Renal ATP Research Articles

Fucoidan Improves Early Stage Diabetic Nephropathy via the Gut Microbiota-Mitochondria Axis in High-Fat Diet-Induced Diabetic Mice.

Diabetic nephropathy (DN) is a common microvascular complication of diabetes. Fucoidan, a polysaccharide containing fucose and sulfate group, ameliorates DN. However, the underlying mechanism has not been fully understood. This study aimed to explore the effects and mechanism of fucoidan on DN in high-fat diet-induced diabetic mice. A total of 90 C57BL/6J mice were randomly assigned to six groups (n = 15) as follows: normal control (NC), diabetes mellitus (DM), metformin (MTF), low-dose fucoidan (LFC), medium-dose fucoidan (MFC), and high-dose fucoidan (HFC). A technique based on fluorescein isothiocyanate (FITC-sinistin) elimination kinetics measured percutaneously was applied to determine the glomerular filtration rate (GFR). After 24 weeks, the mice were sacrificed and an early stage DN model was confirmed by GFR hyperfiltration, elevated urinary creatinine, normal urinary albumin, tubulointerstitial fibrosis, and glomerular hypertrophy. Fucoidan significantly improved the GFR hyperfiltration and renal fibrosis. An enriched SCFAs-producing bacteria and increased acetic concentration in cecum contents were found in fucoidan groups, as well as increased renal ATP levels and improved mitochondrial dysfunction. The renal inflammation and fibrosis were ameliorated through inhibiting the MAPKs pathway. In conclusion, fucoidan improved early stage DN targeting the microbiota-mitochondria axis by ameliorating mitochondrial oxidative stress and inhibiting the MAPKs pathway.

Renal Mitochondrial ATP Transporter Ablation Ameliorates Obesity-Induced CKD.

This study sheds light on the central role of adenine nucleotide translocase 2 (ANT2) in the pathogenesis of obesity-induced CKD. Our data demonstrate that ANT2 depletion in renal proximal tubule cells (RPTCs) leads to a shift in their primary metabolic program from fatty acid oxidation to aerobic glycolysis, resulting in mitochondrial protection, cellular survival, and preservation of renal function. These findings provide new insights into the underlying mechanisms of obesity-induced CKD and have the potential to be translated toward the development of targeted therapeutic strategies for this debilitating condition. The impairment in ATP production and transport in RPTCs has been linked to the pathogenesis of obesity-induced CKD. This condition is characterized by kidney dysfunction, inflammation, lipotoxicity, and fibrosis. In this study, we investigated the role of ANT2, which serves as the primary regulator of cellular ATP content in RPTCs, in the development of obesity-induced CKD. We generated RPTC-specific ANT2 knockout ( RPTC-ANT2-/- ) mice, which were then subjected to a 24-week high-fat diet-feeding regimen. We conducted comprehensive assessment of renal morphology, function, and metabolic alterations of these mice. In addition, we used large-scale transcriptomics, proteomics, and metabolomics analyses to gain insights into the role of ANT2 in regulating mitochondrial function, RPTC physiology, and overall renal health. Our findings revealed that obese RPTC-ANT2-/- mice displayed preserved renal morphology and function, along with a notable absence of kidney lipotoxicity and fibrosis. The depletion of Ant2 in RPTCs led to a fundamental rewiring of their primary metabolic program. Specifically, these cells shifted from oxidizing fatty acids as their primary energy source to favoring aerobic glycolysis, a phenomenon mediated by the testis-selective Ant4. We propose a significant role for RPTC-Ant2 in the development of obesity-induced CKD. The nullification of RPTC-Ant2 triggers a cascade of cellular mechanisms, including mitochondrial protection, enhanced RPTC survival, and ultimately the preservation of kidney function. These findings shed new light on the complex metabolic pathways contributing to CKD development and suggest potential therapeutic targets for this condition.

Time-Dependent Differences in Vancomycin Sensitivity of Macrophages Underlie Vancomycin-Induced Acute Kidney Injury.

Although vancomycin (VCM)-frequently used to treat drug-resistant bacterial infections-often induces acute kidney injury (AKI), discontinuation of the drug is the only effective treatment; therefore, analysis of effective avoidance methods is urgently needed. Here, we report the differences in the induction of AKI by VCM in 1/2-nephrectomized mice depending on the time of administration. Despite the lack of difference in the accumulation of VCM in the kidney between the light (ZT2) and dark (ZT14) phases, the expression of AKI markers due to VCM was observed only in the ZT2 treatment. Genomic analysis of the kidney suggested that the time of administration was involved in VCM-induced changes in monocyte and macrophage activity, and VCM had time-dependent effects on renal macrophage abundance, ATP activity, and interleukin (IL)-1β expression. Furthermore, the depletion of macrophages with clodronate abolished the induction of IL-1β and AKI marker expression by VCM administration at ZT2. This study provides evidence of the need for time-dependent pharmacodynamic considerations in the prevention of VCM-induced AKI as well as the potential for macrophage-targeted AKI therapy. SIGNIFICANCE STATEMENT: There is a time of administration at which vancomycin (VCM)-induced renal injury is more and less likely to occur, and macrophages are involved in this difference. Therefore, there is a need for time-dependent pharmacodynamic considerations in the prevention of VCM-induced acute kidney injury as well as the potential for macrophage-targeted acute kidney injury therapy.

Virgin Camellia Seed Oil Improves Glycolipid Metabolism in the Kidney of High Fat-Fed Rats through AMPK-SREBP Pathway

Camellia seed oil (CO) is used as edible oil in southern China because of its excellent fatty acid composition and abundant bioactive compounds. Chronic kidney disease (CKD) is one of the most common chronic degenerative diseases in China, and active compounds in vegetable oil, like virgin olive oil, have been demonstrated to be efficacious in the management of CKD. In this study, virgin CO was refined using a standard process. The refining had minimal impact on the fatty acid composition, but significantly reduced the presence of bioactive compounds like polyphenols in CO. Sprague-Dawley (SD) rats fed with high fat diet (Group G) were treated with either virgin (Group Z) or refined CO (Group R). The oral administration of CO alleviated lipid accumulation and decreased body and kidney weight gain. Furthermore, treatment with virgin CO increased the renal ATP content. The renal expression levels of AMPK and key enzymes involved in fatty acid oxidation (CPT-1 and ACOX1) and glycolysis (HK, PFK, PK and GAPDH) were up-regulated in Group Z, thereby enhancing the ATP production. Virgin CO treatment downregulated the expression level of SREBP2 and its downstream target genes, such as ACC, FAS, and HMGCR, which reduced lipid synthesis. These findings indicate that virgin CO improves glycolipid metabolism and restores energy homeostasis in the kidneys of rats fed with a high-fat diet by modulating the AMPK–SREBP-signaling pathway, suggesting the potential of active compounds in virgin CO for managing the renal failure associated with glycolipid dysmetabolism.

Sodium Hydrosulfide Treatment During Porcine Kidney Ex Vivo Perfusion and Transplantation.

In rodents, hydrogen sulfide (H2S) reduces ischemia-reperfusion injury and improves renal graft function after transplantation. Here, we hypothesized that the benefits of H2S are conserved in pigs, a more clinically relevant model. Adult porcine kidneys retrieved immediately or after 60 min of warm ischemia (WI) were exposed to 100 µM sodium hydrosulfide (NaHS) (1) during the hypothermic ex vivo perfusion only, (2) during WI only, and (3) during both WI and ex vivo perfusion. Kidney perfusion was evaluated with dynamic contrast-enhanced MRI. MRI spectroscopy was further employed to assess energy metabolites including ATP. Renal biopsies were collected at various time points for histopathological analysis. Perfusion for 4 h pig kidneys with Belzer MPS UW + NaHS resulted in similar renal perfusion and ATP levels than perfusion with UW alone. Similarly, no difference was observed when NaHS was administered in the renal artery before ischemia. After autotransplantation, no improvement in histologic lesions or cortical/medullary kidney perfusion was observed upon H2S administration. In addition, AMP and ATP levels were identical in both groups. In conclusion, treatment of porcine kidney grafts using NaHS did not result in a significant reduction of ischemia-reperfusion injury or improvement of kidney metabolism. Future studies will need to define the benefits of H2S in human, possibly using other molecules as H2S donors.

Protective effect of eriodictyol against hyperglycemia-induced diabetic nephropathy in rats entails antioxidant and anti-inflammatory effects mediated by activating Nrf2

The pathogenesis of diabetic nephropathy (DN) involves cellular activation of oxidative stress and inflammation. Eriodictyol is a citrus-derived flavonoid with multiple pharmacological and protective effects in various conditions. The protective role of Eriodictyol against diabetes and diabetic nephropathy is less investigated. The current research aimed to explore the role of eriodictyol in protecting against DN prompted by streptozotocin in male rats and investigate some possible mechanisms of action. Diabetes was brought about in rats by an i.p injection of a lone dose (65 mg/kg). Five groups of rats were included (n = 8 each) as control (non-diabetic), eriodictyol (20 mg/kg, orally), STZ-diabetic, STZ + eriodictyol (20 mg/kg, orally), and STZ + eriodictyol (20 mg/kg, orally) + ML385 (30 µg/kg, i.p.). Kidney histology and the levels of some markers of kidney function, renal oxidative stress, and renal inflammation were analyzed in all groups of rats. Treatment with eriodictyol prevented the damage in the renal glomeruli and tubules and reduced renal immune cell infiltration in STZ-treated animals. It also spiked urinary creatinine excretion and reduced urine volume and urinary levels of albumin, monocyte chemoattractant protein 1 (MCP-1), urinary kidney injury molecule-1 (KIM-1), and nephrin in these diabetic rats. In addition, eriodictyol stimulated the nuclear protein accumulation of Nrf2 and boosted the expression of superoxide dismutase (SOD), glutathione (GSH), heme oxygenase-1 (HO-1), and catalase (CAT) in the diabetic rat kidneys. In concomitance, it reduced the nuclear levels of NF-κB and levels of interleukine-6 (IL-6), malondialdehyde (MDA), and tumor necrosis factor-α (TNF-α) and attenuated the reduction in renal ATP levels and the increase in the mitochondria transition pore opening (mtTPT). However, the administration of eriodictyol did not affect rats’ body weights and fasting glucose and insulin levels but significantly reduced serum levels of cholesterol, triglycerides, LDL-c, and oxidized LDL-c (ox-LDL-c). In conclusion, eriodictyol prevents STZ-induced nephropathy by a hypolipidemic effect and concomitant antioxidant and anti-inflammatory effects mediated by activating Nrf2/NF-κB/antioxidant axis.

Long-term dietary restriction ameliorates ageing-related renal fibrosis in male mice by normalizing mitochondrial functions and autophagy.

As the kidneys age, gradual changes in the structures and functions of mitochondria occur. Dietary restriction (DR) can play a protective role in ageing-associated renal decline, however the exact mechanisms involved are still unclear. This study aims to clarify the beneficial effects of long-term DR on renal ageing and to explore the potential mechanisms of mitochondrial homeostasis. Eight-week-old C57BL/6 male mice (n = 30) were randomly divided into three groups, Young-AL (AL, ad libitum), Aged-AL, and Aged-DR (60% intake of AL). Mice were sacrificed at age of 7months (Young) or 22months (Aged). Heavier body and kidney weights were associated with ageing, but DR reduced these increases in aged mice. Ageing caused extensive tubulointerstitial fibrosis and glomerulosclerosis in the kidney. Giant mitochondria with looser and irregular crista were observed in Aged-AL kidneys. DR retarded these morphological alterations in aged kidneys. In addition, DR reversed the increase of MDA caused by ageing. Renal ATP level was elevated by DR treatment. Mitochondrial-related proteins were analysed to elucidate this association. Ageing downregulated the renal levels of VDAC, FOXO1, SOD2, LC3I and II, and upregulated the renal levels of MFN2 and PINK1. In contrast, DR elevated the levels of VDAC, FOXO1, and LC3I and reduced the ratio of LC3II to LC3I in aged kidneys. To conclude, impaired mitochondria, increased oxidative stress, and severe fibrosis were noticed in the aged kidneys, and DR improved these changes by increasing functional mitochondria and promoting autophagic clearance.

Proximal tubular epithelia-specific transcriptomics of diabetic mice treated with dapagliflozin

Based on recent clinical trials using sodium-glucose co-transporter 2 inhibitor (SGLT2i) demonstrating the significant improvement of outcomes of diabetic kidney disease (DKD), the paradigm shift from “glomerulocentric” to “tubule centric” pathophysiology in DKD progression has been highlighted. Several responsible mechanisms for renoprotective effects by SGLT2i have been proposed recently, but the changes in proximal tubule-specific gene expression by SGLT2i in diabetic mice have not been elucidated. We report the analysis of the proximal tubular-specific pathway, demonstrating the downregulation of oxidative phosphorylation in dapagliflozin-treated db/db mice, a type 2 diabetic model. After 8-week treatment of dapagliflozin for db/db mice having a proximal tubule-specific tdTomato reporter, tdTomato-positive cells were isolated by FACS. Pathway analysis of RNA sequencing of isolated tubular epithelia revealed that oxidative phosphorylation was downregulated in dapagliflozin-treated mice. However, depletion of renal tissue ATP content in db/db mice was ameliorated by dapagliflozin administration. Pimonidazole staining demonstrated renal cortical tissue hypoxia in db/db mice, which was improved by dapagliflozin administration. This study suggests that dapagliflozin can ameliorate the excessive oxygen and ATP consumption, and subsequent tissue hypoxia in the diabetic kidney, which may explain, in part, the responsible mechanisms of the renoprotective effects of dapagliflozin.

Percutaneous transluminal renal angioplasty attenuates poststenotic kidney mitochondrial damage in pigs with renal artery stenosis and metabolic syndrome.

Percutaneous transluminal renal angioplasty (PTRA) has been used to treat renovascular disease (RVD), a chronic condition characterized by renal ischemia and metabolic abnormalities. Mitochondrial injury has been implicated as a central pathogenic mechanism in RVD, but whether it can be reversed by PTRA remains uncertain. We hypothesized that PTRA attenuates mitochondrial damage, renal injury, and dysfunction in pigs with coexisting renal artery stenosis (RAS) and metabolic syndrome (MetS). Four groups of pigs (n = 6 each) were studied after 16 weeks of diet-induced MetS and RAS (MetS + RAS), MetS + RAS treated 4 weeks earlier with PTRA, and Lean and MetS Sham controls. Single-kidney renal blood flow (RBF) and glomerular filtration rate (GFR) were assessed in vivo with multidetector computed tomography, and renal tubular mitochondrial structure and function and renal injury ex vivo. PTRA successfully restored renal artery patency, but mean arterial pressure remained unchanged. Stenotic kidney RBF and GFR, which fell in MetS + RAS compared to MetS, rose after PTRA. PTRA attenuated MetS + RAS-induced mitochondrial structural abnormalities in tubular cells and peritubular capillary endothelial cells, decreased mitochondrial H2 02 production, and increased renal cytochrome-c oxidase-IV activity and ATP production. PTRA also improved cortical microvascular and peritubular capillary density and ameliorated tubular injury and tubulointerstitial fibrosis in the poststenotic kidney. Importantly, renal mitochondrial damage correlated with poststenotic injury and dysfunction. Renal revascularization attenuated mitochondrial injury and improved renal hemodynamics and function in swine poststenotic kidneys. This study suggests a novel mechanism by which PTRA might be relatively effective in ameliorating mitochondrial damage and improving renal function in coexisting MetS and RAS.

Abstract 16: Progressive Alterations Of Mitochondrial Bioenergetics In The Kidney During The Development Of Salt-induced Hypertension

Hypertension is a complex disease and a leading cause of morbidity and mortality globally. Although oxidative stress and mitochondrial dysfunction have been found in the kidney in various models of hypertension, progressive alteration of mitochondrial oxidative phosphorylation (OxPhos) in the kidney during the development of salt-sensitive hypertension has not been characterized. The present study determined changes of OxPhos in kidneys of Dahl salt-sensitive (SS) rats before (0.4% NaCl diet; LS) and after switching to a high salt diet (4.0% NaCl; HS) during the development of hypertension. Mitochondria were isolated from the outer medulla (OM) and cortex of the kidney of SS rats fed a LS diet since weaning and studied at days 3, 7, 14 & 21 of a HS diet feeding. Oxygen consumption rates (OCR) were measured in mitochondria energized with pyruvate + malate as substrates for three different respiratory states using an Oroboros Oxygraph-2k Instrument. This includes i) leak state (in the absence of ADP), ii) ADP-stimulated state, and iii) uncoupled state (in the presence of an uncoupler FCCP). A biphasic pattern of ADP-stimulated OCR with progressive uncoupling was observed in both the renal OM and cortex. Mitochondrial efficiency for ATP synthesis was increased in the early phases of hypertension (3 & 7 days) but was severely compromised in the established phases of hypertension (14 & 21 days). This decreased mitochondrial efficiency was associated with uncoupling of OxPhos and high levels of oxidative stress which we hypothesized were due to mitochondrial ROS stimulation of membrane NOXs. To test this, experiments were performed in SS rats with double knock out (DKO) of the cytosolic subunit of NOX2 (p67 phox ) and NOX4 (SS p67phox-/-/Nox4-/- ). DKO SS rats were fed a HS diet and OCR of renal cortical and OM mitochondria was determined at days 7 and 14. In contrast to SS rats, the DKO SS rats fed a HS diet showed no significant differences in mitochondrial OCR in the cortex or OM, nor to a control group maintained on a LS diet. HS diet in SS rats initially increases the efficiency of renal cortical and medullary mitochondrial ATP production (days 1-7) followed by an enhanced ROS production with mitochondrial uncoupling and reduced efficiency of ATP production by the third week.

SGLT2 Inhibition Mediates Protection from Diabetic Kidney Disease by Promoting Ketone Body-Induced mTORC1 Inhibition

SGLT2 inhibitors offer strong renoprotection in subjects with diabetic kidney disease (DKD). But the mechanism for such protection is not clear. Here, we report that in damaged proximal tubules of high-fat diet-fed ApoE-knockout mice, a model of non-proteinuric DKD, ATP production shifted from lipolysis to ketolysis dependent due to hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1). We further found that empagliflozin raised endogenous ketone body (KB) levels, and thus its use or treatment with 1,3-butanediol, a KB precursor, prevented decreases in renal ATP levels and organ damage in the mice. The renoprotective effect of empagliflozin was abolished by gene deletion of Hmgcs2, a rate-limiting enzyme of ketogenesis. Furthermore, KBs attenuated mTORC1-associated podocyte damage and proteinuria in diabetic db/db mice. Our findings show that SGLT2 inhibition-associated renoprotection is mediated by anelevation of KBs that in turn corrects mTORC1 hyperactivation that occurs in non-proteinuric and proteinuric DKD.

Roles of Mitochondrial Dysfunction in Maleic Acid‐induced Acute Kidney Injury

BackgroundAcute kidney injury (AKI) is a growing public health burden globally. Mitochondrial function plays the key role in the progress of AKI. Maleic acid (MA) has been proofed to induce various renal dysfunctions include AKI. In our previous study, there were MA affinity proteins, including ATP synthase subunits, mitochondrial glyceraldehydes‐3‐phosphate, been identified in human kidney proximal tubular cells (HK2 cells) (1). The goal of this study is investigating mitochondria roles in AKI using our MA‐induced mitochondrial dysfunction AKI modelMethodsFor the in vitro study, HK2 cells were induced with MA with 2mM and 5mM for 24 hours separately. For the in vivo study, we induced AKI in c57BL/6 mice by intraperitoneal MA injection with 400mg/kg body weight and sacrificed after 24 hours of injection.ResultsWe found that the MA treatment induces HK2 cell death with dosage responsive in MA 2mM (p<0.01) and MA 5mM (p<0.001). Serum BUN and creatinine were all increased (p<0.05) in the MA‐treated mice when compared to the vehicle treated mice. The histological analysis showed increased tubule injury index Jablonski score (p<0.001), tubular dilatation areas (p<0.001) and greater casting area (p<0.001) in MA‐treated as compared to vehicle‐treated mice. AKI marker, Kidney Injury Molecule‐1 was increased (p<0.001) in MA‐treated as compared to vehicle‐treated mice. The flow cytometry analysis indicated increased apoptosis (p<0.001), cell reactive oxidative stress (ROS) (p<0.001), mitochondrial ROS (p<0.001), and decreased mitochondria membrane potential (p<0.001) in MA‐treated compared to vehicle‐treated in vitro. In O2 respiration, MA‐treated HK2 cells decreased the capacity of electro transfer system activity (p<0.001) and nonphosphorylating LEAK respiration (electron flow coupled to proton pumping to compensate for proton leaks) (p<0.001), compared to vehicle‐treated. There was also significantly decreased ATP production in MA‐treated compared to vehicle‐treated in vitro. In mitochondria structure, the cristae of mitochondria were lost in MA‐treated compared to vehicle‐treated under transmission electron miscroscope examination. The gene expression was confirmed with the significant lower of ATPase6 in MA‐treated compared to vehicle‐treated. Moreover, renal mitochondrial protein of ATP subunit C (p<0.05) were significantly decreased in MA‐treated compared to vehicle treated in vivo.ConclusionThere results demonstrated that MA‐induced AKI through targeting renal proximal tubular mitochondrial ATP Synthase. Mitochondria may represent a novel therapeutic target for MA‐induced AKI.Support or Funding Information

Abstract 142: Purinergic P2 X Receptors and a T 1 Receptors Share Post - Receptor Signaling Pathways Regulating Renal Afferent Arterioles in Angiotensin I I ( Ang I I) Dependent Hypertension

In Ang II dependent hypertension, the increased afferent arteriolar resistance (AAR) is assumed to be primarily due to the increased intrarenal levels of Ang II activating AT1 receptors. Because the renal interstitial ATP levels increase with increased renal perfusion pressure (RPP), purinergic P2X receptors (P2XR) also influence AAR. However, the interactions between the respective receptors has not been fully established. Accordingly, experiments were performed to test the hypothesis that P2XR and AT1R share post-receptor signaling pathways which allow dominance of P2XR to control AAR. Experiments using the in vitro juxtamedullary nephron preparation allowed direct visualization of afferent arteriolar diameters (AAD). Normotensive and Ang II infused hypertensive rats showed AAD responses to increases in RPP from 100 to 140 mmHg by decreasing AAD by 26% ± 10% and 19% ± 4% demonstrating autoregulation. In kidneys from normotensive rats, superfusion at 100 mmHg RPP with 5 μM ATP induced a rapid vasoconstriction from a control of 14.5 ± 0.9 μm to 11.2 ± 0.7 μm (n=8, P < 0.05). P2X1 receptor inhibition with NF449 (1 μM) increased AAD by 28% ± 7% returning it close to control values (14.0 ± 0.8 μm). P2X7 receptor inhibition with A438079 (1 μM) did not significantly alter the AAD (12.2 ± 0.6 μm), but combined treatment with P2X1R plus P2X7R inhibitors fully returned the AAD to control values (14.8 ± 0.7 μm), reflecting a dominant role of P2X1R in mediating ATP afferent vasoconstriction in normotensive kidneys. In Ang II infused hypertensive rats, at RPP 140 mmHg, treatment with AT1R inhibitor by SML1394 (1 μM) increased AAD by 10% ± 7% (13.44 ± 0.73 μm vs. 12.21 ± 0.63 μm, n=10, P<0.05). Treatment with P2X1R inhibitor increased AAD by 21% ± 14% (14.42 ± 0.51 μm vs. 11.94 ± 1.20 μm, n=5, P<0.05), and treatment with the P2X7R inhibitor also dilated AAD by 15% ± 3% (12.98 ± 0.64 μm vs. 11.26 ± 0.61 μm, n=5, P<0.05) reflecting a greater role of P2X7R in hypertension. Importantly, treatment with P2X1R inhibitor plus P2X7R inhibitor fully restored AAD to control levels (15.00 ± 0.65 μm vs. 12.02 ± 0.70 μm, n=5, P<0.05). The results suggest that in Ang II dependent hypertension, P2X1R, P2X7R and AT1R act through post-receptor converging signaling pathways allowing dominance of P2XR to regulate AAR.

5-HT1F receptor regulates mitochondrial homeostasis and its loss potentiates acute kidney injury and impairs renal recovery.

Our laboratory previously reported that agonists of the 5-hydoxytryptamine 1F (5-HT1F) receptor induce renal mitochondrial biogenesis (MB) and that stimulation of the 5-HT1F receptor following ischemia/reperfusion (I/R)-induced acute kidney injury (AKI) accelerated the recovery of renal function in mice. The goal of this study was to examine the contribution of the 5-HT1F receptor in the regulation of renal mitochondrial homeostasis and renal function in naïve and injured mice. Although 5-HT1F receptor knockout (KO) mice were healthy and fertile, and did not exhibit renal dysfunction, renal mitochondrial DNA copy number and mitochondrial fission gene expression increased at 10 wk of age. The 5-HT1F receptor KO mice exhibited greater proximal tubular injury and diminished renal recovery after I/R-induced AKI compared with wild-type mice. These findings were associated with persistent suppression of renal cortical MB and ATP levels after injury. In summary, the 5-HT1F receptor is a component of physiological MB regulation in the kidney, and its absence potentiates renal injury and impedes recovery.

Activation of the cholinergic anti-inflammatory pathway by GTS-21 attenuates cisplatin-induced acute kidney injury in mice.

Acute kidney injury (AKI) is the most common side effect of cisplatin, a widely used chemotherapy drug. Although AKI occurs in up to one third of cancer patients receiving cisplatin, effective renal protective strategies are lacking. Cisplatin targets renal proximal tubular epithelial cells leading to inflammation, reactive oxygen species, tubular cell injury, and eventually cell death. The cholinergic anti-inflammatory pathway is a vagus nerve-mediated reflex that suppresses inflammation via α7 nicotinic acetylcholine receptors (α7nAChRs). Our previous studies demonstrated the renoprotective and anti-inflammatory effects of cholinergic agonists, including GTS-21. Therefore, we examined the effect of GTS-21 on cisplatin-induced AKI. Male C57BL/6 mice received either saline or GTS-21 (4mg/kg, i.p.) twice daily for 4 days before cisplatin and treatment continued through euthanasia; 3 days post-cisplatin mice were euthanized and analyzed for markers of renal injury. GTS-21 significantly reduced cisplatin-induced renal dysfunction and injury (p<0.05). GTS-21 significantly attenuated renal Ptgs2/COX-2 mRNA and IL-6, IL-1β, and CXCL1 protein expression, as well as neutrophil infiltration after cisplatin. GTS-21 blunted cisplatin-induced renal ERK1/2 activation, as well as renal ATP depletion and apoptosis (p<0.05). GTS-21 suppressed the expression of CTR1, a cisplatin influx transporter and enhanced the expression of cisplatin efflux transporters MRP2, MRP4, and MRP6 (p<0.05). Using breast, colon, and lung cancer cell lines we showed that GTS-21 did not inhibit cisplatin’s tumor cell killing activity. GTS-21 protects against cisplatin-AKI by attenuating renal inflammation, ATP depletion and apoptosis, as well as by decreasing renal cisplatin influx and increasing efflux, without impairing cisplatin-mediated tumor cell killing. Our results support further exploring the cholinergic anti-inflammatory pathway for preventing cisplatin-induced AKI.

Metabolic adaptability in hexavalent chromium-treated renal tissue: an in vivo study.

BackgroundHexavalent chromium [Cr(VI)], an environmental pollutant that originates mostly from anthropogenic sources, is a serious threat to human health. After entering into cells, Cr(VI) is capable of producing excessive free radicals and causing tissue damage. The present study aims to reveal the toxic manifestation of Cr(VI) on the metabolic activity of renal tissue.MethodsMale Swiss albino mice were treated orally with potassium dichromate (K2Cr2O7) at a dose of 10 mg/kg body weight for a period of 30 days. Important tricarboxylic acid (TCA) cycle enzyme activities like isocitrate dehydrogenase, succinate dehydrogenase and malate dehydrogenase, as well as the activities of enzymes involved in oxidative phosphorylation such as Nicotinamide adenine dinucleotide (NADH) dehydrogenase, were measured. Additionally, transaminase and protease (pronase, cathepsin and trypsin) activities, tissue protein and free amino nitrogen were estimated in renal tissue. Glucose-6-phosphatase, glucose-6-phosphate dehydrogenase and alkaline phosphatase activities, as well as lactic acid, pyruvic acid and chromium contents, of kidneys were determined following standard protocols. Kidney histology was performed by hematoxylin and eosin staining.ResultsCr(VI) suppresses the rate-limiting enzymes of the TCA cycle and oxidative phosphorylation indicating an inhibition of renal ATP production. It decreases protease activity by eliminating the protein substrates and alters the gluconeogenic pathway. Cr(VI) worsens the normophysiological attributes of renal tissue by enhancing the activity of alkaline phosphatase, pointing towards kidney disease. Histopathological observations confirmed these biochemical results through the presence of chronic tubular nephritis and altered glomerular structure. Cr(VI) retention occurs to a greater extent in renal tissue, which intensifies the toxic manifestation of this pollutant in the kidney.ConclusionsCr(VI) disrupts the metabolic interaction between carbohydrates and proteins in mammalian renal tissue.

Evaluating the significance of hypoglycemia in promoting insulin induced hypertension by using a glucose clamp model

Background: Hypoglycemia unawareness is a detrimental risk factor in diabetic patients leading to serious complications if left untreated. Previous studies from our group determined that recurrent insulin induced hypoglycemia (RIIH) promotes hypertension.Objective: We hypothesize that RIIH not insulin by itself is the source of hypertension and end organ damage in diabetic patients.Methods: Male Sprague-Dawley-rats (200-250g, n=18) were provided with glucose food (125g glucose/kg body weight) and glucose water (0.1g glucose/100g body weight/ml). They were treated with subcutaneous insulin injections (7U/Kg) and blood glucose was monitored intermittently. Daily blood pressure was measured using the tail cuff method. Interstitial samples of ATP and angiotensinII (AngII) were collected by renal microdialysis and analyzed using luciferin-luciferase bioluminescent assay and EIA respectively. Reactive oxygen and nitrogen species in hearts and kidneys were analyzed using Electron Paramagnetic Resonance Spectroscopy (EPR).Results: Renal interstitial ATP levels increased from 90.2± 4.7ng/µl to 99.6± 8.7ng/µl (not significant) and AngII from 0.15± 0.02ng/ml to 0.13± 0.05ng/ml (not significant) from day 0 to 14. There was no significant change in mean arterial pressure (121.3 ± 1.4 mmHg on day 0 to 127.8 ± 1.4mmHg on day 14). Oxidative stress was reduced compared to RIIH model, which was evident from the EPR spectra.Conclusion: We demonstrated that hypertension induced end organ damage in diabetics is due to insulin induced hypoglycemia not insulin alone (by itself).

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.

Obesity-induced changes in kidney mitochondria and endoplasmic reticulum in the presence or absence of leptin.

We investigated obesity-induced changes in kidney lipid accumulation, mitochondrial function, and endoplasmic reticulum (ER) stress in the absence of hypertension, and the potential role of leptin in modulating these changes. We compared two normotensive genetic mouse models of obesity, leptin-deficient ob/ob mice and hyperleptinemic melanocortin-4 receptor-deficient mice (LoxTB MC4R-/-), with their respective lean controls. Compared with controls, ob/ob and LoxTB MC4R-/- mice exhibit significant albuminuria, increased creatinine clearance, and high renal triglyceride content. Renal ATP levels were decreased in both obesity models, and mitochondria isolated from both models showed alterations that would lower mitochondrial ATP production. Mitochondria from hyperleptinemic LoxTB MC4R-/- mice kidneys respired NADH-generating substrates (including palmitate) at lower rates due to an apparent decrease in complex I activity, and these mitochondria showed oxidative damage. Kidney mitochondria of leptin-deficient ob/ob mice showed normal rates of respiration with no evidence of oxidative damage, but electron transfer was partially uncoupled from ATP synthesis. A fourfold induction of C/EBP homologous protein (CHOP) expression indicated induction of ER stress in kidneys of hyperleptinemic LoxTB MC4R-/- mice. In contrast, ER stress was not induced in kidneys of leptin-deficient ob/ob mice. Our findings show that obesity, in the absence of hypertension, is associated with renal dysfunction in mice but not with major renal injury. Alterations to mitochondria that lower cellular ATP levels may be involved in obesity-induced renal injury. The type and severity of mitochondrial and ER dysfunction differs depending upon the presence or absence of leptin.

ENOS deficiency causes podocyte injury with mitochondrial abnormality

The contribution of endothelial nitric oxide synthase (eNOS) to podocyte integrity remains unclear. This study therefore examined podocytes and mitochondrial abnormalities in eNOS deficient mice. Absence of eNOS caused glomerular hypertrophy, along with occasional glomerular sclerosis and mesangiolysis. While many glomeruli did not have such advanced lesions, ultrastructural analysis showed cellular hypertrophy, vacuolization, lysosomal enlargement, and microvillus formation in podocytes of eNOS knockout (KO) mice. Increased oxidative stress was associated with mitochondrial abnormalities, including an increase in number, coupled with a reduction in size, of mitochondria in podocytes of eNOS-KO mice. While the levels of expression of several mitochondrial proteins were not altered, the d-17 mutation in mitochondrial DNA was significantly associated with the eNOS deficiency. Renal ATP level in the renal cortex and mitochondrial respiration in the primary podocytes were significantly lower in eNOS-KO mice, suggesting that renal mitochondria may be functionally impaired. Podocytes cultured with endothelial conditioned medium lacking NO consistently showed a greater degree of mitochondrial fragmentation and an increase in mitochondrial oxidative stress, with these mitochondrial alterations rescued by an NO donor. In conclusion, eNOS may be necessary to maintain podocyte integrity, especially mitochondrial function.