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ABSTRACT: Background: The folkloric use of Indigofera aspalathoides (Vahl) in anti-inflammatory and anti-cancer treatment dates back to circa 300 BC. However, reports on scientific validation for folkloric usage are not available yet. Purpose: The objective of the study is to assess the cytotoxic potential of I. aspalathoides and to provide a rationale for its ethnopharmacological use with lime juice (ascorbic acid) as an adjuvant. Methods: Using Soxhlet, the active principles of I. aspalathoides were extracted using methanol and ethanol as solvent and studied using gas chromatography coupled with mass spectrometry (GC–MS). We assessed the in vitro antioxidant efficacy by studying the DPPH radical scavenging activity, ferrous reducing antioxidant capacity (FRAC), and ABTS cation radical scavenging activity. Using MTT assay, the cytotoxic activity of SV-ME and EE against human cervical cancer (HeLa) and normal murine fibroblast (L929) cells were evaluated. The cytotoxicity and apoptosis induction was studied by dual acridine orange/ethidium bromide (AO/EB) fluorescent probe and Hoechst dye. Mitochondria membrane potential (Δψm) was assessed using JC1. Ascorbic acid (AA) additive/synergism was studied by co-treating HeLa cells with AA along with SV-ME and SV-EE at IC50 concentration. Results: SV-ME and EE exert higher DPPH radical scavenging at 200 μg/mL. Both the extracts showed effective ABTS radical cation scavenging activity and ferrous reducing antioxidant capacity. Further, SV-ME and SV-EE treatment affected the cell viability and induced apoptosis, reduced cell viability with a concomitant increase in nuclear condensation and DNA fragmentation in a dose-dependent manner in cancer (HeLa) cells, but not in normal (L929) cells. Both the extracts increased the mitochondria membrane potential (Δψm) depolarization and altered the cellular redox state in HeLa cells. In addition, adjuvant ascorbic acid (AA) treatment increased the cytotoxic efficacy of the SV-ME and SV-EE in HeLa cells. Conclusion: I. aspalathoides used in the Siddha medicine for antitumor treatment have a very potent cytotoxic effect against cervical cancer cells (HeLa) in vitro. The cytotoxic and antitumor effect may have plausible involvement of Nrf2-ARE signaling pathway by restoration of cellular redox homeostasis.

Diabetic kidney disease (DKD) can lead to end-stage kidney disease (ESKD) and mortality; however, few mechanistic biomarkers are available for high-risk patients, especially those without macroalbuminuria. Urine from participants with diabetes from the Chronic Renal Insufficiency Cohort (CRIC) study, the Singapore Study of Macro-angiopathy and Micro-vascular Reactivity in Type 2 Diabetes (SMART2D), and the American Indian Study determined whether urine adenine/creatinine ratio (UAdCR) could be a mechanistic biomarker for ESKD. ESKD and mortality were associated with the highest UAdCR tertile in the CRIC study and SMART2D. ESKD was associated with the highest UAdCR tertile in patients without macroalbuminuria in the CRIC study, SMART2D, and the American Indian study. Empagliflozin lowered UAdCR in nonmacroalbuminuric participants. Spatial metabolomics localized adenine to kidney pathology, and single-cell transcriptomics identified ribonucleoprotein biogenesis as a top pathway in proximal tubules of patients without macroalbuminuria, implicating mTOR. Adenine stimulated matrix in tubular cells via mTOR and stimulated mTOR in mouse kidneys. A specific inhibitor of adenine production was found to reduce kidney hypertrophy and kidney injury in diabetic mice. We propose that endogenous adenine may be a causative factor in DKD.

Chronic kidney disease (CKD) has a strong genetic component; however, the underlying pathways are not well understood. Dahl salt-sensitive (SS)/Jr rats spontaneously develop CKD with age and are used to investigate the genetic determinants of CKD. However, there are currently several genetically diverse Dahl SS rats maintained at various institutions and the extent to which some exhibit age-related CKD is unclear. We assessed glomerulosclerosis (GS) and tubulointerstitial fibrosis (TIF) in 3- and 6-mo-old male and female SS/JrHsdMcwi, BN/NHsd/Mcwi [Brown-Norway (BN)], and consomic SS-Chr 1 BN /Mcwi (SS.BN1) rats, in which chromosome 1 from the BN rat was introgressed into the genome of the SS/JrHsdMcwi rat. Rats were fed a 0.4% NaCl diet. GS (31 ± 3% vs. 7 ± 1%) and TIF (2.3 ± 0.2 vs. 0.5 ± 0.1) were significantly greater in 6-mo-old compared with 3-mo-old SS/JrHsdMcwi rats, and CKD was exacerbated in males. GS was minimal in 6- and 3-mo-old BN (3.9 ± 0.6% vs. 1.2 ± 0.4%) and SS.BN1 (2.4 ± 0.5% vs. 1.0 ± 0.3%) rats, and neither exhibited TIF. In SS/JrHsdMcwi and SS.BN1 rats, mean arterial blood pressure was significantly greater in 6-mo-old compared with 3-mo-old SS/JrHsdMcwi (162 ± 4 vs. 131 ± 2 mmHg) but not SS.BN1 (115 ± 2 vs. 116 ± 1 mmHg) rats. In 6-mo-old SS/JrHsdMcwi rats, blood pressure was significantly greater in females. RNA-sequencing analysis revealed that inflammatory pathways were upregulated in isolated medullary thick ascending tubules in 7-wk-old SS/JrHsdMcwi rats, before the development of tubule pathology, compared with SS.BN1 rats. In summary, SS/JrHsdMcwi rats exhibit robust age-related progression of medullary thick ascending limb abnormalities, CKD, and hypertension, and gene(s) on chromosome 1 have a major pathogenic role in such changes. NEW & NOTEWORTHY This study shows that the robust age-related progression of kidney disease in Dahl SS/JrHsdMcw rats maintained on a normal-salt diet is abolished in consomic SS.BN1 rats. Evidence that medullary thick ascending limb segments of SS/JrHsdMcw rats are structurally abnormal and enriched in proinflammatory pathways before the development of protein casts provides new insights into the pathogenesis of kidney disease in this model.

Following acute kidney injury (AKI), renal tubular cells may stimulate fibroblasts in a paracrine fashion leading to interstitial fibrosis, but the paracrine factors and their regulation under this condition remain elusive. Here we identify a macroautophagy/autophagy-dependent FGF2 (fibroblast growth factor 2) production in tubular cells. Upon induction, FGF2 acts as a key paracrine factor to activate fibroblasts for renal fibrosis. After ischemic AKI in mice, autophagy activation persisted for weeks in renal tubular cells. In inducible, renal tubule-specific atg7 (autophagy related 7) knockout (iRT- atg7 -KO) mice, autophagy deficiency induced after AKI suppressed the pro-fibrotic phenotype in tubular cells and reduced fibrosis. Among the major cytokines, tubular autophagy deficiency in iRT- atg7 -KO mice specifically diminished FGF2. Autophagy inhibition also attenuated FGF2 expression in TGFB1/TGF-β1 (transforming growth factor, beta 1)-treated renal tubular cells. Consistent with a paracrine action, the culture medium of TGFB1-treated tubular cells stimulated renal fibroblasts, and this effect was suppressed by FGF2 neutralizing antibody and also by fgf2 - or atg7 -deletion in tubular cells. In human, compared with non-AKI, the renal biopsies from post-AKI patients had higher levels of autophagy and FGF2 in tubular cells, which showed significant correlations with renal fibrosis. These results indicate that persistent autophagy after AKI induces pro-fibrotic phenotype transformation in tubular cells leading to the expression and secretion of FGF2, which activates fibroblasts for renal fibrosis during maladaptive kidney repair. Abbreviations: 3-MA: 3-methyladnine; ACTA2/α-SMA: actin alpha 2, smooth muscle, aorta; ACTB/β-actin: actin, beta; AKI: acute kidney injury; ATG/ Atg : autophagy related; BUN: blood urea nitrogen; CCN2/CTGF: cellular communication network factor 2; CDKN2A/p16: cyclin dependent kinase inhibitor 2A; CKD: chronic kidney disease; CM: conditioned medium; COL1A1: collagen, type I, alpha 1; COL4A1: collagen, type IV, alpha 1; CQ: chloroquine; ECM: extracellular matrix; eGFR: estimated glomerular filtration rate; ELISA: enzyme-linked immunosorbent assay; FGF2: fibroblast growth factor 2; FN1: fibronectin 1; FOXO3: forkhead box O3; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HAVCR1/KIM-1: hepatitis A virus cellular receptor 1; IHC: immunohistochemistry; IRI: ischemia-reperfusion injury; ISH: in situ hybridization; LTL: lotus tetragonolobus lectin; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PDGFB: platelet derived growth factor, B polypeptide; PPIB/cyclophilin B: peptidylprolyl isomerase B; RT-qPCR: real time-quantitative PCR; SA-GLB1/β-gal: senescence-associated galactosidase, beta 1; SASP: senescence-associated secretory phenotype; sCr: serum creatinine; SQSTM1/p62: sequestosome 1; TASCC: TOR-autophagy spatial coupling compartment; TGFB1/TGF-β1: transforming growth factor, beta 1; VIM: vimentin.

Mitochondria are essential for the activity, function and viability of eukaryotic cells and mitochondrial dysfunction is involved in the pathogenesis of acute kidney injury (AKI) and chronic kidney disease, as well as in abnormal kidney repair after AKI. Multiple quality control mechanisms, including antioxidant defence, protein quality control, mitochondrial DNA repair, mitochondrial dynamics, mitophagy and mitochondrial biogenesis, have evolved to preserve mitochondrial homeostasis under physiological and pathological conditions. Loss of these mechanisms may induce mitochondrial damage and dysfunction, leading to cell death, tissue injury and, potentially, organ failure. Accumulating evidence suggests a role of disturbances in mitochondrial quality control in the pathogenesis of AKI, incomplete or maladaptive kidney repair and chronic kidney disease. Moreover, specific interventions that target mitochondrial quality control mechanisms to preserve and restore mitochondrial function have emerged as promising therapeutic strategies to prevent and treat kidney injury and accelerate kidney repair. However, clinical translation of these findings is challenging owing to potential adverse effects, unclear mechanisms of action and a lack of knowledge of the specific roles and regulation of mitochondrial quality control mechanisms in kidney resident and circulating cell types during injury and repair of the kidney.

Lysophosphatidic acid (LPA) increases platelet-derived growth factor-B (PDGFB) and connective tissue growth factor (CTGF) production and secretion by proximal tubule (PT) cells through LPA2 receptor-G q α-α v β 6 -integrin-mediated activation of transforming growth factor-β1 (TGFB1). LPA2, β 6 -integrin, PDGFB, and CTGF increase in kidneys after ischemia-reperfusion injury (IRI), coinciding with fibrosis. The TGFB1 receptor antagonist SD-208 prevents increases of β 6 -integrin, TGFB1-SMAD signaling, and PDGFB/CTGF expression after IRI and ameliorates fibrosis (Geng H, Lan R, Singha PK, Gilchrist A, Weinreb PH, Violette SM, Weinberg JM, Saikumar P, Venkatachalam MA. Am J Pathol 181: 1236-1249, 2012; Geng H, Lan R, Wang G, Siddiqi AR, Naski MC, Brooks AI, Barnes JL, Saikumar P, Weinberg JM, Venkatachalam MA. Am J Pathol 174: 1291-1308, 2009). We report now that LPA1 receptor signaling through epidermal growth factor receptor (EGFR)-ERK1/2-activator protein-1 cooperates with LPA2-dependent TGFB1 signaling to additively increase PDGFB/CTGF production and secretion by PT cells. Conversely, inhibition of both pathways results in greater suppression of PDGFB/CTGF production and secretion and promotes greater PT cellular differentiation than inhibiting one pathway alone. Antagonism of the LPA-generating enzyme autotaxin suppressed signaling through both pathways. After IRI, kidneys showed not only more LPA2, nuclear SMAD2/3, and PDGFB/CTGF but also increased LPA1 and autotaxin proteins, together with enhanced EGFR/ERK1/2 activation. Remarkably, the TGFB1 receptor antagonist SD-208 prevented all of these abnormalities excepting increased LPA2. SD-208 inhibits only one arm of LPA signaling: LPA2-G q α-α v β 6 -integrin-dependent production of active TGFB1 and its receptor-bound downstream effects. Consequently, far-reaching protection by SD-208 against IRI-induced signaling alterations and tubule-interstitial pathology is not fully explained by our data. TGFB1-dependent feedforward modulation of LPA1 signaling is one possibility. SD-208 effects may also involve mitigation of injury caused by IRI-induced TGFB1 signaling in endothelial cells and monocytes. Our results have translational implications for using TGFB1 receptor antagonists, LPA1 and LPA2 inhibitors concurrently, and autotaxin inhibitors in acute kidney injury to prevent the development of chronic kidney disease.

The role of insulin receptor (IR) activated by hyperinsulinemia in obesity-induced kidney injury is not well understood. We hypothesized that activation of kidney proximal tubule epithelial IR contributes to obesity-induced kidney injury. We administered normal-fat diet (NFD) or high-fat diet (HFD) to control and kidney proximal tubule IR-knockout (KPTIRKO) mice for 4 months. Renal cortical IR expression was decreased by 60% in male and female KPTIRKO mice. Baseline serum glucose, serum creatinine, and the ratio of urinary albumin to creatinine (ACR) were similar in KPTIRKO mice compared to those of controls. On HFD, weight gain and increase in serum cholesterol were similar in control and KPTIRKO mice; blood glucose did not change. HFD increased the following parameters in the male control mice: renal cortical contents of phosphorylated IR and Akt, matrix proteins, urinary ACR, urinary kidney injury molecule-1-to-creatinine ratio, and systolic blood pressure. Renal cortical generation of hydrogen sulfide was reduced in HFD-fed male control mice. All of these parameters were ameliorated in male KPTIRKO mice. Interestingly, female mice were resistant to HFD-induced kidney injury in both genotypes. We conclude that HFD-induced kidney injury requires renal proximal tubule IR activation in male mice.

The proximal tubule relies on oxidative mitochondrial metabolism to meet its energy needs and has limited capacity for glycolysis, which makes it uniquely susceptible to damage during AKI, especially after ischemia and anoxia. Under these conditions, mitochondrial ATP production is initially decreased by several mechanisms, including fatty acid-induced uncoupling and inhibition of respiration related to changes in the shape and volume of mitochondria. Glycolysis is initially insufficient as a source of ATP to protect the cells and mitochondrial function, but supplementation of tricarboxylic acid cycle intermediates augments anaerobic ATP production, and improves recovery of mitochondrial oxidative metabolism. Incomplete recovery is characterized by defects of respiratory enzymes and lipid metabolism. During the transition to CKD, tubular cells atrophy but maintain high expression of glycolytic enzymes, and there is decreased fatty acid oxidation. These metabolic changes may be amenable to a number of therapeutic interventions., Competing Interests: M.A. Venkatachalam reports honoraria from Surrozen. All remaining authors have nothing to disclose., (Copyright © 2021 by the American Society of Nephrology.)

Unilateral ischemia-reperfusion (UIR) injury leads to progressive renal atrophy and tubulointerstitial fibrosis (TIF) and is commonly used to investigate the pathogenesis of the acute kidney injury-chronic kidney disease transition. Although it is well known that contralateral nephrectomy (CNX), even 2 wk post-UIR injury, can improve recovery, the physiological mechanisms and tubular signaling pathways mediating such improved recovery remain poorly defined. Here, we examined the renal hemodynamic and tubular signaling pathways associated with UIR injury and its reversal by CNX. Male Sprague-Dawley rats underwent left UIR or sham UIR and 2 wk later CNX or sham CNX. Blood pressure, left renal blood flow (RBF), and total glomerular filtration rate were assessed in conscious rats for 3 days before and over 2 wk after CNX or sham CNX. In the presence of a contralateral uninjured kidney, left RBF was lower ( P < 0.05) from 2 to 4 wk following UIR (3.6 ± 0.3 mL/min) versus sham UIR (9.6 ± 0.3 mL/min). Without CNX, extensive renal atrophy, TIF, and tubule dedifferentiation, but minimal pimonidazole and hypoxia-inducible factor-1α positivity in tubules, were present at 4 wk post-UIR injury. Conversely, CNX led ( P < 0.05) to sustained increases in left RBF (6.2 ± 0.6 mL/min) that preceded the increases in glomerular filtration rate. The CNX-induced improvement in renal function was associated with renal hypertrophy, more redifferentiated tubules, less TIF, and robust pimonidazole and hypoxia-inducible factor-1α staining in UIR injured kidneys. Thus, contrary to expectations, indexes of hypoxia are not observed with the extensive TIF at 4 wk post-UIR injury in the absence of CNX but are rather associated with the improved recovery of renal function and structure following CNX.

Introduction: Diabetic kidney disease (DKD) is the most prevalent complication in diabetic patients, which contributes to high morbidity and mortality. Urine and plasma metabolomics studies have been demonstrated to provide valuable insights for DKD. However, limited information on spatial distributions of metabolites in kidney tissues have been reported., Objectives: In this work, we employed an ambient desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) coupled to a novel bioinformatics platform (METASPACE) to characterize the metabolome in a mouse model of DKD., Methods: DESI-MSI was performed for spatial untargeted metabolomics analysis in kidneys of mouse models (F1 C57BL/6J-Ins2Akita male mice at 17 weeks of age) of type 1 diabetes (T1D, n = 5) and heathy controls (n = 6)., Results: Multivariate analyses (i.e., PCA and PLS-DA (a 2000 permutation test: P < 0.001)) showed clearly separated clusters for the two groups of mice on the basis of 878 measured m/z's in kidney cortical tissues. Specifically, mice with T1D had increased relative abundances of pseudouridine, accumulation of free polyunsaturated fatty acids (PUFAs), and decreased relative abundances of cardiolipins in cortical proximal tubules when compared with healthy controls., Conclusion: Results from the current study support potential key roles of pseudouridine and cardiolipins for maintaining normal RNA structure and normal mitochondrial function, respectively, in cortical proximal tubules with DKD. DESI-MSI technology coupled with METASPACE could serve as powerful new tools to provide insight on fundamental pathways in DKD.

Screening of several TNBC cell lines showed altered Smad2 and Smad3 protein levels compared to normal mammary epithelial cells, suggesting the possibility that it could play an important role in the escape of cancer cells from TGF-β mediated growth inhibition. To assess the functional relevance of these endogenous molecules, Smad2 or Smad3 expression was knocked down individually and assessed their effects on pro-oncogenic properties of TGF-β. Smad3 deficiency reduced growth and invasion capacity of breast cancer cells in comparison to Smad2 which had no effect. Smad3 deficiency was also found to be associated with a reduction in the expressions of TMEPAI/PMEPA1 and EMT inducing transcription factors, E-Cadherin and increased expression of cell cycle inhibitors and Vimentin. On the other hand, Smad2 deficiency had opposite effect on these regulators. Interestingly, the decreased growth, invasion and associated gene expressions were largely reversed by overexpressing TMEPAI in Smad3 knockdown cells, suggesting that Smad3-TMEPAI axis may be involved in subverting growth suppressive effects of TGF-β into growth promotion. Similarly, altered levels of Smad proteins and TMEPAI were also noted in primary TNBC tumor tissues. Analysis of the existing databases provided additional support in terms of TMEPAI and Smad2 expression impacting the survival of TNBC patients. Taken together, our data demonstrate a novel role for Smad3 in cancer transformation and cancer progression through TMEPAI and further suggest that selective targeting of TGF-β-Smad3-TMEPAI axis may be beneficial in triple negative breast cancer therapy and prevention., Competing Interests: CONFLICTS OF INTEREST No potential conflicts of interest were disclosed by the authors., (Copyright: © 2019 Singha et al.)

Acute kidney injury (AKI) and chronic kidney disease (CKD) are interconnected. Although AKI-to-CKD transition has been intensively studied, the information of AKI on CKD is very limited. Nonetheless, AKI, when occurring in patients with CKD, is known to be more severe and difficult to recover. CKD is associated with significant changes in cell signaling in kidney tissues, including the activation of transforming growth factor-β, p53, hypoxia-inducible factor, and major developmental pathways. At the cellular level, CKD is characterized by mitochondrial dysfunction, oxidative stress, and aberrant autophagy. At the tissue level, CKD is characterized by chronic inflammation and vascular dysfunction. These pathologic changes may contribute to the heightened sensitivity of, and nonrecovery from, AKI in patients with CKD., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

During recovery by regeneration after AKI, proximal tubule cells can fail to redifferentiate, undergo premature growth arrest, and become atrophic. The atrophic tubules display pathologically persistent signaling increases that trigger production of profibrotic peptides, proliferation of interstitial fibroblasts, and fibrosis. We studied proximal tubules after ischemia-reperfusion injury (IRI) to characterize possible mitochondrial pathologies and alterations of critical enzymes that govern energy metabolism. In rat kidneys, tubules undergoing atrophy late after IRI but not normally recovering tubules showed greatly reduced mitochondrial number, with rounded profiles, and large autophagolysosomes. Studies after IRI of kidneys in mice, done in parallel, showed large scale loss of the oxidant-sensitive mitochondrial protein Mpv17L. Renal expression of hypoxia markers also increased after IRI. During early and late reperfusion after IRI, kidneys exhibited increased lactate and pyruvate content and hexokinase activity, which are indicators of glycolysis. Furthermore, normally regenerating tubules as well as tubules undergoing atrophy exhibited increased glycolytic enzyme expression and inhibitory phosphorylation of pyruvate dehydrogenase. TGF-β antagonism prevented these effects. Our data show that the metabolic switch occurred early during regeneration after injury and was reversed during normal tubule recovery but persisted and became progressively more severe in tubule cells that failed to redifferentiate. In conclusion, irreversibility of the metabolic switch, taking place in the context of hypoxia, high TGF-β signaling and depletion of mitochondria characterizes the development of atrophy in proximal tubule cells and may contribute to the renal pathology after AKI., (Copyright © 2016 by the American Society of Nephrology.)

Acute kidney injury (AKI) is a major renal disease associated with high mortality rate and increasing prevalence. Decades of research have suggested numerous chemical and biological agents with beneficial effects in AKI. In addition, cell therapy and molecular targeting have been explored for reducing kidney tissue damage and promoting kidney repair or recovery from AKI. Mechanistically, these approaches may mitigate oxidative stress, inflammation, cell death, and mitochondrial and other organellar damage, or activate cytoprotective mechanisms such as autophagy and pro-survival factors. However, none of these findings has been successfully translated into clinical treatment of AKI. In this review, we analyze these findings and propose experimental strategies for the identification of renoprotective agents or methods with clinical potential. Moreover, we propose the consideration of combination therapy by targeting multiple targets in AKI., Competing Interests: Declarations All authors declare that they have no commercial or other conflicting interests., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The cytoprotective effects of glycine against cell death have been recognized for over 28 years. They are expressed in multiple cell types and injury settings that lead to necrosis, but are still not widely appreciated or considered in the conceptualization of cell death pathways. In this paper, we review the available data on the expression of this phenomenon, its relationship to major pathophysiologic pathways that lead to cell death and immunomodulatory effects, the hypothesis that it involves suppression by glycine of the development of a hydrophilic death channel of molecular dimensions in the plasma membrane, and evidence for its impact on disease processes in vivo.

Objective: To assess the 1-year renal functional changes in patients undergoing partial nephrectomy with intra-operative renal biopsies., Patients and Methods: A total of 40 patients with a single renal mass deemed fit for a partial nephrectomy were recruited prospectively between January 2009 and October 2010. We performed renal biopsies of normal renal parenchyma and collected serum markers before, during and after surgically induced renal clamp ischaemia during the partial nephrectomy. We then followed patients clinically with interval serum creatinine and physical examination., Results: Peri-operative data from 40 patients showed a transient increase in creatinine levels which did not correlate with ischaemia time. Renal ultrastructural changes were generally mild and included mitochondrial swelling, which resolved at the post-perfusion biopsy. A total of 37 patients had 1-year follow-up data. Creatinine at 1 year increased by 0.121 mg/dL, which represents a 12.99% decrease in renal function from baseline (preoperative creatinine 0.823 mg/dL, estimated glomerular filtration rate = 93.9 mL/min/1.73 m(2) ). The only factors predicting creatinine change on multivariate analysis were patient age, race and ischaemia type, with cold ischaemia being associated with higher creatinine level. Importantly, the duration of ischaemia did not show any significant correlation with renal function change, either as a continuous variable (P = 0.452) or as a categorical variable (P = 0.792)., Conclusions: Our data suggest that limited ischaemia is generally well tolerated in the setting of partial nephrectomy and does not directly correspond to long-term renal functional decline. For surgeons performing partial nephrectomy, the kidney can be safely clamped to ensure optimum oncological outcomes., Competing Interests: Conflicts of Interest: None declared., (© 2015 The Authors BJU International © 2015 BJU International Published by John Wiley & Sons Ltd.)

A report by Neelisetty et al. suggests that TGFBR2 deletion from matrix-producing interstitial cells results in decreased transforming growth factor-β (TGF-β) signaling in the cells but does not decrease renal fibrosis after injury. Considered in the context of TGF-β signaling in different cell types involved in renal fibrosis and the existence of other ligands that may produce fibrosis, these findings are provocative, but owing to technical issues of recombination efficiency in inducible models of Cre-lox gene deletion, further studies are needed.

The transition of AKI to CKD has major clinical significance. As reviewed here, recent studies show that a subpopulation of dedifferentiated, proliferating tubules recovering from AKI undergo pathologic growth arrest, fail to redifferentiate, and become atrophic. These abnormal tubules exhibit persistent, unregulated, and progressively increasing profibrotic signaling along multiple pathways. Paracrine products derived therefrom perturb normal interactions between peritubular capillary endothelium and pericyte-like fibroblasts, leading to myofibroblast transformation, proliferation, and fibrosis as well as capillary disintegration and rarefaction. Although signals from injured endothelium and inflammatory/immune cells also contribute, tubule injury alone is sufficient to produce the interstitial pathology required for fibrosis. Localized hypoxia produced by microvascular pathology may also prevent tubule recovery. However, fibrosis is not intrinsically progressive, and microvascular pathology develops strictly around damaged tubules; thus, additional deterioration of kidney structure after the transition of AKI to CKD requires new acute injury or other mechanisms of progression. Indeed, experiments using an acute-on-chronic injury model suggest that additional loss of parenchyma caused by failed repair of AKI in kidneys with prior renal mass reduction triggers hemodynamically mediated processes that damage glomeruli to cause progression. Continued investigation of these pathologic mechanisms should reveal options for preventing renal disease progression after AKI., (Copyright © 2015 by the American Society of Nephrology.)

Most patients with essential hypertension do not exhibit substantial renal damage. Renal autoregulation by preventing glomerular transmission of systemic pressures has been postulated to mediate this resistance. Conversely, malignant nephrosclerosis (MN) has been postulated to develop when severe hypertension exceeds a critical ceiling. If the concept is valid, even modest blood pressure (BP) reductions to below this threshold regardless of antihypertensive class (1) should prevent MN and (2) lead to the healing of the already developed MN lesions. Both predicates were tested using BP radiotelemetry in the stroke-prone spontaneously hypertensive rats receiving 1% NaCl as drinking fluid for 4 weeks. Severe hypertension (final 2 weeks average systolic BP, >200 mm Hg) and MN (histological damage score 36±5; n=27) developed in the untreated stroke-prone spontaneously hypertensive rats but were prevented by all antihypertensive classes (enalapril [n=15], amlodipine [n=13], or a hydralazine/hydrochlorothiazide combination [n=15]) if the final 2-week systolic BP remained <190 mm Hg. More impressively, modest systolic BP reductions to 160 to 180 mm Hg (hydralazine/hydrochlorothiazide regimen) initiated at ≈4 weeks in additional untreated rats after MN had already developed (injury score 35±4 in the right kidney removed before therapy) led to a striking resolution of the vascular and glomerular MN injury over 2 to 3 weeks (post-therapy left kidney injury score 9±2, P<0.0001; n=27). Proteinuria also declined rapidly from 122±9.5 mg/24 hours before therapy to 20.5±3.6 mg 1 week later. These data clearly demonstrate the barotrauma-mediated pathogenesis of MN and the striking capacity for spontaneous and rapid repair of hypertensive kidney damage if new injury is prevented., (© 2014 American Heart Association, Inc.)

TMEPAI (transmembrane prostate androgen-induced) is amplified at genomic, transcript and protein levels in triple-negative breast cancers and promotes TGF-β dependent growth, motility and invasion. Tumor promotion by TMEPAI depends on two different but related actions on TGF-β signaling. Firstly, TMEPAI binds and sequesters regulatory Smads2/3 and thereby decreases growth suppressive signaling by TGF-β. Secondly, increased expression of TMEPAI decreases PTEN (phosphatase and tensin homolog) abundance, and thereby increases TGF-β dependent tumor promotive PI3K/Akt signaling. These actions of TMEPAI give rise to increased cell proliferation and motility. Moreover, signaling alterations produced by high TMEPAI were associated with oncogenic Snail expression and lung metastases. Finally, an inverse correlation between TMEPAI and PTEN levels was confirmed in triple negative breast cancer tumor samples. Together, our findings suggest that TMEPAI has dually critical roles to promote TGF-β dependent cancer cell growth and metastasis. Thus, redirected TGF-β signaling through TMEPAI may play a pivotal role in TGF-β mediated tumor promotion.

Preexisting CKD may affect the severity of and/or recovery from AKI. We assessed the impact of prior graded normotensive renal mass reduction on ischemia-reperfusion-induced AKI. Rats underwent 40 minutes of ischemia 2 weeks after right uninephrectomy and surgical excision of both poles of the left kidney (75% reduction of renal mass), right uninephrectomy (50% reduction of renal mass), or sham reduction of renal mass. The severity of AKI was comparable among groups, which was reflected by similarly increased serum creatinine (SCr; approximately 4.5 mg/dl) at 2 days, tubule necrosis at 3 days, and vimentin-expressing regenerating tubules at 7 days postischemia-reperfusion. However, SCr remained elevated compared with preischemia-reperfusion values, and more tubules failed to differentiate during late recovery 4 weeks after ischemia-reperfusion in rats with 75% renal mass reduction relative to other groups. Tubules that failed to differentiate continued to produce vimentin, exhibited vicarious proliferative signaling, and expressed less vascular endothelial growth factor but more profibrotic peptides. The disproportionate failure of regenerating tubules to redifferentiate in rats with 75% renal mass reduction associated with more severe capillary rarefaction and greater tubulointerstitial fibrosis. Furthermore, initially normotensive rats with 75% renal mass reduction developed hypertension and proteinuria, 2-4 weeks postischemia-reperfusion. In summary, severe (>50%) renal mass reduction disproportionately compromised tubule repair, diminished capillary density, and promoted fibrosis with hypertension after ischemia-reperfusion-induced AKI in rats, suggesting that accelerated declines of renal function may occur after AKI in patients with preexisting CKD., (Copyright © 2014 by the American Society of Nephrology.)

Kim et al. show that isoflurane uses a tubule-based transforming growth factor-β/CD73-dependent process that generates adenosine to protect mice from ischemic acute kidney injury (AKI) with effects to prevent the 'no-reflow phenomenon' and decrease inflammation. While direct cytoprotection occurred in culture, extensive research suggests that in vivo adenosine protection from rodent ischemic AKI is mediated by a mutually cooperative mechanism involving blood flow, inflammation, and innate immunity through multiple adenosine receptors with promiscuous actions on diverse cell types.

Shen et al. exploit glycobiology to dampen transforming growth factor-β (TGF-β) signaling and ameliorate renal fibrosis after ureteral obstruction. Core fucosylation of N-linked oligosaccharides in TGF-β receptors is required for receptor function. Adenoviruses expressing Fut8-fucosyl transferase-shRNA inhibited receptor fucosylation, decreased tubule TGF-β signaling, and reduced fibrosis. Fut8-shRNA interferes with core fucosylation of other receptors also. Regardless, this first attempt to capitalize on a new aspect of TGF-β receptor function provides a basis for further research.

Tolerance of the human kidney to ischemia is controversial. Here, we prospectively studied the renal response to clamp ischemia and reperfusion in humans, including changes in putative biomarkers of AKI. We performed renal biopsies before, during, and after surgically induced renal clamp ischemia in 40 patients undergoing partial nephrectomy. Ischemia duration was >30 minutes in 82.5% of patients. There was a mild, transient increase in serum creatinine, but serum cystatin C remained stable. Renal functional changes did not correlate with ischemia duration. Renal structural changes were much less severe than observed in animal models that used similar durations of ischemia. Other biomarkers were only mildly elevated and did not correlate with renal function or ischemia duration. In summary, these data suggest that human kidneys can safely tolerate 30-60 minutes of controlled clamp ischemia with only mild structural changes and no acute functional loss.

Therapy resistance can be attributed to acquisition of anti-apoptotic mechanisms by the cancer cells. Therefore, developing approaches that trigger non-apoptotic cell death in cancer cells to compensate for apoptosis resistance will help to treat cancer effectively. Triple-negative breast cancers (TNBC) are among the most aggressive and therapy resistant to breast tumors. Here we report that manumycin A (Man A), an inhibitor of farnesyl protein transferase, reduces cancer cell viability through induction of non-apoptotic, non-autophagic cytoplasmic vacuolation death in TNBC cells. Man A persistently induced cytoplasmic vacuolation and cell death through the expression of microtubule-associated protein 1 light chain 3 (LC3) and p62 proteins along with endoplasmic reticulum (ER) stress markers, Bip and CHOP, and accumulation of ubiquitinated proteins. As inhibitors of apoptosis and autophagy failed to block cytoplasmic vacuolation and its associated protein expression or cell death, it appears that these processes are not involved in the death induced by Man A. Ability of thiol antioxidant, NAC in blocking Man A-induced vacuolation, death and its related protein expression suggests that sulfhydryl homeostasis may be the target of Man A. Surprisingly, normal human mammary epithelial cells failed to undergo cytoplasmic vacuolation and cell death, and grew normally in presence of Man A. In conjunction with its in vitro effects, Man A also reduced tumor burden in vivo in xenograft models that showed extensive cytoplasmic vacuoles and condensed nuclei with remarkable increase in the vacuolation-associated protein expression together with increase of p21, p27, PTEN and decrease of pAkt. Interestingly, Man A-mediated upregulation of p21, p27 and PTEN and downregulation of pAkt and tumor growth suppression were also mimicked by LC3 knockdown in MDA-MB-231 cells. Overall, these results suggest novel therapeutic actions by Man A through the induction of non-apoptotic and non-autophagic cytoplasmic vacuolation death by probably affecting ER stress, LC3 and p62 pathways in TNBC but not in normal mammary epithelial cells.

After ischemia-reperfusion injury (IRI), kidney tubules show activated transforming growth factor β (TGF-β) signaling and increased expression of profibrotic peptides, platelet-derived growth factor-B (PDGF-B) and connective tissue growth factor (CTGF). If tubule repair after IRI is incomplete, sustained paracrine activity of these peptides can activate interstitial fibroblast progenitors and cause fibrosis. We show that lysophosphatidic acid (LPA), a ubiquitous phospholipid that is increased at sites of injury and inflammation, signals through LPA2 receptors and Gαq proteins of cultured proximal tubule cells to transactivate latent TGF-β in a Rho/Rho-kinase and αvβ6 integrin-dependent manner. Active TGF-β peptide then initiates signaling to increase the production and secretion of PDGF-B and CTGF. In a rat model of IRI, increased TGF-β signaling that was initiated early during reperfusion did not subside during recovery, but progressively increased, causing tubulointerstitial fibrosis. This was accompanied by correspondingly increased LPA2 and β6 integrin proteins and elevated tubule expression of TGF-β1, together with PDGF-B and CTGF. Treatment with a pharmacological TGF-β type I receptor antagonist suppressed TGF-β signaling, decreased the expression of β6 integrin, PDGF-B, and CTGF, and ameliorated fibrosis. We suggest that LPA-initiated autocrine signaling is a potentially important mechanism that gives rise to paracrine profibrotic signaling in injured kidney tubule cells., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

We investigated the signaling basis for tubule pathology during fibrosis after renal injury. Numerous signaling pathways are activated physiologically to direct tubule regeneration after acute kidney injury (AKI) but several persist pathologically after repair. Among these, transforming growth factor (TGF)-β is particularly important because it controls epithelial differentiation and profibrotic cytokine production. We found that increased TGF-β signaling after AKI is accompanied by PTEN loss from proximal tubules (PT). With time, subpopulations of regenerating PT with persistent loss of PTEN (phosphate and tension homolog) failed to differentiate, became growth arrested, expressed vimentin, displayed profibrotic JNK activation, and produced PDGF-B. These tubules were surrounded by fibrosis. In contrast, PTEN recovery was associated with epithelial differentiation, normal tubule repair, and less fibrosis. This beneficial outcome was promoted by TGF-β antagonism. Tubule-specific induction of TGF-β led to PTEN loss, JNK activation, and fibrosis even without prior AKI. In PT culture, high TGF-β depleted PTEN, inhibited differentiation, and activated JNK. Conversely, TGF-β antagonism increased PTEN, promoted differentiation, and decreased JNK activity. Cre-Lox PTEN deletion suppressed differentiation, induced growth arrest, and activated JNK. The low-PTEN state with JNK signaling and fibrosis was ameliorated by contralateral nephrectomy done 2 wk after unilateral ischemia, suggesting reversibility of the low-PTEN dysfunctional tubule phenotype. Vimentin-expressing tubules with low-PTEN and JNK activation were associated with fibrosis also after tubule-selective AKI, and with human chronic kidney diseases of diverse etiology. By preventing tubule differentiation, the low-PTEN state may provide a platform for signals initiated physiologically to persist pathologically and cause fibrosis after injury.

Oxidant stress and compromised microcirculation underlie renal pathophysiology in septic acute kidney injury (AKI). Holthoff et al. report that resveratrol ameliorates these coupled abnormalities. They did not establish the primacy of either defect in septic AKI. However, tubule mitochondrial defects were recently reported to be involved in septic AKI pathogenesis, and resveratrol targets PGC-1α and respiratory enzymes. Together, these findings open new avenues for research into long-unresolved issues in the pathophysiology of septic AKI.

Several adenosine receptor subtypes on endothelial, epithelial, mesangial, and inflammatory cells have been implicated in ischemic acute kidney injury, a life-threatening condition that frequently complicates the care of hospitalized patients. In this issue of the JCI, Grenz and coworkers provide novel insight into how preservation of postischemic renal perfusion by endothelial cell adenosine A2B receptors is antagonized by adenosine reuptake into proximal tubule cells by equilibrative nucleotide transporter 1, which can be inhibited by dipyridamole. The work suggests that adenosine A2B receptor agonists and inhibition of equilibrative nucleoside transporters by dipyridamole may have therapeutic potential in ischemic acute kidney injury, a condition for which there are currently no specific therapeutic interventions.

We showed earlier that 15 deoxy Delta(12,14) prostaglandin J2 (15d-PGJ2) inactivates Drp1 and induces mitochondrial fusion [1]. However, prolonged incubation of cells with 15d-PGJ2 resulted in remodeling of fused mitochondria into large swollen mitochondria with irregular cristae structure. While initial fusion of mitochondria by 15d-PGJ2 required the presence of both outer (Mfn1 and Mfn2) and inner (OPA1) mitochondrial membrane fusion proteins, later mitochondrial changes involved increased degradation of the fusion protein OPA1 and ubiquitination of newly synthesized OPA1 along with decreased expression of Mfn1 and Mfn2, which likely contributed to the loss of tubular rigidity, disorganization of cristae, and formation of large swollen degenerated dysfunctional mitochondria. Similar to inhibition of Drp1 by 15d-PGJ2, decreased expression of fission protein Drp1 by siRNA also resulted in the loss of fusion proteins. Prevention of 15d-PGJ2 induced mitochondrial elongation by thiol antioxidants prevented not only loss of OPA1 isoforms but also its ubiquitination. These findings provide novel insights into unforeseen complexity of molecular events that modulate mitochondrial plasticity., (Copyright 2010 Elsevier Inc. All rights reserved.)

TMEPAI is a transforming growth factor-beta (TGF-beta)-induced transmembrane protein that is overexpressed in several cancers. How TMEPAI expression relates to malignancy is unknown. Here, we report high expression of TMEPAI in estrogen receptor/progesterone receptor-negative and human epidermal growth factor receptor-2-negative breast cancer cell lines and primary breast cancers that was further increased by TGF-beta treatment. Basal and TGF-beta-induced expression of TMEPAI were inhibited by the TGF-beta receptor antagonist SB431542 and overexpression of Smad7 or a dominant-negative mutant of Alk-5. TMEPAI knockdown attenuated TGF-beta-induced growth and motility in breast cancer cells, suggesting a role for TMEPAI in growth promotion and invasiveness. Further, TMEPAI knockdown decreased breast tumor mass in a mouse xenograft model in a manner associated with increased expression of phosphatase and tensin homologue (PTEN) and diminished phosphorylation of Akt. Consistent with the effects through the phosphatidylinositol 3-kinase pathway, tumors with TMEPAI knockdown exhibited elevated levels of the cell cycle inhibitor p27kip1 and attenuated levels of DNA replication and expression of hypoxia-inducible fator 1alpha and vascular endothelial growth factor. Together, these results suggest that TMEPAI functions in breast cancer as a molecular switch that converts TGF-beta from a tumor suppressor to a tumor promoter.

Recently published epidemiological and outcome analysis studies have brought to our attention the important role played by acute kidney injury (AKI) in the progression of chronic kidney disease (CKD) to end-stage renal disease (ESRD). AKI accelerates progression in patients with CKD; conversely, CKD predisposes patients to AKI. This research gives credence to older, well-thought-out wisdom that recovery from AKI is often not complete and is marked by residual structural damage. It also mirrors older experimental observations showing that unilateral nephrectomy, a surrogate for loss of nephrons by disease, compromises structural recovery and worsens tubulointerstitial fibrosis after ischemic AKI. Moreover, review of a substantial body of work on the relationships among reduced renal mass, hypertension, and pathology associated with these conditions suggests that impaired myogenic autoregulation of blood flow in the setting of hypertension, the arteriolosclerosis that results, and associated recurrent ischemic AKI in microscopic foci play important roles in the development of progressively increasing tubulointerstitial fibrosis. How nutrition, an additional factor that profoundly affects renal disease progression, influences these events needs reevaluation in light of information on the effects of calories vs. protein and animal vs. vegetable protein on injury and progression. Considerations based on published and emerging data suggest that a pathology that develops in regenerating tubules after AKI characterized by failure of differentiation and persistently high signaling activity is the proximate cause that drives downstream events in the interstitium: inflammation, capillary rarefaction, and fibroblast proliferation. In light of this information, we advance a comprehensive hypothesis regarding the pathophysiology of AKI as it relates to the progression of kidney disease. We discuss the implications of this pathophysiology for developing efficient therapeutic strategies to delay progression and avert ESRD.

Arachidonic acid derived endogenous electrophile 15d-PGJ2 has gained much attention in recent years due to its potent anti-proliferative and anti-inflammatory actions mediated through thiol modification of cysteine residues in its target proteins. Here, we show that 15d-PGJ2 at 1 microM concentration converts normal mitochondria into large elongated and interconnected mitochondria through direct binding to mitochondrial fission protein Drp1 and partial inhibition of its GTPase activity. Mitochondrial elongation induced by 15d-PGJ2 is accompanied by increased assembly of Drp1 into large oligomeric complexes through plausible intermolecular interactions. The role of decreased GTPase activity of Drp1 in the formation of large oligomeric complexes is evident when Drp1 is incubated with a non-cleavable GTP analog, GTPgammaS or by a mutation that inactivated GTPase activity of Drp1 (K38A). The mutation of cysteine residue (Cys644) in the GTPase effector domain, a reported target for modification by reactive electrophiles, to alanine mimicked K38A mutation induced Drp1 oligomerization and mitochondrial elongation, suggesting the importance of cysteine in GED to regulate the GTPase activity and mitochondrial morphology. Interestingly, treatment of K38A and C644A mutants with 15d-PGJ2 resulted in super oligomerization of both mutant Drp1s indicating that 15d-PGJ2 may further stabilize Drp1 oligomers formed by loss of GTPase activity through covalent modification of middle domain cysteine residues. The present study documents for the first time the regulation of a mitochondrial fission activity by a prostaglandin, which will provide clues for understanding the pathological and physiological consequences of accumulation of reactive electrophiles during oxidative stress, inflammation and degeneration., (2010 Elsevier Inc. All rights reserved.)

Development of the mitochondrial permeability transition (MPT) can importantly contribute to lethal cell injury from both necrosis and apoptosis, but its role varies considerably with both the type of cell and type of injury, and it can be strongly opposed by the normally abundant endogenous metabolites ADP and Mg(2+). To better characterize the MPT in kidney proximal tubule cells and assess its contribution to injury to them, we have refined and validated approaches to follow the process in whole kidney proximal tubules and studied its regulation in normoxic tubules and after hypoxia-reoxygenation (H/R). Physiological levels of ADP and Mg(2+) greatly decreased sensitivity to the MPT. Inhibition of cyclophilin D by cyclosporine A (CsA) effectively opposed the MPT only in the presence of ADP and/or Mg(2+). Nonesterified fatty acids (NEFA) had a large role in the decreased resistance to the MPT seen after H/R irrespective of the available substrate or the presence of ADP, Mg(2+), or CsA, but removal of NEFA was less effective at restoring normal resistance to the MPT in the presence of electron transport complex I-dependent substrates than with succinate. The data indicate that the NEFA accumulation that occurs during both hypoxia in vitro and ischemic acute kidney injury in vivo is a critical sensitizing factor for the MPT that overcomes the antagonistic effect of endogenous metabolites and cyclophilin D inhibition, particularly in the presence of complex I-dependent substrates, which predominate in vivo.

Thiol reactive cyclopentenone prostaglandin, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ2), induced a novel, nonapoptotic and microtubule-associated protein 1 light chain 3 (MAP1 LC3) dependent but nonautophagic form of cell death in colon, breast and prostate cancer cell lines, characterized by extensive cytoplasmic vacuolation with dilatation of endoplasmic reticulum (ER). Disruption of sulfhydryl homeostasis, which resulted in ER stress, accumulation of ubiquitinated proteins and subsequent ER dilation, contributed to peroxisome proliferator-activated receptor gamma (PPARgamma)-independent cell death by 15d-PGJ2. Absence of intracellular organelles in these vacuoles, shown by electron microscopy and unique fragmentation of lamin B, suggested this form of cell death to be different from autophagy and apoptosis. Cell death induced by 15d-PGJ2 is prevented by cycloheximide and actinomycin D, suggesting a requirement of new protein synthesis for death with cytoplasmic vacuolation. Here, we report for the first time that upregulation and processing of autophagy marker LC3 is an important event in nonautophagic cytoplasmic vacuolation and cell death. Notably, knockdown of LC3 conferred significant protection against 15d-PGJ2-induced cytoplasmic vacuolation and cell death, suggesting a novel role of LC3 in a death process other than autophagy.

We studied autocrine transforming growth factor (TGF)beta signaling in kidney epithelium. Cultured proximal tubule cells showed regulated signaling that was high during log-phase growth, low during contact-inhibited differentiation, and rapidly increased during regeneration of wounded epithelium. Autoregulation of signaling correlated with TGFbeta receptor and Smad7 levels, but not with active TGFbeta, which was barely measurable in the growth medium. Confluent differentiated cells with low receptor and high Smad7 levels exhibited blunted responses to saturating concentrations of exogenously provided active TGFbeta, suggesting that TGFbeta signaling homeostasis was achieved by cell density-dependent modulation of signaling intermediates. Antagonism of Alk5 kinase, the TGFbeta type I receptor, dramatically accelerated the induction of differentiation in sparse, proliferating cultures and permitted better retention of differentiated features in regenerating cells of wounded, confluent cultures. Alk5 antagonism accelerated the differentiation of cells in proximal tubule primary cultures while simultaneously increasing their proliferation. Consequently, Alk5-inhibited primary cultures formed confluent, differentiated monolayers faster than untreated cultures. Furthermore, treatment with an Alk5 antagonist promoted kidney repair reflected by increased tubule differentiation and decreased tubulo-interstitial pathology during the recovery phase following ischemic injury in vivo. Our results show that autocrine TGFbeta signaling in proliferating proximal tubule cells exceeds the levels that are necessary for physiological regeneration. To that end, TGFbeta signaling is redundant and maladaptive during tubule repair by epithelial regeneration.

Loss of Ca(2+) homeostasis, often in the form of cytoplasmic increases, leads to cell injury. Depending upon cell type and the intensity of Ca(2+) toxicity, the ensuing pathology can be reversible or irreversible. Although multiple destructive processes are activated by Ca(2+), lethal outcomes are determined largely by Ca(2+)-induced mitochondrial permeability transition. This form of damage is primarily dependent upon mitochondrial Ca(2+) accumulation, which is regulated by the mitochondrial membrane potential. Retention of the mitochondrial membrane potential during Ca(2+) increases favors mitochondrial Ca(2+) uptake and overload, resulting in mitochondrial permeability transition and cell death. In contrast, dissipation of mitochondrial membrane potential reduces mitochondrial Ca(2+) uptake, retards mitochondrial permeability transition, and delays death, even in cells with large Ca(2+) increases. The rates of mitochondrial membrane potential dissipation and mitochondrial Ca(2+) uptake may determine cellular sensitivity to Ca(2+) toxicity under pathological conditions, including ischemic injury.

Inhibition of complex I has been considered to be an important contributor to mitochondrial dysfunction in tissues subjected to ischemia-reperfusion. We have investigated the role of complex I in a severe energetic deficit that develops in kidney proximal tubules subjected to hypoxia-reoxygenation and is strongly ameliorated by supplementation with specific citric acid cycle metabolites, including succinate and the combination of -ketoglutarate plus malate. NADH: ubiquinone reductase activity in the tubules was decreased by only 26% during 60-min hypoxia and did not change further during 60-min reoxygenation. During titration of complex I activity with rotenone, progressive reduction of NAD+ to NADH was detected at >20% complex I inhibition, but substantial decreases in ATP levels and mitochondrial membrane potential did not occur until >70% inhibition. NAD+ was reduced to NADH during hypoxia, but the NADH formed was fully reoxidized during reoxygenation, consistent with the conclusion that complex I function was not limiting for recovery. Extensive degradation of cytosolic and mitochondrial NAD(H) pools occurred during either hypoxia or severe electron transport inhibition by rotenone, with patterns of metabolite accumulation consistent with catabolism by both NAD+ glycohydrolase and pyrophosphatase. This degradation was strongly blocked by alpha-ketoglutarate plus malate. The data demonstrate surprisingly little sensitivity of these cells to inhibition of complex I and high levels of resistance to development of complex I dysfunction during hypoxia-reoxygenation and indicate that events upstream of complex I are important for the energetic deficit. The work provides new insight into fundamental aspects of mitochondrial pathophysiology in proximal tubules during acute renal failure.