computational models were developed by integrating the kinetics and regulation of mitochondrial substrate transport and oxidation, TCA cycle, and ETC/OxPhos, incorporating published data and the novel mitochondrial bioenergetics data from our laboratory. Intrinsic model parameters such as Michaelis‐Menten constants were assumed identical and fixed for the heart and kidney, while extrinsic model parameters such as maximal enzymatic rates were separately estimated based on experimental data for the heart and kidney mitochondrial bioenergetics.ResultsConsistent with the data, model simulations of the ADP‐induced state 3 responses of the heart and kidney cortex and OM mitochondria yielded dramatically different values, which for a given organ were also very distinct for different substrates. In addition, heart mitochondria energized with succinate without rotenone (blocker of complex I) and saturated [ADP] failed to produce a robust state 3 response. This was in contrast to kidney cortex and OM mitochondria for which responses to succinate±rotenone were similar, indicating that reverse electron transfer via complex I played less prominent regulatory role in the kidney than the heart. In addition, oxaloacetate, a TCA cycle intermediate, was shown to accumulate faster in the heart compared to the kidney, thereby potently inhibiting succinate oxidation more in the heart than the kidney. As such, the model was able to quantitatively characterize how various substrate combinations account for the differential contributions of different substrate oxidation pathways within the mitochondria towards OxPhos and ATP synthesis in the heart and kidney cortex and OM.ConclusionModeling of these data provided a deeper quantitative understanding of key determinants of kinetic and molecular mechanisms involved in the differential regulation of substrate‐dependent mitochondrial bioenergetics in the heart and kidney cortex and OM.

Oxidized alkyl phospholipids stimulate sodium transport in proximal tubules via a nongenomic PPARγ-dependent pathway

Oxidized phospholipids have been shown to exhibit pleiotropic effects in numerous biological contexts. For example, 1-O-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine (azPC), an oxidized phospholipid formed from alkyl phosphatidylcholines, is a peroxisome proliferator–activated receptor gamma (PPARγ) nuclear receptor agonist. Although it has been reported that PPARγ agonists including thiazolidinediones can induce plasma volume expansion by enhancing renal sodium and water retention, the role of azPC in renal transport functions is unknown. In the present study, we investigated the effect of azPC on renal proximal tubule (PT) transport using isolated PTs and kidney cortex tissues and also investigated the effect of azPC on renal sodium handling in vivo. We showed using a microperfusion technique that azPC rapidly stimulated Na+/HCO3− cotransporter 1 (NBCe1) and luminal Na+/H+ exchanger (NHE) activities in a dose-dependent manner at submicromolar concentrations in isolated PTs from rats and humans. The rapid effects (within a few minutes) suggest that azPC activates NBCe1 and NHE via nongenomic signaling. The stimulatory effects were completely blocked by specific PPARγ antagonist GW9662, ERK kinase inhibitor PD98059, and CD36 inhibitor sulfosuccinimidyl oleate. Treatment with an siRNA against PPAR gamma completely blocked the stimulation of both NBCe1 and NHE by azPC. Moreover, azPC induced ERK phosphorylation in rat and human kidney cortex tissues, which were completely suppressed by GW9662 and PD98059 treatments. These results suggest that azPC stimulates renal PT sodium-coupled bicarbonate transport via a CD36/PPARγ/mitogen-activated protein/ERK kinase/ERK pathway. We conclude that the stimulatory effects of azPC on PT transport may be partially involved in volume expansion.

Cadmium induces the expression of Interleukin-6 through Heme Oxygenase-1 in HK-2 cells and Sprague-Dawley rats

Cadmium is toxic to the kidney through mechanisms involving oxidative stress and inflammation. We studied reciprocal crosstalk among the oxidative stress, inflammation, and the nuclear Nrf2 pathway in cadmium-induced nephrotoxicity on HK-2 human renal proximal tubular epithelial cells. Cadmium chloride (CdCl2) caused cell viability loss, Reactive Oxygen Species (ROS) generation, glutathione reduction, and Interleukin-6 (IL-6) expression, accompanied by Nrf2 activation and Heme Oxygenase-1 (HO-1) expression. Pharmacological treatments demonstrated cytotprotective and anti-inflammatory effects of Nrf2 activation. Intriguingly, inhibition of HO-1 activity mitigated cell viability loss and IL-6 expression in CdCl2-treated cells. Parallel attenuation by HO-1 inhibitor was demonstrated in cadmium-induced ROS generation and glutathione reduction. CdCl2-treated cells also increased levels of ferrous iron, cGMP, Mitogen-Activated Protein Kinases phosphorylation, as well as NF-κB DNA-binding activity. These increments were mitigated by antioxidant N-Acetyl Cysteine, HO-1 inhibitor SnPP, and PKG inhibitor KT5823, and were mimicked by the Carbon Monoxide-releasing compound. In the kidney cortex of CdCl2-exposed Sprague-Dawley rats, we found similar renal injury, histological changes, ROS generation, IL-6 expression, and accompanied pro-oxidant and pro-inflammatory changes. These observations indicated that cadmium-induced nephrotoxicity was associated with oxidative stress and inflammation, and HO-1 likely acts as a linking molecule to induce nephrotoxicity-associated IL-6 expression upon cadmium exposure.

Substrate-dependent differential regulation of mitochondrial bioenergetics in the heart and kidney cortex and outer medulla

The kinetics and efficiency of mitochondrial oxidative phosphorylation (OxPhos) can depend on the choice of respiratory substrates. Furthermore, potential differences in this substrate dependency among different tissues are not well-understood. Here, we determined the effects of different substrates on the kinetics and efficiency of OxPhos in isolated mitochondria from the heart and kidney cortex and outer medulla (OM) of Sprague-Dawley rats. The substrates were pyruvate+malate, glutamate+malate, palmitoyl-carnitine+malate, alpha-ketoglutarate+malate, and succinate±rotenone at saturating concentrations. The kinetics of OxPhos were interrogated by measuring mitochondrial bioenergetics under different ADP perturbations. Results show that the kinetics and efficiency of OxPhos are highly dependent on the substrates used, and this dependency is distinctly different between heart and kidney. Heart mitochondria showed higher respiratory rates and OxPhos efficiencies for all substrates in comparison to kidney mitochondria. Cortex mitochondria respiratory rates were higher than OM mitochondria, but OM mitochondria OxPhos efficiencies were higher than cortex mitochondria. State 3 respiration was low in heart mitochondria with succinate but increased significantly in the presence of rotenone, unlike kidney mitochondria. Similar differences were observed in mitochondrial membrane potential. Differences in H2O2 emission in the presence of succinate±rotenone were observed in heart mitochondria and to a lesser extent in OM mitochondria, but not in cortex mitochondria. Bioenergetics and H2O2 emission data with succinate±rotenone indicate that oxaloacetate accumulation and reverse electron transfer may play a more prominent regulatory role in heart mitochondria than kidney mitochondria. These studies provide novel quantitative data demonstrating that the choice of respiratory substrates affects mitochondrial responses in a tissue-specific manner.

Abstract 11322: Role of Centhaquine (lyfaquin ® ) in the Protection of Renal Tissues After Acute Kidney Injury

Background: Acute Kidney Injury (AKI) is frequently associated with an in-hospital complication of sepsis, heart conditions, hemorrhagic shock, and surgery. However, at present, no specific drug to treat AKI is available. Centhaquine (Lyfaquin ® ) is a safe and effective first-in-class resuscitative agent for hypovolemic shock. We are investigating whether centhaquine could protect kidneys from acute injury during hypovolemic shock. Hypothesis: AKI is known to be associated with shock-related morbidity and mortality. We hypothesize that centhaquine would have a direct effect on alleviating the AKI and reducing shock-related mortality. Methods: Renal arteries of rats were clamped and de-clamped to induce AKI like ischemia/reperfusion model, and hemorrhage was carried out by withdrawing blood for 30 min from the femoral artery. Rats were resuscitated with centhaquine (0.02 mg/kg) for 10 min. Mean Arterial Pressure (MAP), heart rate (HR), and renal blood flow (RBF) were monitored for 120 min. Results: An improved RBF (p&lt;0.003) in centhaquine compared to vehicle rats was observed after resuscitation. While MAP and HR were similar in centhaquine and vehicle groups. At 120 min post-resuscitation, significantly reduced blood lactate level (p=0.0064) in centhaquine than the vehicle was seen. Higher damage in renal tissues in the vehicle than centhaquine was observed by histopathological analysis. Western blots showed higher HIF-1α (p=0.0152) and lower NGAL (p=0.01626) levels in centhaquine vs. vehicle. While significantly higher (p&lt;0.045) expression of HIF-1α and lower expression of Bax (p&lt;0.044) in kidney cortex and medulla of centhaquine rats were observed after immunofluorescence analysis. On the other hand, expression of PHD 3 (p&lt;0.0001) was higher, while the expression of Cytochrome C (p=0.01429) was lower in the cortex of centhaquine than the vehicle. Also, the in situ PCR data showed higher mitochondrial biogenesis in centhaquine than in the vehicle. Conclusions: Centhaquine (Lyfaquin ® ) increases renal blood flow and augments hypoxia response, which probably helps reduce tissue damage and apoptosis following hemorrhagic shock induced AKI, and hence the potential of centhaquine could be explored further to prevent/treat AKI.

Abstract 12660: Novel Model of Oxalate Diet Induced Chronic Kidney Disease in Dahl-Salt-Sensitive Rats

Background: Diet induced models of chronic kidney disease (CKD) may offer several advantages vs surgical models in terms of clinical relevance and animal welfare. Oxalate is a plant-based, terminal toxic metabolite that is eliminated by the kidneys through glomerular filtration and tubular secretion. Increased load of dietary oxalate leads to supersaturation, calcium oxalate crystal formation, renal tubular obstruction and eventually CKD. Dahl-salt-sensitive rats (Dahl-S) are a common strain used to study for hypertensive renal disease however characterization of other diet induced models on this background would allow for comparative studies of CKD within the same strain. We hypothesized that Dahl-S rats on a low-salt, oxalate rich diet would have increased renal injury and will serve as a novel rat model to study CKD. Methods/Results: Ten week-old male Dahl-S rats were fed either 0.2% salt normal chow (SS-NC) or a 0.2% salt diet containing 0.67% sodium oxalate (SS-OX) for five weeks (n=6-8/group). Real-time PCR demonstrated increased expression of inflammatory marker IL-6 (235% vs SS-NC, p&lt;0.0001) and fibrotic marker Timp-1 metalloproteinase (142% vs SS-NC, p&lt;0.0001) in the renal cortex of SS-OX rat kidneys compared to SS-NC. Immunohistochemistry of kidney tissue demonstrated increase in CD-68 levels, a marker of macrophage infiltration in SS-OX rats (275% vs SS-NC, p&lt;0.001). In addition, SS-OX rats displayed increased 24-hour urinary protein excretion (UPE) (97% vs SS-NC, p&lt;0.01) as well as significant elevations of plasma Cystatin C (135% vs SS-NC, p&lt;0.01). Furthermore, oxalate diet induced hypertension (23% increase in systolic blood pressure vs. SS-NC, p&lt;0.05) and renin-angiotensin-aldosterone system (RAAS) profiling (via LC-MS/MS) in the SS-OX plasma showed significant (p&lt;0.05) increases in angiotensin [1-5] (128% vs SS-NC) and angiotensin I (56% vs SS-NC) as well as suppression of the steroid aldosterone (-54% vs SS-NC). Conclusion: Oxalate diet induces significant renal inflammation, fibrosis, renal dysfunction, decreased aldosterone secretion as well as RAAS activation and hypertension in Dahl-S rats and provides a novel diet-induced model to study hypertension and CKD.