Hidden Dangers: A Gastrointestinal Stromal Tumor Concealed Inside of a Meckel's Diverticulum.

Meckel's diverticulum (MD) is one of the most common congenital abnormalities of the gastrointestinal tract, affecting approximately two percent of the population. Rarely, Meckel's diverticula have been found to harbor various tumors, which go unnoticed until later in their course. The clinical presentation varies among each individual, and tumors have often metastasized or caused diverticular rupture at the time of diagnosis. This is a case of a 55-year-old male with a past medical history of alcohol abuse and asthma who presented to the emergency department with abdominal pain. He denied any fever, chills, chest pain, nausea, changes in urinary patterns, recent travel, or sick contacts. He is a non-smoker but has been a heavy drinker for many years. On physical exam, he was found to have diffuse abdominal tenderness with pain greatest in the epigastric region and no bowel sounds. He was afebrile but tachycardic at 112 bpm, hypertensive at 168/98 mmHg, and tachypneic at 38 bpm. Labs showed a markedly elevated white blood cell count, hemoglobin and platelet count, as well as metabolic acidosis and elevated lactate levels. Abdominal CT showed a mechanical small bowel obstruction with unclear etiology. Of note was a 7.2 cm thick-walled collection in the right lower quadrant having no clear communication with any bowel loops. Despite aggressive hydration and supportive care, his abdominal exam continued to worsen, prompting an exploratory laparotomy. During the laparotomy, a perforated MD with frank succus was found. On pathology, the affected segment of the bowel revealed a CD117 and CD34 positive spindle cell gastrointestinal stromal tumor (GIST) with mild cytological atypia, no necrosis, and no regional lymph node involvement. Cultures of peritoneal fluid were positive for Klebsiella oxytoca, and the patient was started on meropenem and doxycycline. The patient showed significant improvement with the appropriate administration of antibiotics and was eventually discharged to follow-up with hematology/oncology as an outpatient for further management and monitoring of his GIST tumor. This case is unique as there are only a few reported cases of patients developing GIST inside of MDs. Despite the high five-year survival rate of typically localized GIST tumors, the complications (such as perforation in the case of our patient) caused by tumor growth inside a MD are detrimental if not diagnosed promptly. Not only does perforation increase the risk of metastasis but also the risk of peritonitis and other complications. This case calls for more research on standardization of care for patients who have MD to prevent malignant transformations as well as potential prophylactic excision of incidental MD findings in adult patients.

Prospective validation of a novel renal activity index of lupus nephritis.

Objectives The renal activity index for lupus (RAIL) score was developed in children with lupus nephritis as a weighted sum of six urine biomarkers (UBMs) (neutrophil gelatinase-associated lipocalin, monocyte chemotactic protein 1, ceruloplasmin, adiponectin, hemopexin and kidney injury molecule 1) measured in a random urine sample. We aimed at prospectively validating the RAIL in adults with lupus nephritis. Methods Urine from 79 adults was collected at the time of kidney biopsy to assay the RAIL UBMs. Using receiver operating characteristic curve analysis, we evaluated the accuracy of the RAIL to discriminate high lupus nephritis activity status (National Institutes of Health activity index (NIH-AI) score >10), from low/moderate lupus nephritis activity status (NIH-AI score ≤10). Results In this mixed racial cohort, high lupus nephritis activity was present in 15 patients (19%), and 71% had proliferative lupus nephritis. Use of the identical RAIL algorithm developed in children resulted in only fair prediction of lupus nephritis activity status of adults (area under the receiver operating characteristic curve (AUC) 0.62). Alternative weightings of the six RAIL UBMs as suggested by logistic regression yielded excellent accuracy to predict lupus nephritis activity status (AUC 0.88). Accuracy of the model did not improve with adjustment of the UBMs for urine creatinine or albumin, and was little influenced by concurrent kidney damage. Conclusions The RAIL UBMs provide excellent prediction of lupus nephritis activity in adults. Age adaption of the RAIL is warranted to optimize its discriminative validity to predict high lupus nephritis activity status non-invasively.

Urinary biomarkers of kidney injury are associated with all-cause mortality in the Women's Interagency HIV Study (WIHS).

OBJECTIVES: Chronic kidney disease (CKD) is common in HIV-infected individuals, and is associated with mortality in both the HIV-infected and general populations. Urinary markers of tubular injury have been associated with future kidney disease risk, but associations with mortality are unknown. METHODS: We evaluated the associations of urinary interleukin-18 (IL-18), liver fatty acid binding protein (L-FABP), kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL) and the albumin-to-creatinine ratio (ACR) with 10-year, all-cause death in 908 HIV-infected women. Serum cystatin C was used to estimate the glomerular filtration rate (eGFRcys). RESULTS: There were 201 deaths during 9269 person-years of follow-up. After demographic adjustment, compared with the lowest tertile, the highest tertiles of IL-18 [hazard ratio (HR) 2.54; 95% confidence interval (CI) 1.75-3.68], KIM-1 (HR 2.04; 95% CI 1.44-2.89), NGAL (HR 1.50; 95% CI 1.05-2.14) and ACR (HR 1.63; 95% CI 1.13-2.36) were associated with higher mortality. After multivariable adjustment including adjustment for eGFRcys, only the highest tertiles of IL-18 (HR 1.88; 95% CI 1.29-2.74) and ACR (HR 1.46; 95% CI 1.01-2.12) remained independently associated with mortality. Findings for KIM-1 were borderline (HR 1.41; 95% CI 0.99-2.02). We found a J-shaped association between L-FABP and mortality. Compared with persons in the lowest tertile, the HR for the middle tertile of L-FABP was 0.67 (95% CI 0.46-0.98) after adjustment. Associations were stronger when IL-18, ACR and L-FABP were simultaneously included in models. CONCLUSIONS: Among HIV-infected women, some urinary markers of tubular injury are associated with mortality risk, independently of eGFRcys and ACR. These markers represent potential tools with which to identify early kidney injury in persons with HIV infection.

Influence of particle shape on plasma protein adsorption and macrophage uptake.

The purpose of this study was to evaluate the plasma protein adsorption behavior onto different LIPOMER nanoparticles, especially looking for the first time, if the particle shape affects the protein adsorption pattern. The potential in vivo fate is discussed and compared with previous in vivo animal studies. The two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was used for identification of adsorbed plasma proteins. Qualitative similar patterns were obtained from the protein adsorption analysis and four apolipoproteins with considerable quantitative differences were identified. Besides the quantitative differences in the adsorbed apolipoproteins, in vitro uptake in the human macrophage cell line U-937 of histocytic lymphoma organ revealed significantly lower uptake of the irregular glycerol monostearate LIPOMER nanoparticles. Therefore, protein adsorption does not seem to play a role in the splenotropic behavior in the sense, that adsorption of opsonins, especially spleen-specific opsonins are required for the uptake. The splenotropic uptake might be favored because all LIPOMER nanoparticles did not adsorb opsonins at all, mediating competitive uptake by liver macrophages. Differences in the in vivo uptake by the spleen were attributed to differences in particle shape with potential super position effect by the quantitative differences in the adsorbed proteins.

Cardio-renal syndrome: new perspective in diagnostics.

Chronic heart failure and chronic renal failure are at epidemic proportions. These patients have significantly altered cardiac, renal, and all-cause outcomes. Much of the current research has focused on treating these individual organs in isolation. Although there are positive data on outcomes with neurohormonal modulation, they, however, remain underused. At present, data lacks for novel treatment options, while evidence continues to point at significantly worsened prognosis. Current diagnostic tools that detect acute changes in renal function or renal injury appear retrospective, which often hinder meaningful diagnostic and therapeutic decisions. This review is aimed at exploring the importance of accurate assessment of renal function for the heart failure patient by providing a synopsis on cardio-renal physiology and establishing the possibility of novel approaches in bridging the divide.

Pharmacological targeting of C5a receptors during organ preservation improves kidney graft survival.

Cadaveric renal transplants suffer frequently from delayed graft function, which is associated with increased risk for long-term graft survival loss. One-third of kidney grafts that are stored in current organ preservation solutions experience delayed graft function, demonstrating the urgent need for improvement. Although ischaemic graft injury is complex in nature, complement activation is considered important to the process. Here we show that pharmacological targeting of the complement 5a receptor (C5aR) during cold ischaemia has a protective effect on early kidney graft survival, inflammation and apoptosis in a mouse model of syngeneic kidney transplantation. Graft survival of kidneys that were stored in University of Wisconsin solution in the presence of a C5aR antagonist increased from 29% to 57%. Increased graft survival was associated with less tubular damage and apoptosis, protection from sustained C5aR expression and decreased production of tumour necrosis factor-alpha and macrophage inflammatory protein-2. In a translational approach, we determined C5aR expression in paediatric living-related and cadaveric allografts. C5aR expression was significantly higher in all compartments of kidneys from cadaveric compared with kidneys from living-related donors. C5aR expression in cadaveric kidneys correlated positively with cold ischaemia time, renal dysfunction and the frequency of apoptotic tubular cells, suggesting a novel role for C5a in delayed graft function pathogenesis. Supplementing organ preservation solutions with C5aR inhibitors may improve early graft function following cadaveric kidney transplantation.

Liver fatty acid-binding protein as a biomarker of acute kidney injury after cardiac surgery.

Acute kidney injury (AKI) is a major complication of cardiac bypass surgery. We examined whether levels of liver fatty acid-binding protein (L-FABP) can be an early biomarker for ischemic injury by measuring this protein in the urine of 40 pediatric patients prior to and following cardiopulmonary bypass surgery. AKI was defined as a 50% increase in the serum creatinine from baseline, which was normally not seen until 24-72 h after surgery. Enzyme-linked immunosorbent assay analysis showed increased L-FABP levels (factored for creatinine excretion) of about 94- and 45-fold at 4 and 12 h, respectively, following surgery in the 21 patients who developed AKI with western blot analysis, confirming L-FABP identity. Univariate logistic regression analyses showed that both bypass time and urinary L-FABP were significant independent risk indicators for AKI. After excluding bypass time from the model and using a stepwise multivariate logistic regression analysis, urinary L-FABP levels at 4 h after surgery were an independent risk indicator with the area under the receiver-operating characteristic curve 0.810, sensitivity 0.714, and specificity 0.684 for a 24-fold increase in urinary L-FABP. Our study shows that urinary L-FABP levels represent a sensitive and predictive early biomarker of AKI after cardiac surgery.

Urinary biomarkers in the early diagnosis of acute kidney injury.

A change in the serum creatinine is not sensitive for an early diagnosis of acute kidney injury. We evaluated urinary levels of matrix metalloproteinase-9 (MMP-9), N-acetyl-beta-D-glucosaminidase (NAG), and kidney injury molecule-1 (KIM-1) as biomarkers for the detection of acute kidney injury. Urine samples were collected from 44 patients with various acute and chronic kidney diseases, and from 30 normal subjects in a cross-sectional study. A case-control study of children undergoing cardio-pulmonary bypass surgery included urine specimens from each of 20 patients without and with acute kidney injury. Injury was defined as a greater than 50% increase in the serum creatinine within the first 48 h after surgery. The biomarkers were normalized to the urinary creatinine concentration at 12, 24, and 36 h after surgery with the areas under the receiver-operating characteristic curve compared for performance. In the cross-sectional study, the area under the curve for MMP-9 was least sensitive followed by KIM-1 and NAG. Combining all three biomarkers achieved a perfect score diagnosing acute kidney injury. In the case-control study, KIM-1 was better than NAG at all time points, but combining both was no better than KIM-1 alone. Urinary MMP-9 was not a sensitive marker in the case-control study. Our results suggest that urinary biomarkers allow diagnosis of acute kidney injury earlier than a rise in serum creatinine.

Do all children need intervention after exposure to tsunami?

Children are uniquely vulnerable in the context of a major natural disaster like tsunami. Post disaster intervention studies in children are few, especially from developing countries like India. An intervention programme for children was developed and conducted at Srinivasapuram, a coastal hamlet in Chennai, a year after tsunami. Sixty-five children who participated in all the six intervention modules were compared to 70 children who did not participate in the intervention. All the children were assessed before and after intervention using the Youth Self Report (YSR) form of Child Behaviour Check List (CBCL) in addition to PTSD symptoms. Children in the two groups were comparable in socio-demographic factors and exposure to tsunami. Prevalence of family psychopathology was more in the intervention group. Only hyperactivity problems were significantly reduced after intervention (z = 2.41 p = 0.016). Children in the intervention group appreciated expression of positive emotions (F = 8.044 p = 0.005) and were also more likely to desist from smoking (F = 6.102 p = 0.003) compared to the control group. The majority of the children are likely to be resilient and only children with pre-existing vulnerability require specific and specialized interventions.

Urine NGAL and IL-18 are predictive biomarkers for delayed graft function following kidney transplantation.

Delayed graft function (DGF) due to tubule cell injury frequently complicates deceased donor kidney transplants. We tested whether urinary neutrophil gelatinase-associated lipocalin (NGAL) and interleukin-18 (IL-18) represent early biomarkers for DGF (defined as dialysis requirement within the first week after transplantation). Urine samples collected on day 0 from recipients of living donor kidneys (n = 23), deceased donor kidneys with prompt graft function (n = 20) and deceased donor kidneys with DGF (n = 10) were analyzed in a double blind fashion by ELISA for NGAL and IL-18. In patients with DGF, peak postoperative serum creatinine requiring dialysis typically occurred 2-4 days after transplant. Urine NGAL and IL-18 values were significantly different in the three groups on day 0, with maximally elevated levels noted in the DGF group (p < 0.0001). The receiver-operating characteristic curve for prediction of DGF based on urine NGAL or IL-18 at day 0 showed an area under the curve of 0.9 for both biomarkers. By multivariate analysis, both urine NGAL and IL-18 on day 0 predicted the trend in serum creatinine in the posttransplant period after adjusting for effects of age, gender, race, urine output and cold ischemia time (p < 0.01). Our results indicate that urine NGAL and IL-18 represent early, predictive biomarkers of DGF.

Urinary IL-18 is an early predictive biomarker of acute kidney injury after cardiac surgery.

Acute kidney injury (AKI) is a frequent complication of cardiopulmonary bypass (CPB). The lack of early biomarkers for AKI has impaired our ability to intervene in a timely manner. Urinary neutrophil gelatinase-associated lipocalin (NGAL) is recently demonstrated as an early biomarker of AKI after CPB, increasing 25-fold within 2 h and declining 6 h after surgery. In the present study, we tested whether interleukin-18 (IL-18) is a predictive biomarker for AKI in the same group of patients following CPB. Exclusion criteria included pre-existing renal insufficiency and nephrotoxin use. Serial urine samples were analyzed by enzyme-linked immunosorbent assay for IL-18 in 20 patients who developed AKI (defined as a 50% or greater increase in serum creatinine after CPB) and 35 controls (age, race, and gender-matched patients who did not develop AKI after CPB). Using serum creatinine, AKI was detected only 48-72 h after CPB. In contrast, urine IL-18 increased at 4-6 h after CPB, peaked at over 25-fold at 12 h, and remained markedly elevated up to 48 h after CPB. The performance of IL-18 as demonstrated by area under the receiver operating characteristics curve for diagnosis of AKI at 4, 12, and 24 h after CPB was 61, 75, and 73% respectively. Also, on multivariate analysis, both IL-18 and NGAL were independently associated with number of days in AKI among cases. Our results indicate that IL-18 is an early, predictive biomarker of AKI after CPB, and that NGAL and IL-18 are increased in tandem after CPB. The combination of these two biomarkers may allow for the reliable early diagnosis and prognosis of AKI at all times after CPB, much before the rise in serum creatinine.

Basiliximab induction improves the outcome of renal transplants in children and adolescents.

Thirty-two children and adolescents received their renal transplant at the Montefiore Medical Center, in New York, between October 1996 and May 2000. Twenty-four patients received basiliximab, in addition to tacrolimus and steroids (basiliximab group). The remaining eight patients received only tacrolimus and steroids (non-basiliximab group). The 1-year patient survival rate was 100% in both groups. The 1-year graft survival rate was 87.5% for the basiliximab group and 75% for the non-basiliximab group (P=0.45). The rates of acute rejection in the basiliximab and non-basiliximab groups were 26% and 43%, respectively (P=0.36). However, in recipients with

The von Hippel-Lindau gene product inhibits renal cell apoptosis via Bcl-2-dependent pathways.

Previous studies have reported a protective role for the von Hippel-Lindau (VHL) gene products against pro-apoptotic cellular stresses, but the mechanisms remain unclear. In this study, we examined the role of VHL in renal cells subjected to chemical hypoxia, using four VHL-negative and two VHL-positive cell lines. VHL-negative renal carcinoma cells underwent apoptosis following chemical hypoxia (short-term glucose deprivation and antimycin treatment), as evidenced by morphologic changes and internucleosomal DNA cleavage. Reintroduction of VHL expression prevented this apoptosis. VHL-negative cells displayed a significant (greater than 5-fold) activation of caspase 9 and release of cytochrome c into the cytosol following chemical hypoxia. In contrast, VHL-positive cells showed minimal caspase 9 activation, and absence of cytochrome c release under the same conditions. Caspase 8 was only minimally activated in both VHL-negative and -positive cells. In addition, VHL-positive cells displayed a striking up-regulation of Bcl-2 expression (5-fold) following chemical hypoxia. Antisense oligonucleotides to Bcl-2 significantly down-regulated Bcl-2 protein expression in VHL-positive cells and rendered them sensitive to apoptosis. Overexpression of Bcl-2 in VHL-negative cells conferred resistance to apoptosis. Our results suggest that VHL protects renal cells from apoptosis via Bcl-2-dependent pathways.

Albumin overload induces apoptosis in LLC-PK(1) cells.

The degree of albuminuria is a well-known adverse prognostic indicator in human glomerular diseases. However, the mechanisms by which albuminuria by itself contributes to tubulointerstitial injury and progression of renal disease remain unclear. We tested the hypothesis that apoptosis may represent one of the mechanisms by which tubule epithelial cells are damaged after albumin overload in vitro. Cultured LLC-PK(1) proximal tubule cells were incubated with varying concentrations of BSA. This resulted in a dose- and duration-dependent induction of apoptosis, as evidenced by internucleosomal DNA cleavage (DNA laddering and nick-end labeling), externalization of plasma membrane phosphatidylserine (annexin labeling), and characteristic morphological changes (cell shrinkage and nuclear condensation). Albumin overload also resulted in a dose-dependent upregulation of Fas and Fas-associated protein with death domain (FADD), and activation of caspase 8. Incubation with the caspase 8 inhibitor IETD ameliorated the albumin-induced apoptosis. Collectively, our results indicate that albumin overload induces apoptosis of cultured LLC-PK(1) cells, mediated at least in part by the Fas-FADD-caspase 8 pathway.

The von Hippel-Lindau tumor suppressor gene protects cells from UV-mediated apoptosis.

The familial cancer syndrome, von Hippel-Lindau (VHL) disease, characterized by a predisposition to renal cell carcinoma and certain other tumor types, is caused by mutational inactivation of the VHL tumor suppressor gene. Loss of VHL gene function is detected also in the vast majority of sporadic renal cell carcinomas. Previous reports have determined a protective role for VHL in response to serum withdrawal and glucose deprivation. In this study, the effect of UV irradiation on VHL-negative and VHL-positive renal carcinoma cells was examined. VHL-negative 786-O renal carcinoma cells underwent apoptosis following UV irradiation. In contrast, reintroduction of wild-type VHL expression protected 786-O cells from UV-mediated cell death. p53 and Bax levels were equivalent in VHL-negative and VHL-positive 786-O cells. Strikingly, cyclin-dependent kinase inhibitors p21 and p27 underwent proteasome-dependent degradation in VHL-negative 786-O cells following UV treatment. However, p21 and p27 protein levels were stable in VHL-positive cells. Also, levels of the anti-apoptotic proteins, Bcl-2 and Bcl-xL were elevated in VHL-positive cells, consistent with the protection from apoptotic stimuli. UV treatment led to increased S phase in VHL-negative, but not VHL-positive cells. Thus, following UV irradiation, diminution of p21 and p27 levels resulted in a hyperproliferative state in VHL-negative cells, leading to apoptosis. These results suggest that loss of VHL function promotes apoptosis and may provide selective pressure toward cells that are able to escape apoptosis, leading to tumorigenesis.

Translational regulation of Na-K-ATPase subunit mRNAs by glucocorticoids.

Glucocorticoids (GC) regulate Na-K-ATPase-subunit mRNA transcription. However, GC-induced increases in Na-K-ATPase activity are not always paralleled by changes in subunit mRNA abundance. We therefore examined posttranscriptional mechanisms of subunit gene regulation by GC. cDNA-derived mRNAs encoding alpha 1-, alpha 3-, and beta 1-subunits were tested for stability and translation efficiency in a cell-free lysate, in the presence of hydrocortisone (HC) or dexamethasone (Dex). No effect of HC on subunit mRNA stability was noted. Translation efficiency of alpha1- and alpha 3-mRNAs, but not of beta 1-mRNA, was significantly increased by HC and Dex. Deletion of the 5'untranslated region (5'UT) of alpha 1-mRNA abolished this effect. Translation of a chimeric beta 1-mRNA, constructed by transposing the 5'UT of alpha 1 onto the coding region of beta1, was enhanced by HC. Transposition of a putative steroid-modulatory element conserved in the 5'UT of all alpha isoforms (ACAGGACCC) onto the coding region of beta 1-mRNA rendered it responsive to HC. A synthetic primer containing the ACAGGACCC sequence abolished the effect of HC on alpha 1- and chimeric beta 1-mRNAs. Our results indicate that GC can directly enhance Na-K-ATPase translation in vitro in a subunit-specific manner, via a putative GC-modulatory element situated in a predicted loop structure within the 5'UT of alpha-mRNAs.

HPTLC determination of ketorolac tromethamine.

A High Performance Thin Layer Chromatography (HPTLC) method for quantification of ketorolac tromethamine, a non-narcotic and non-steroidal agent was developed. The mobile phase composition was chloroform-ethyl acetate-glacial acetic acid (3:8:0.1, v/v/v). Spectrodensitometric analysis of ketorolac tromethamine was carried out at 323 nm. The calibration curve was linear in the range of 200-700 ng. The mean values of slope, intercept and correlation coefficient were, 2941, 749583, 0.99. The method was validated for method precision, system precision, marketed sample analysis and recovery studies. The % CV for method precision studies was 1.98 (n = 6) and system precision study was 1.83 (n = 6). The average recovery was found to be 99.2%. Acid and base degraded products were adequately separated from the drug. The method was successfully used for the determination of drug from saliva. The results indicate that the method is simple, specific, selective and reliable for quantitative analysis of ketorolac tromethamine as bulk drug and from formulations. It can also be applied for the stability study of the drug and analysis of drug in biological fluids.

Simultaneous determination of lignocaine hydrochloride and phenylephrine hydrochloride by HPTLC.

A high performance thin layer chromatographic (HPTLC) method for the simultaneous quantification of lignocaine hydrochloride (LIG) and phenylephrine hydrochloride (PHE) is described. The mobile phase consisted of ethyl acetate-methanol ammonia (4:1:0.4 v/v/v). The densitometric determination of LIG and PHE was carried out at 262 nm and 291 nm, respectively. The calibration curves of LIG and PHE were linear in the range of 8-18 microg and 4-9 microg, respectively. The method was validated with respect to system precision, method precision, recoveries, intra-day and inter-day variation. The system was applied for the simultaneous determination of LIG and PHE from a new drug delivery system. The results indicate that the method is simple, specific, selective and reliable for simultaneous quantitative determination of LIG and PHE as bulk drug and from formulations.

Feasibility of an in vitro microbiological model as an alternative to the rabbit eye model.

Ocular inserts of gentamicin sulfate with polyvinyl alcohol (PVA) 1.5%, 2.0%, and 2.5% and a combination of methyl cellulose 2% and Eudragit NE 30D 30%, 35%, and 40% w/w of methyl cellulose were fabricated by a casting technique. The inserts were sterilized by gamma radiation at 25 kGy and tested for sterility. The microbiological efficacy of the ocular inserts against Staphylococcus aureus ATCC 6538P and Pseudomonas aeruginosa NCIM 2200 was evaluated by developing an in vitro microbiological model and an in vivo noninvasive rabbit eye model. Parameters of the in vitro microbiological model were varied, and the results correlated with a noninvasive rabbit eye model. The in vitro model proved to be a viable alternative to the rabbit eye model in evaluating the microbiological efficacy of gentamicin sulfate ocular inserts.

Partial ATP depletion induces Fas- and caspase-mediated apoptosis in MDCK cells.

Brief periods of in vitro hypoxia/ischemia induce apoptosis of cultured renal epithelial cells, but the underlying mechanisms remain unknown. We show that partial ATP depletion (approximately 10-65% of control) results in a duration-dependent induction of apoptosis in Madin-Darby canine kidney (MDCK) cells, as evidenced by internucleosomal DNA cleavage (DNA laddering and in situ nick end labeling), morphological changes (cell shrinkage), and plasma membrane alterations (externalization of phosphatidylserine). The ATP-depleted cells display a significant upregulation of Fas, Fas ligand, and the Fas-associating protein with death domain (FADD). Exogenous application of stimulatory Fas monoclonal antibodies also induces apoptosis in nonischemic MDCK cells, indicating that they retain Fas-dependent pathways of programmed cell death. Furthermore, cleavage of poly(ADP)ribose polymerase (PARP) is evident after ATP depletion, indicating activation of caspases. Indeed, the apoptotic cells display a significant increase in caspase-8 (FLICE) activity. Finally, apoptosis induced by ATP depletion is ameliorated by pretreatment with inhibitors of caspase-8 (IETD), caspase-1 (YVAD), or caspase-3 (DEVD) but is not affected by inhibitors of serine proteases (TPCK). Our results indicate that partial ATP depletion of MDCK cells results in apoptosis and that Fas- and caspase-mediated pathways may play a critical role.