Practicality of intermittent fasting in humans and its effect on oxidative stress and genes related to aging and metabolism.

Caloric restriction has consistently been shown to extend life span and ameliorate aging-related diseases. These effects may be due to diet-induced reactive oxygen species acting to up-regulate sirtuins and related protective pathways, which research suggests may be partially inhibited by dietary anti-oxidant supplementation. Because caloric restriction is not sustainable long term for most humans, we investigated an alternative dietary approach, intermittent fasting (IF), which is proposed to act on similar biological pathways. We hypothesized that a modified IF diet, where participants maintain overall energy balance by alternating between days of fasting (25% of normal caloric intake) and feasting (175% of normal), would increase expression of genes associated with aging and reduce oxidative stress and that these effects would be suppressed by anti-oxidant supplementation. To assess the tolerability of the diet and to explore effects on biological mechanisms related to aging and metabolism, we recruited a cohort of 24 healthy individuals in a double-crossover, double-blinded, randomized clinical trial. Study participants underwent two 3-week treatment periods-IF and IF with anti-oxidant (vitamins C and E) supplementation. We found strict adherence to study-provided diets and that participants found the diet tolerable, with no adverse clinical findings or weight change. We detected a marginal increase (2.7%) in SIRT3 expression due to the IF diet, but no change in expression of other genes or oxidative stress markers analyzed. We also found that IF decreased plasma insulin levels (1.01 µU/mL). Although our study suggests that the IF dieting paradigm is acceptable in healthy individuals, additional research is needed to further assess the potential benefits and risks.

Should MD-PhD programs encourage graduate training in disciplines beyond conventional biomedical or clinical sciences?

The goal of MD-PhD training programs is to produce physician-scientists with unique capacities to lead the future biomedical research workforce. The current dearth of physician-scientists with expertise outside conventional biomedical or clinical sciences raises the question of whether MD-PhD training programs should allow or even encourage scholars to pursue doctoral studies in disciplines that are deemed nontraditional, yet are intrinsically germane to major influences on health. This question is especially relevant because the central value and ultimate goal of the academic medicine community is to help attain the highest level of health and health equity for all people. Advances in medical science and practice, along with improvements in health care access and delivery, are steps toward health equity, but alone they will not come close to eliminating health inequalities. Addressing the complex health issues in our communities and society as a whole requires a biomedical research workforce with knowledge, practice, and research skills well beyond conventional biomedical or clinical sciences. To make real progress in advancing health equity, educational pathways must prepare physician-scientists to treat both micro and macro determinants of health. The authors argue that MD-PhD programs should allow and encourage their scholars to cross boundaries into less traditional disciplines such as epidemiology, statistics, anthropology, sociology, ethics, public policy, management, economics, education, social work, informatics, communications, and marketing. To fulfill current and coming health care needs, nontraditional MD-PhD students should be welcomed and supported as valuable members of our biomedical research workforce.

Ablation of TRIP-Br2, a regulator of fat lipolysis, thermogenesis and oxidative metabolism, prevents diet-induced obesity and insulin resistance.

Obesity develops as a result of altered energy homeostasis favoring fat storage. Here we describe a new transcription co-regulator for adiposity and energy metabolism, SERTA domain containing 2 (TRIP-Br2, also called SERTAD2). TRIP-Br2-null mice are resistant to obesity and obesity-related insulin resistance. Adipocytes of these knockout mice showed greater stimulated lipolysis secondary to enhanced expression of hormone sensitive lipase (HSL) and ß3-adrenergic (Adrb3) receptors. The knockout mice also have higher energy expenditure because of increased adipocyte thermogenesis and oxidative metabolism caused by upregulating key enzymes in their respective processes. Our data show that a cell-cycle transcriptional co-regulator, TRIP-Br2, modulates fat storage through simultaneous regulation of lipolysis, thermogenesis and oxidative metabolism. These data, together with the observation that TRIP-Br2 expression is selectively elevated in visceral fat in obese humans, suggests that this transcriptional co-regulator is a new therapeutic target for counteracting the development of obesity, insulin resistance and hyperlipidemia.

Association of KLK3 (PSA) genetic variants with prostate cancer risk and PSA levels.

Genome-wide association studies have identified genetic markers in kallikrein-related peptidase 3 (KLK3) associated with prostate cancer. However, some of these markers are also associated with prostate-specific antigen (PSA) levels, so it is unclear whether the polymorphisms are causal or if the association with risk is solely due to detection bias through PSA screening. PSA is a biologically active serine protease, cleaving insulin-like growth factor-binding protein. We examined the association of single-nucleotide polymorphisms (SNPs) in KLK3 with prostate cancer risk, disease-specific survival and pre-diagnostic PSA levels in a case-control study nested within the Physicians' Health Study, which began in 1982, with over 27 years of follow-up. We genotyped SNPs spanning the entire KLK3 locus to capture common variation at high resolution. Six polymorphisms were significantly associated with prostate cancer incidence (P < 0.05); the odds ratios per minor allele ranged from 0.88 to 0.73. For four of these, the odds ratios were lower when restricting to cases diagnosed in the pre-PSA screening era (before 1989). The four alleles significantly associated with lower PSA levels were also associated with lower prostate cancer risk. KLK3 variants were not significantly associated with stage at diagnosis, risk of lethal cancer or survival. Our results suggest that detection bias due to the association of KLK3 variants with PSA levels cannot completely explain the association with prostate cancer risk. Understanding the mechanism by which genetic variation in KLK3 affects prostate cancer risk has important implications for study of the biological role of PSA in prostate tumorigenesis.

The Oxford IgA nephropathy clinicopathological classification is valid for children as well as adults.

To study the predictive value of biopsy lesions in IgA nephropathy in a range of patient ages we retrospectively analyzed the cohort that was used to derive a new classification system for IgA nephropathy. A total of 206 adults and 59 children with proteinuria over 0.5 g/24 h/1.73 m(2) and an eGFR of stage-3 or better were followed for a median of 69 months. At the time of biopsy, compared with adults children had a more frequent history of macroscopic hematuria, lower adjusted blood pressure, and higher eGFR but similar proteinuria. Although their outcome was similar to that of adults, children had received more immunosuppressants and achieved a lower follow-up proteinuria. Renal biopsies were scored for variables identified by an iterative process as reproducible and independent of other lesions. Compared with adults, children had significantly more mesangial and endocapillary hypercellularity, and less segmental glomerulosclerosis and tubulointerstitial damage, the four variables previously identified to predict outcome independent of clinical assessment. Despite these differences, our study found that the cross-sectional correlation between pathology and proteinuria was similar in adults and children. The predictive value of each specific lesion on the rate of decline of renal function or renal survival in IgA nephropathy was not different between children and adults.

The Oxford classification of IgA nephropathy: pathology definitions, correlations, and reproducibility.

Pathological classifications in current use for the assessment of glomerular disease have been typically opinion-based and built on the expert assumptions of renal pathologists about lesions historically thought to be relevant to prognosis. Here we develop a unique approach for the pathological classification of a glomerular disease, IgA nephropathy, in which renal pathologists first undertook extensive iterative work to define pathologic variables with acceptable inter-observer reproducibility. Where groups of such features closely correlated, variables were further selected on the basis of least susceptibility to sampling error and ease of scoring in routine practice. This process identified six pathologic variables that could then be used to interrogate prognostic significance independent of the clinical data in IgA nephropathy (described in the accompanying article). These variables were (1) mesangial cellularity score; percentage of glomeruli showing (2) segmental sclerosis, (3) endocapillary hypercellularity, or (4) cellular/fibrocellular crescents; (5) percentage of interstitial fibrosis/tubular atrophy; and finally (6) arteriosclerosis score. Results for interobserver reproducibility of individual pathological features are likely applicable to other glomerulonephritides, but it is not known if the correlations between variables depend on the specific type of glomerular pathobiology. Variables identified in this study withstood rigorous pathology review and statistical testing and we recommend that they become a necessary part of pathology reports for IgA nephropathy. Our methodology, translating a strong evidence-based dataset into a working format, is a model for developing classifications of other types of renal disease.

The Oxford classification of IgA nephropathy: rationale, clinicopathological correlations, and classification.

IgA nephropathy is the most common glomerular disease worldwide, yet there is no international consensus for its pathological or clinical classification. Here a new classification for IgA nephropathy is presented by an international consensus working group. The goal of this new system was to identify specific pathological features that more accurately predict risk of progression of renal disease in IgA nephropathy, thus enabling both clinicians and pathologists to improve individual patient prognostication. In a retrospective analysis, sequential clinical data were obtained on 265 adults and children with IgA nephropathy who were followed for a median of 5 years. Renal biopsies from all patients were scored by pathologists blinded to the clinical data for pathological variables identified as reproducible by an iterative process. Four of these variables: (1) the mesangial hypercellularity score, (2) segmental glomerulosclerosis, (3) endocapillary hypercellularity, and (4) tubular atrophy/interstitial fibrosis were subsequently shown to have independent value in predicting renal outcome. These specific pathological features withstood rigorous statistical analysis even after taking into account all clinical indicators available at the time of biopsy as well as during follow-up. The features have prognostic significance and we recommended they be taken into account for predicting outcome independent of the clinical features both at the time of presentation and during follow-up. The value of crescents was not addressed due to their low prevalence in the enrolled cohort.

TRIP-Br2 promotes oncogenesis in nude mice and is frequently overexpressed in multiple human tumors.

BACKGROUND: Members of the TRIP-Br/SERTAD family of mammalian transcriptional coregulators have recently been implicated in E2F-mediated cell cycle progression and tumorigenesis. We, herein, focus on the detailed functional characterization of the least understood member of the TRIP-Br/SERTAD protein family, TRIP-Br2 (SERTAD2). METHODS: Oncogenic potential of TRIP-Br2 was demonstrated by (1) inoculation of NIH3T3 fibroblasts, which were engineered to stably overexpress ectopic TRIP-Br2, into athymic nude mice for tumor induction and (2) comprehensive immunohistochemical high-throughput screening of TRIP-Br2 protein expression in multiple human tumor cell lines and human tumor tissue microarrays (TMAs). Clinicopathologic analysis was conducted to assess the potential of TRIP-Br2 as a novel prognostic marker of human cancer. RNA interference of TRIP-Br2 expression in HCT-116 colorectal carcinoma cells was performed to determine the potential of TRIP-Br2 as a novel chemotherapeutic drug target. RESULTS: Overexpression of TRIP-Br2 is sufficient to transform murine fibroblasts and promotes tumorigenesis in nude mice. The transformed phenotype is characterized by deregulation of the E2F/DP-transcriptional pathway through upregulation of the key E2F-responsive genes CYCLIN E, CYCLIN A2, CDC6 and DHFR. TRIP-Br2 is frequently overexpressed in both cancer cell lines and multiple human tumors. Clinicopathologic correlation indicates that overexpression of TRIP-Br2 in hepatocellular carcinoma is associated with a worse clinical outcome by Kaplan-Meier survival analysis. Small interfering RNA-mediated (siRNA) knockdown of TRIP-Br2 was sufficient to inhibit cell-autonomous growth of HCT-116 cells in vitro. CONCLUSION: This study identifies TRIP-Br2 as a bona-fide protooncogene and supports the potential for TRIP-Br2 as a novel prognostic marker and a chemotherapeutic drug target in human cancer.

Identification of PP2A as a novel interactor and regulator of TRIP-Br1.

TRIP-Br proteins are a novel family of transcriptional coregulators involved in E2F-mediated cell cycle progression. Three of the four mammalian members of TRIP-Br family, including TRIP-Br1, are known oncogenes. We now report the identification of the Balpha regulatory subunit of serine/threonine protein phosphatase 2A (PP2A) as a novel TRIP-Br1 interactor, based on an affinity binding assay coupled with mass spectrometry. A GST-TRIP-Br1 fusion protein associates with catalytically active PP2A-ABalphaC holoenzyme in vitro. Coimmunoprecipitation confirms this association in vivo. Immunofluorescence staining with a monoclonal antibody against TRIP-Br1 reveals that endogenous TRIP-Br1 and PP2A-Balpha colocalize mainly in the cytoplasm. Consistently, immunoprecipitation followed by immunodetection with anti-phosphoserine antibody suggest that TRIP-Br1 exists in a serine-phosphorylated form. Inhibition of PP2A activity by okadaic acid or transcriptional silencing of the PP2A catalytic subunit by small interfering RNA results in downregulation of total TRIP-Br1 protein levels but upregulation of serine-phosphorylated TRIP-Br1. Overexpression of PP2A catalytic subunit increases TRIP-Br1 protein levels and TRIP-Br1 co-activated E2F1/DP1 transcription. Our data support a model in which association between PP2A-ABalphaC holoenzyme and TRIP-Br1 in vivo in mammalian cells represents a novel mechanism for regulating the level of TRIP-Br1 protooncoprotein.

CRM1-mediated nuclear export is required for 26 S proteasome-dependent degradation of the TRIP-Br2 proto-oncoprotein.

Overexpression of the proto-oncogene TRIP-Br2 (SERTAD2) has been shown to induce E2F activity and promote tumorigenesis, whereas ablation of TRIP-Br2 arrests cell proliferation. Timely degradation of many cell cycle regulators is fundamental to the maintenance of proper cell cycle progression. Here we report novel mechanism(s) that govern the tight regulation of TRIP-Br2 levels during cell cycle progression. TRIP-Br2 was observed to be a short-lived protein in which the expression level peaks at the G(1)/S boundary. TRIP-Br2 accumulated in cells treated with 26 S proteasome inhibitors. Co-immunoprecipitation studies revealed that TRIP-Br2 forms ubiquitin conjugates. In silico analysis identified a putative leucine-rich nuclear export signal (NES) motif that overlaps with the PHD-Bromo interaction domain in the acidic C-terminal transactivation domain (TAD) of TRIP-Br2. This NES motif is highly conserved in widely divergent species and in all TRIP-Br family members. TRIP-Br2 was shown to be stabilized in G(2)/M phase cells through nuclear entrapment, either by deletion of the acidic C-terminal TAD, which includes the NES motif, or by leptomycin B-mediated inhibition of the CRM1-dependent nuclear export machinery. Mutation of leucine residue 238 of this NES motif abolished the interaction between CRM1 and TRIP-Br2, as well as the nuclear export of TRIP-Br2 and its subsequent 26 S proteasome-dependent degradation. These data suggest that CRM1-mediated nuclear export may be required for the proper execution of ubiquitin-proteasome-dependent degradation of TRIP-Br2.

Pathogenic role of NF-kappaB activation in tubulointerstitial inflammatory lesions in human lupus nephritis.

In vitro and in vivo experimental studies suggest that the transcription factor NF-kappaB plays a role in tubulointerstitial injury. We investigated possible cellular and molecular mechanisms involving NF-kappaB activation in the progression of tubulointerstitial lesions in human lupus nephritis (LN). Paraffin-embedded renal biopsies from 50 patients with LN and six control patients with minimal change disease (MCD) were examined by Southwestern histochemistry for in situ detection of active NF-kappaB and AP-1. Immunohistochemistry was performed to examine the expression of NF-kappaB, AP-1, and NF-kappaB regulatory proteins (IkappaB-alpha, p-IkappaB-alpha, and IKK-alpha proteins), as well as NF-kappaB and AP-1 downstream target proinflammatory molecules (ICAM-1, TNF-alpha, IL-1beta, IL-6, and GM-CSF) and NF-kappaB upstream signaling molecules (CD40 and CD40L). We observed extensive upregulation of activated NF-kappaB in renal tubular cells and interstitial cells, in parallel with overactivation of transcription factor AP-1 in LN, as compared with normal controls and MCD. Tubular expression of activated NF-kappaB correlated well with the degree of tubulointerstitial histopathological indices and/or renal function. Tubulointerstitial IKK-alpha expression was specifically upregulated in LN. IkappaB-alpha and p-IkappaB-alpha were detected only in interstitial cells in LN. Tubulointerstitial expression levels of NF-kappaB and AP-1 downstream inflammatory molecules and NF-kappaB upstream signaling molecules CD40 and CD40L were markedly enhanced in LN as compared with MCD or normal controls and were associated with tubulointerstitial histopathological indices and/or renal function. The results suggest that altered IKK-alpha expression and NF-kappaB activation along with AP-1 overexpression may play a pathogenic role in tubulointerstitial injury in human LN mediated through a network of downstream proinflammatory molecules.

Racial and genetic factors in IgA nephropathy.

Racial and ethnic variations in the incidence of IgA nephropathy (IgAN) could imply both genetic and environmental influences that exist in a complex and poorly understood interplay to modify the expression of the IgAN clinical phenotype. Progress in identifying genetic factors that influence either susceptibility to IgAN or its progression has been slow. Recent progress using family based approaches (genome-wide scan for linkage and family based genetic association studies) to study the genetic basis for susceptibility to familial and sporadic IgAN strongly point to clinical and genetic heterogeneity in the entity we presently call IgAN. The inconsistent findings reported from case-control genetic association studies may be explained by new understanding of the haplotype block structure of the human genome. Rapid improvements in available and developing technologies in the postgenomic era are needed and are expected to accelerate progress in understanding genetic factors underlying IgAN.

Exploiting the TRIP-Br family of cell cycle regulatory proteins as chemotherapeutic drug targets in human cancer.

TRIP-Br1 and TRIP-Br2 are potent cell growth promoting factors that function as components of the E2F1/DP1 transcription complex to integrate positive growth signals provided by PHD zinc finger- and/or bromodomain-containing transcription factors. TRIP-Br1 has been demonstrated to be an oncogene. We recently reported that antagonism of the TRIP-Br integrator function by synthetic decoy peptides that compete with TRIP-Br for binding to PHD zinc finger- and/or bromodomain-containing proteins elicit an anti-proliferative effect and induces caspase-3-independent sub-diploidization in cancer cells in vitro. We now demonstrate the chemotherapeutic potential of TRIP-Br decoy peptides for the treatment of cutaneous and intracavitary lesions in vitro as well as in vivo in representative human nasopharyngeal cancer (CNE2), cervical cancer (Ca Ski) and melanoma (MeWo) cancer cell lines. In vitro, BrdU incorporation, colony formation assays and cell cycle analysis confirmed that TRIP-Br decoy peptides possess strong anti-proliferative effects and induce nuclear sub-diploidization in cancer cells. In vivo, CNE2, Ca Ski and MeWo-derived chick embryo chorioallantoic membrane (CAM) tumor xenografts were used to evaluate the effect of topically applied TRIP-Br peptides. Confocal microscopy and flow cytometric analysis demonstrated that cells comprising the tumor xenografts efficiently internalized topically applied FITC-labeled peptides. Fifty muM of TRIP-Br1 decoy peptide significantly suppressed the growth of NPC2-derived human nasopharyngeal tumors, while 50 muM of TRIP-Br2 decoy peptide significantly inhibited tumor growth in all three CAM tumor xenograft models. Two hundred muM of TRIP-Br1 decoy peptide significantly inhibited MeWo-derived tumors. These results suggest that the TRIP-Br integrator function may represent a novel chemotherapeutic target for the treatment of human cutaneous and intracavitary proliferative lesions.

The TRIP-Br family of transcriptional regulators is essential for the execution of cyclin E-mediated cell cycle progression.

The G1 D-type cyclins, in conjunction with cyclin-dependent kinases Cdk4 and Cdk6, play key roles in the execution of mitogen-induced cellular proliferation. TRIP-Br1, a member of the TRIP-Br family of transcriptional regulators, has been implicated in the regulation of Cdk4/cyclin D activity. To further elucidate the functional role(s) of the TRIP-Br proteins in mitogenic signaling, we have developed the synthetic DNA enzymes E-Br1 and E-Br2 to specifically knock down the serum-inducible expression of TRIP-Br1 and TRIP-Br2, respectively, in WI-38 human fibroblasts in culture, as well as generated TRIP-Br2 null primary embryonic fibroblasts from a novel TRIP-Br2 knockout mouse model. Both strategies consistently reveal that ablation of TRIP-Br1 or TRIP-Br2 expression disrupts mitogenic signaling in a manner that suppresses serum-induced cyclin E expression, S-phase entry and cellular proliferation. We conclude that both TRIP-Br1 and TRIP-Br2 are required for proper transduction of mitogenic signals and execution of serum-inducible cell cycle progression.

Renal cell apoptosis and proliferation may be linked to nuclear factor-kappaB activation and expression of inducible nitric oxide synthase in patients with lupus nephritis.

The mechanism of renal cell apoptosis involves transcriptional activation of the inducible nitric oxide synthase (iNOS) gene by nuclear factor (NF)-kappaB. The role of apoptosis in mediating tubulointerstitial injury in human lupus nephritis (LN) remains unclear. We examined the relationship between alterations in NF-kappaB activation and iNOS expression levels and the degree of apoptosis in both glomerular and tubulointerstitial compartments of subjects with LN. Studies were done in renal biopsies from 49 patients with LN and 10 normal kidney tissues. Apoptotic and proliferating cells were identified by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and staining with anti-proliferating cell nuclear antigen antibody, respectively. Nuclear factor-kappaB and iNOS expression was examined by Southwestern histochemistry and immunohistochemistry, respectively. Glomerular cell apoptosis and proliferation increased concomitantly in LN. Glomerular apoptosis correlated with the activity index, the degree of proliferation, and the level of glomerular overexpression of iNOS and activated NF-kappaB in LN. Tubular cell apoptosis correlated with the activity and chronicity indices, the degree of tubular atrophy, and decline in renal function at the time of biopsy. Tubular expression of iNOS and activated NF-kappaB correlated with tubular cell proliferation in LN. Nuclear factor-kappaB activation accompanied overexpression of iNOS in both glomerular and tubulointerstitium compartments in LN. Apoptosis of renal cells associated with NF-kappaB activation and iNOS overexpression may play an important role in mediating chronic renal injury, especially tubulointerstitial lesions that may manifest clinically as progressive renal insufficiency.

In situ glomerular expression of activated NF-kappaB in human lupus nephritis and other non-proliferative proteinuric glomerulopathy.

Nuclear Factor-kappaB (NF-kappaB) has been suggested to play a role in the cellular and molecular mechanisms underlying glomerular injury. We investigated the potential role of NF-kappaB activation in the pathogenesis of glomerular injury in 31 patients with class III-V lupus nephritis (LN), 14 patients with non-proliferative proteinuric glomerulopathy and six normal controls. The expression of NF-kappaB subunits p65 and p50, and the NF-kappaB regulated proinflammatory mediators tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1) as well as CD68 and synaptopodin was examined by Southwestern histochemistry (SWH) or immunohistochemistry. In contrast to non-proliferative glomerulopathy and normal controls, NF-kappaB activation (both p65 and p50) was enhanced in glomerular endothelial, mesangial cells or infiltrating cells in class IV LN, along with upregulation of TNF-alpha, IL-1beta, IL-6 and ICAM-1 expression. Glomerular endothelial and mesangial activation of NF-kappaB and mesangial ICAM-1 expression correlated with disease activity and the level of glomerular macrophage infiltration. Podocyte NF-kappaB overactivation (predominantly p65) paralleled podocyte expression of TNF-alpha and IL-1beta in patients with LN and non-proliferative glomerulopathy. Podocyte staining scores of NF-kappaB and p65 were positively correlated with the severity of proteinuria in LN and non-proliferative glomerulopathy. These results suggest a pathogenic role for NF-kappaB in glomerular injury by multiple mechanisms.

TRIP-Br links E2F to novel functions in the regulation of cyclin E expression during cell cycle progression and in the maintenance of genomic stability.

The TRIP-Br family of transcriptional regulators (TRIP-Br1 and TRIP-Br2) has been proposed to function at E2F-responsive promoters to integrate regulatory signals provided by PHD zinc finger- and/or bromodomain-containing transcription factors. To characterize the TRIP-Br "integrator" function(s), we have employed decoy peptides (*Br1 and *Br2) to antagonize the interaction between TRIP-Br1 or TRIP-Br2 and the PHD zinc finger and/or bromodomain of other transcription factors. Antagonism of the TRIP-Br integrator function elicits anti-proliferative effects through the transcriptional downregulation of a subset of E2F-responsive genes in vivo, and induces aberrant cyclin E accumulation, leading to Geminin deregulation and caspase-3-independent cellular sub-diploidization. The observed cyclin E deregulation is attributed to the downregulation of Fbxw7, which encodes the Fbw7 receptor subunit of the SCF(FBW7) ubiquitin ligase (E3) responsible for targeting cyclin E for proteolysis. Fbxw7 is identified herein as an E2F-responsive and TRIP-Br coregulated gene. Our results demonstrate a physiologic role for TRIP-Br in coupling E2F to novel functions in the regulation of cyclin E expression during cell cycle progression to ensure the proper execution of DNA replication and the maintenance of genomic stability.

Coupled induction of iNOS and p53 upregulation in renal resident cells may be linked with apoptotic activity in the pathogenesis of progressive IgA nephropathy.

In situ hybridization, immunohistochemistry, and TUNEL staining were applied to renal biopsy specimens of immunoglobulin A nephropathy (IgAN) patients to determine the expression of nitric oxide synthase (iNOS) (mRNA and protein), p53, and their potential roles in renal cell apoptosis in relation to the development of pathologic lesions. Fifty-one cases were categorized into four subgroups (A-D) according to the presence of progressive histopathological features. A cell type-specific and differential overexpression of iNOS mRNA and protein was demonstrated in glomerular cells in subgroups (A-C) and was found to correlate well with the upregulation of p53 protein by glomerular endothelium and epithelium in early- and advanced-stage disease. In the tubulointerstitium, induction of iNOS products was evident in damaged tubules in late-stage disease, in parallel with the upregulation of p53 protein levels in these tubules. Increased TUNEL staining observed in glomeruli with progressive lesions and tubules with degenerative changes positively correlated with the expression levels of iNOS and p53 in glomerular endothelium, epithelium, and their overexpression in damaged tubules. Clinicopathologic correlations demonstrated that induction of iNOS products in renal cells was associated with indices of poor renal prognosis in human IgAN. The coupled induction of iNOS and p53 upregulation in intrinsic renal cells of IgAN may be linked with both pro- and anti-apoptotic activities, thus playing an important role in mediating progressive renal injury and determining renal outcome in human IgAN.