Proximal tubule LPA1 and LPA2 receptors use divergent signaling pathways to additively increase profibrotic cytokine secretion.

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-Gqα-αvß6-integrin-mediated activation of transforming growth factor-ß1 (TGFB1). LPA2, ß6-integrin, PDGFB, and CTGF increase in kidneys after ischemia-reperfusion injury (IRI), coinciding with fibrosis. The TGFB1 receptor antagonist SD-208 prevents increases of ß6-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-Gqα-αvß6-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.

Increased Smad3 and reduced Smad2 levels mediate the functional switch of TGF-ß from growth suppressor to growth and metastasis promoter through TMEPAI/PMEPA1 in triple negative breast cancer.

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-ß 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-ß. 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-ß 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-ß-Smad3-TMEPAI axis may be beneficial in triple negative breast cancer therapy and prevention.

Co-targeting ALK and EGFR parallel signaling in oral squamous cell carcinoma.

Squamous cell carcinoma (SCC) comprises 90% of all head and neck cancers and has a poor survival rate due to late-stage disease that is refractive to traditional therapies. Epidermal growth factor receptor (EGFR) is over-expressed in greater than 80% of head and neck SCC (HNSCC). However, EGFR targeted therapies yielded little to no efficacy in clinical trials. This study investigated the efficacy of co-targeting EGFR and the anaplastic lymphoma kinase (ALK) whose promoter is hypomethylated in late-stage oral SCC (OSCC). We observed increased ALK activity in late-stage human OSCC tumors and invasive OSCC cell lines. We also found that while ALK inhibition alone had little effect on proliferation, co-targeting ALK and EGFR significantly reduced OSCC cell proliferation in vitro. Further analysis showed significant efficacy of combined treatment in HSC3-derived xenografts resulting in a 30% decrease in tumor volumes by 14days (p<0.001). Western blot analysis showed that co-targeting ALK and EGFR significantly reduced EGFR phosphorylation (Y1148) in HSC3 cells but not Cal27 cells. ALK and EGFR downstream signaling interactions are also demonstrated by Western blot analysis in which lone EGFR and ALK inhibitors attenuated AKT activity whereas co-targeting ALK and EGFR completely abolished AKT activation. No effects were observed on ERK1/2 activation. STAT3 activity was significantly induced by lone ALK inhibition in HSC3 cells and to a lower extent in Cal27 cells. Together, these data illustrate that ALK inhibitors enhance anti-tumor activity of EGFR inhibitors in susceptible tumors that display increased ALK expression, most likely through abolition of AKT activation.

Mitochondrial Pathology and Glycolytic Shift during Proximal Tubule Atrophy after Ischemic AKI.

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-ß 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-ß signaling and depletion of mitochondria characterizes the development of atrophy in proximal tubule cells and may contribute to the renal pathology after AKI.

TGF-ß induced TMEPAI/PMEPA1 inhibits canonical Smad signaling through R-Smad sequestration and promotes non-canonical PI3K/Akt signaling by reducing PTEN in triple negative breast cancer.

TMEPAI (transmembrane prostate androgen-induced) is amplified at genomic, transcript and protein levels in triple-negative breast cancers and promotes TGF-ß dependent growth, motility and invasion. Tumor promotion by TMEPAI depends on two different but related actions on TGF-ß signaling. Firstly, TMEPAI binds and sequesters regulatory Smads2/3 and thereby decreases growth suppressive signaling by TGF-ß. Secondly, increased expression of TMEPAI decreases PTEN (phosphatase and tensin homolog) abundance, and thereby increases TGF-ß 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-ß dependent cancer cell growth and metastasis. Thus, redirected TGF-ß signaling through TMEPAI may play a pivotal role in TGF-ß mediated tumor promotion.

Lysophosphatidic acid increases proximal tubule cell secretion of profibrotic cytokines PDGF-B and CTGF through LPA2- and Gαq-mediated Rho and αvß6 integrin-dependent activation of TGF-ß.

After ischemia-reperfusion injury (IRI), kidney tubules show activated transforming growth factor ß (TGF-ß) 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-ß in a Rho/Rho-kinase and αvß6 integrin-dependent manner. Active TGF-ß peptide then initiates signaling to increase the production and secretion of PDGF-B and CTGF. In a rat model of IRI, increased TGF-ß 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 ß6 integrin proteins and elevated tubule expression of TGF-ß1, together with PDGF-B and CTGF. Treatment with a pharmacological TGF-ß type I receptor antagonist suppressed TGF-ß signaling, decreased the expression of ß6 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.

PTEN loss defines a TGF-ß-induced tubule phenotype of failed differentiation and JNK signaling during renal fibrosis.

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)-ß is particularly important because it controls epithelial differentiation and profibrotic cytokine production. We found that increased TGF-ß 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-ß antagonism. Tubule-specific induction of TGF-ß led to PTEN loss, JNK activation, and fibrosis even without prior AKI. In PT culture, high TGF-ß depleted PTEN, inhibited differentiation, and activated JNK. Conversely, TGF-ß 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.

Estrogen receptor-beta mediates the protective effects of aromatase induction in the MMTV-Her-2/neu x aromatase double transgenic mice.

Breast cancers amplified for the tyrosine kinase receptor Her-2/neu constitute ~30% of advanced breast cancer cases, and are characterized by hormone independence and aggressive growth, implicating this pathway in breast oncogenesis. The induction of Her-2/neu leads to tumor development in 60% of transgenic mice. We have previously examined the effects of estrogen in the MMTV-Her-2/neu background by generating the MMTV-Her-2/neu x aromatase double transgenic mouse strain. MMTV-Her-2/neu x aromatase mice developed fewer mammary tumors than the Her-2/neu parental strain. Our present data show the induction of several estrogen-related genes, including the tumor suppressors BRCA1 and p53, and a decrease in several angiogenic factors. The phosphorylated forms of MAPK p42/44 and AKT were lower in the MMTV-Her-2/neu x aromatase double transgenic mice compared to the MMTV-Her-2/neu parental strain; conversely, phospho-p38 levels were higher in the double transgenic strain. The ERß-selective antagonist THC reversed these changes. The regulation of these factors by ERß was confirmed in clones of MCF7 breast cancer cells overexpressing Her-2/neu in combination with ERß, suggesting that ERß may play a direct role in regulating MAPK and AKT pathways. In summary, the data suggest that ERß may play a major role in decreasing tumorigenesis and that it may affect breast cancer cell proliferation and survival by altering MAPK and AKT activation as well as modulation of tumor suppressor and angiogenesis factors. Treatment with selective ERß agonist may provide therapeutic advantages for the treatment and prevention of breast cancer.

Mitochondrial remodeling following fission inhibition by 15d-PGJ2 involves molecular changes in mitochondrial fusion protein OPA1.

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.

Transforming growth factor-beta (TGF-beta)-inducible gene TMEPAI converts TGF-beta from a tumor suppressor to a tumor promoter in breast cancer.

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.

A novel wounding device suitable for quantitative biochemical analysis of wound healing and regeneration of cultured epithelium.

We describe the fabrication and use of an in vitro wounding device that denudes cultured epithelium in patterns designed to leave behind strips or islands of cells sufficiently narrow or small to ensure that all the remaining cells become rapidly activated and then migrate, dedifferentiate, and proliferate in near synchrony. The design ensures that signals specific to regenerating cells do not become diluted by quiescent differentiated cells that are not affected by wound-induced activation. The device consists of a flat circular disk of rubber, engraved to produce alternating ridges and grooves in patterns of concentric circles or parallel lines. The disk is mounted at the end of a pneumatically controlled piston assembly. Application of controlled pressure and circular or linear movement of the disk on cultures produced highly reproducible wounding patterns. The near-synchronous regenerative activity of cell bands or islands allowed the collection of samples large enough for biochemical studies to sensitively detect alterations involving mRNA for several early response genes and protein phosphorylation in major signaling pathways. The method is versatile, easy to use and reproducible, and should facilitate biochemical, proteomic, and genomic studies of wound-induced regeneration of cultured epithelium.

Acute kidney injury: a springboard for progression in chronic kidney disease.

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.

Inhibition of mitochondrial division through covalent modification of Drp1 protein by 15 deoxy-Delta(12,14)-prostaglandin J2.

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.

Inhibition of autoregulated TGFbeta signaling simultaneously enhances proliferation and differentiation of kidney epithelium and promotes repair following renal ischemia.

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.

Regulation of caspase-9 activity by differential binding to the apoptosome complex.

Proteolytic processing of procaspase-9 is required for its activation, but processing in itself appears to be insufficient for its activity. We studied caspase activation in a cell-free system and found that incubation of cytosol from rat kidney proximal tubule cells with Cytochrome c (Cyt c) and dATP results in rapid autocatalytic processing of procaspase-9 from ~50 kD to ~38 kD size fragment. Moreover, Cyt c concentration influences the production of alternatively processed forms of caspase-9. At lower Cyt c concentration (0.01-0.05 mg/ml), two fragments of caspase-9 of the size 38 and 40 kD are produced. In contrast, at higher concentrations of Cyt c (>0.1 mg/ml) only 38 kD fragment will prevail. However, our failure to capture processed caspase-9 by affinity labeling or co-elution with Apaf-1 suggested that caspase-9 undergoes a conformational change during its enzymatic action on effector caspases, resulting in its release from the apoptosome complex and inactivation. In support of this hypothesis, catalytic inhibitors of caspase-9 prevented its release from the apoptosome complex without affecting its auto-processing and allowed successful capture of active caspase-9 (38 kD) and its complex by affinity labeling. These observations suggest that complex allosteric interactions with the apoptosome complex influence caspase-9 activity and function by controlling not only the induction of its enzymatic activity, but also its rapid termination.

Calcium in cell injury and death.

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.

Role of apoptosis in hypoxic/ischemic damage in the kidney.

Cell death by hypoxia/ischemia may occur by apoptosis as well as necrosis in experimental models of renal injury both in vivo and in vitro. Necrosis can occur during hypoxia/ischemia as a result of widespread cellular degradation, and during reoxygenation/reperfusion as a consequence of the development of the mitochondrial permeability transition pore (PTP). In vitro models of hypoxia/reoxygenation suggest that apoptotic cell death may occur during reoxygenation as a consequence of mitochondrial release of cytochrome c (Cyt c) during hypoxia. In hypoxic renal cells, Bax and Bak, 2 pro-apoptotic proteins of the Bcl-2 family, collaborate to permeabilize the mitochondrial outer membrane to intermembrane proteins such as Cyt c, although Bax, per se, appears to play the dominant role. Cyt c then acts to trigger the downstream apoptotic cascade. Caspase inhibitors suppress these downstream events, but not Cyt c release. However, the anti-apoptotic Bcl-2 prevents mitochondrial permeabilization and maintains viability. Inflammation is known to play a major role in exacerbating parenchymal damage during reperfusion. Recent studies suggest that the apoptosis-related mechanisms contribute to the inflammatory process. By inhibiting tubular cell apoptosis, by suppressing an apoptotic chain reaction in accumulating inflammatory cells, and by inhibiting caspase-1 processing in injured tissue, caspase inhibitors may reduce inflammation, and thereby reduce the cascading parenchymal injury that is associated with inflammation.

Association of Bax and Bak homo-oligomers in mitochondria. Bax requirement for Bak reorganization and cytochrome c release.

ATP depletion induced by hypoxia or mitochondrial inhibitors results in Bax translocation from cytosol to mitochondria and release of cytochrome c from mitochondria into cytosol in cultured rat proximal tubule cells. Translocated Bax undergoes further conformational changes to oligomerize into high molecular weight complexes (Mikhailov, V., Mikhailova, M., Pulkrabek, D. J., Dong, Z., Venkatachalam, M. A., and Saikumar, P. (2001) J. Biol. Chem. 276, 18361-18374). Here we report that following Bax translocation in ATP-depleted rat proximal tubule cells, Bak, a proapoptotic molecule that normally resides in mitochondria, also reorganizes to form homo-oligomers. Oligomerization of both Bax and Bak occurred independently of Bid cleavage and/or translocation. Western blots of chemically cross-linked membrane extracts showed nonoverlapping "ladders" of Bax and Bak complexes in multiples of approximately 21 and approximately 23 kDa, respectively, consistent with molecular homogeneity within each ladder. This indicated that Bax and Bak complexes were homo-oligomeric. Nevertheless, each oligomer could be co-immunoprecipitated with the other, suggesting a degree of affinity between Bax and Bak that permitted co-precipitation but not cross-linking. Furthermore, dissociation of cross-linked complexes by SDS and renaturation prior to immunoprecipitation did not prevent reassociation of the two oligomeric species. Notably, expression of Bcl-2 prevented not only the oligomerization of Bax and Bak, but also the association between these two proteins in energy-deprived cells. Using Bax-deficient HCT116 and BMK cells, we show that there is stringent Bax requirement for Bak homo-oligomerization and for cytochrome c release during energy deprivation. Using Bak-deficient BMK cells we further show that Bak deficiency is associated with delayed kinetics of Bax translocation but does not affect either the oligomerization of translocated Bax or the leakage of cytochrome c. These results suggest a degree of functional cooperation between Bax and Bak in this form of cell injury, but also demonstrate an absolute requirement of Bax for mitochondrial permeabilization.