James Pullman

Epithelial Notch signaling regulates interstitial fibrosis development in the kidneys of mice and humans

Chronic kidney disease is a leading cause of death in the United States. Tubulointerstitial fibrosis (TIF) is considered the final common pathway leading to end-stage renal disease (ESRD). Here, we used pharmacologic, genetic, in vivo, and in vitro experiments to show that activation of the Notch pathway in tubular epithelial cells (TECs) in patients and in mouse models of TIF plays a role in TIF development. Expression of Notch in renal TECs was found to be both necessary and sufficient for TIF development. Genetic deletion of the Notch pathway in TECs reduced renal fibrosis. Consistent with this, TEC-specific expression of active Notch1 caused rapid development of TIF. Pharmacologic inhibition of Notch activation using a γ-secretase inhibitor ameliorated TIF. In summary, our experiments establish that epithelial injury and Notch signaling play key roles in fibrosis development and indicate that Notch blockade may be a therapeutic strategy to reduce fibrosis and ESRD development.

Defective fatty acid oxidation in renal tubular epithelial cells has a key role in kidney fibrosis development

Renal fibrosis is the histological manifestation of a progressive, usually irreversible process causing chronic and end-stage kidney disease. We performed genome-wide transcriptome studies of a large cohort (n = 95) of normal and fibrotic human kidney tubule samples followed by systems and network analyses and identified inflammation and metabolism as the top dysregulated pathways in the diseased kidneys. In particular, we found that humans and mouse models with tubulointerstitial fibrosis had lower expression of key enzymes and regulators of fatty acid oxidation (FAO) and higher intracellular lipid deposition compared to controls. In vitro experiments indicated that inhibition of FAO in tubule epithelial cells caused ATP depletion, cell death, dedifferentiation and intracellular lipid deposition, phenotypes observed in fibrosis. In contrast, restoring fatty acid metabolism by genetic or pharmacological methods protected mice from tubulointerstitial fibrosis. Our results raise the possibility that correcting the metabolic defect in FAO may be useful for preventing and treating chronic kidney disease.

Transcriptome Analysis of Human Diabetic Kidney Disease

OBJECTIVE Diabetic kidney disease (DKD) is the single leading cause of kidney failure in the U.S., for which a cure has not yet been found. The aim of our study was to provide an unbiased catalog of gene-expression changes in human diabetic kidney biopsy samples. RESEARCH DESIGN AND METHODS Affymetrix expression arrays were used to identify differentially regulated transcripts in 44 microdissected human kidney samples. DKD samples were significant for their racial diversity and decreased glomerular filtration rate (~25–35 mL/min). Stringent statistical analysis, using the Benjamini-Hochberg corrected two-tailed t test, was used to identify differentially expressed transcripts in control and diseased glomeruli and tubuli. Two different web-based algorithms were used to define differentially regulated pathways. RESULTS We identified 1,700 differentially expressed probesets in DKD glomeruli and 1,831 in diabetic tubuli, and 330 probesets were commonly differentially expressed in both compartments. Pathway analysis highlighted the regulation of Ras homolog gene family member A, Cdc42, integrin, integrin-linked kinase, and vascular endothelial growth factor signaling in DKD glomeruli. The tubulointerstitial compartment showed strong enrichment for inflammation-related pathways. The canonical complement signaling pathway was determined to be statistically differentially regulated in both DKD glomeruli and tubuli and was associated with increased glomerulosclerosis even in a different set of DKD samples. CONCLUSIONS Our studies have cataloged gene-expression regulation and identified multiple novel genes and pathways that may play a role in the pathogenesis of DKD or could serve as biomarkers.

Cytosine methylation changes in enhancer regions of core pro-fibrotic genes characterize kidney fibrosis development

BackgroundOne in eleven people is affected by chronic kidney disease, a condition characterized by kidney fibrosis and progressive loss of kidney function. Epidemiological studies indicate that adverse intrauterine and postnatal environments have a long-lasting role in chronic kidney disease development. Epigenetic information represents a plausible carrier for mediating this programming effect. Here we demonstrate that genome-wide cytosine methylation patterns of healthy and chronic kidney disease tubule samples obtained from patients show significant differences.ResultsWe identify differentially methylated regions and validate these in a large replication dataset. The differentially methylated regions are rarely observed on promoters, but mostly overlap with putative enhancer regions, and they are enriched in consensus binding sequences for important renal transcription factors. This indicates their importance in gene expression regulation. A core set of genes that are known to be related to kidney fibrosis, including genes encoding collagens, show cytosine methylation changes correlating with downstream transcript levels.ConclusionsOur report raises the possibility that epigenetic dysregulation plays a role in chronic kidney disease development via influencing core pro-fibrotic pathways and can aid the development of novel biomarkers and future therapeutics.

Expression of Notch pathway proteins correlates with albuminuria, glomerulosclerosis, and renal function

Recent studies indicate that the Notch signaling pathway plays an important role in the development of diabetic kidney disease and focal segmental glomerulosclerosis (FSGS). Here we analyzed the degree of expression and localization of Notch ligands (Jagged1 and Delta1) and activated (cleaved) receptors (Notch1 and Notch2) in healthy human kidneys and in renal biopsies from a wide variety of kidney diseases. These included patients with minimal change disease, membranous nephropathy, lupus nephritis ISN/RPS classes III/IV/V, hypertensive nephrosclerosis, crescentic glomerulonephritis, tubulointerstitial fibrosis, IgA nephropathy, diabetic kidney disease, and FSGS. We found that cleaved Notch1, Notch2, and Jagged1 are expressed on podocytes in proteinuric nephropathies and their level of expression correlated with the amount of proteinuria across all disease groups. The degree of glomerulosclerosis correlated with podocyte expression of cleaved Notch1, while the severity of tubulointerstitial fibrosis and the estimated glomerular filtration rate correlated with expression of cleaved Notch1 in the tubulointerstitium. Hence, our results raise the possibility that Notch pathway activation is a common mechanism in the pathophysiology of a wide range of acquired renal diseases.

Successful rescue therapy with plasmapheresis and intravenous immunoglobulin for acute humoral renal transplant rejection

Plasmapheresis (PP) and intravenous immunoglobulin (IVIg) remove donor-specific antibodies, a cause of acute humoral rejection (AHR). We describe the use of PP and IVIg as rescue therapy for AHR. The records of 143 renal transplants performed between October 1, 2000 and April 1, 2002 were reviewed. Patients who underwent PP and IVIg therapy for AHR were identified. The data reviewed included age, sex, source of transplant, number of human leukocyte antigen mismatches, transplant number, number of PP and IVIg treatments, dose of IVIg, time of AHR, serum creatinine (SCr) level at AHR, SCr level after PP and IVIg at 3 months, days to achieve 30% decline in SCr, and graft survival. Immunosuppression included basiliximab induction, tacrolimus, and prednisone (± sirolimus or mycophenolate mofetil [CellCept, Roche Pharmaceutical, Nutley, NJ]). PP was followed by IVIg infusion. Nine patients were treated for AHR with PP and IVIg. All nine patients demonstrated biopsy-proven AHR. One graft was lost. Mean 3-month and 1-year SCr levels were 1.9 and 1.8, respectively, in the remaining eight patients. AHR in renal transplantation can be effectively treated with PP and IVIg.

Expression of translation initiation factor eIF‐2α is increased in benign and malignant melanocytic and colonic epithelial neoplasms

Stimulation of resting cells by growth factors leads to an increase in the rate of protein synthesis, which is necessary for cell growth and division. Translation initiation factor eIF‐2α is one of the key translation factors mediating the effects of growth factors on protein synthesis. In normal cells, expression of eIF‐2α is increased transiently, but its levels are elevated constitutively in oncogene‐transformed cells. Overexpression of constitutively active eIF‐2α in rodent cells makes them tumorigenic. In this article, the authors report their findings on the increased expression of eIF‐2α in human benign and malignant neoplasms originating from melanocytes and colonic epithelium.

Combining neurohormonal blockade with continuous-flow left ventricular assist device support for myocardial recovery: A single-arm prospective study

BACKGROUND Combining mechanical unloading by a continuous-flow left ventricular assist device (CF-LVAD) and neurohormonal blockade with heart failure medications (HFMED) is an underexplored clinical strategy to promote recovery of cardiac function in patients with advanced heart failure (HF). METHODS We implemented a clinical protocol to achieve maximal neurohormonal blockade after placement of a CF-LVAD and assessed its utility in an LVAD weaning (6,200 rpm) study. Thirty-four subjects were enrolled after CF-LVAD and were managed with aggressive, bi-weekly up-titration of HFMED. RESULTS Twenty-one subjects (8 with coronary artery disease, 13 with idiopathic dilated cardiomyopathy) were included in this LVAD weaning investigation. Overall, combined CF-LVAD and HFMED resulted in significant reverse remodeling with a decrease in left atrial volume index (44.7±16.0 to 31.6±12.1 ml/m(2), p < 0.001) and LV internal diastolic diameter (6.7±1.5 to 6.0±1.6 cm, p = 0.003) and an increase in LV ejection fraction (17.4±6.5 to 33.1±16.2%, p < 0.001) during LVAD weaning (6,200 rpm). Five of 21 (24%) subjects demonstrated recovery of biventricular function. Exploratory analysis showed that recovered subjects had shorter duration HF, less myocardial fibrosis and less myocyte hypertrophy, and were supported at higher LVAD speeds. CONCLUSIONS CF-LVAD support in combination with HFMED leads to significant reverse remodeling in patients with advanced HF. Using this approach, one quarter of patients demonstrated complete recovery of cardiac function. Our results suggest that bridge to recovery in the current device era is a clinically meaningful phenomenon and merits further investigation.

Human and Murine Kidneys Show Gender- and Species-Specific Gene Expression Differences in Response to Injury

The incidence of End Stage Renal Disease (ESRD) is approximately 50% higher in men than women. In order to understand the molecular basis of this gender disparity, we examined sex specific gene expression patterns in control and diseased, human and murine kidney samples. Using the Affymetrix platform we performed comprehensive gene expression analysis on 42 microdissected human kidney samples (glomeruli and tubules). We identified 67 genes with gender biased expression in healthy human kidneys and 24 transcripts in diseased male and female human kidneys. Similar analysis performed in mice using male and female control and doxorubicin induced nephrotic syndrome kidneys identified significantly larger number of differentially expressed transcripts. The majority of genes showing gender biased expression either in diseased human and murine kidneys were different from those differentially expressed in healthy kidneys. Only 9 sexually dimorphic transcripts were common to healthy human and murine kidneys and five showed differential regulation in both human and murine diseased kidneys. In humans, sex biased genes showed statistical enrichment only to sex chromosomes while in mice they were enriched to sex chromosomes and various autosomes. Thus we present a comprehensive analysis of gender biased genes in the kidney. We show that sexually dimorphic genes in the kidney show species specific regulation. Our results also indicate that male and female kidneys respond differently to injury. These studies could provide the basis for the development of new treatment strategies for men and women with kidney disease.

Kidney Cancer Is Characterized by Aberrant Methylation of Tissue-Specific Enhancers That Are Prognostic for Overall Survival

Purpose: Even though recent studies have shown that genetic changes at enhancers can influence carcinogenesis, most methylomic studies have focused on changes at promoters. We used renal cell carcinoma (RCC), an incurable malignancy associated with mutations in epigenetic regulators, as a model to study genome-wide patterns of DNA methylation at a high resolution. Experimental Design: Analysis of cytosine methylation status of 1.3 million CpGs was determined by the HELP assay in RCC and healthy microdissected renal tubular controls. Results: We observed that the RCC samples were characterized by widespread hypermethylation that preferentially affected gene bodies. Aberrant methylation was particularly enriched in kidney-specific enhancer regions associated with H3K4Me1 marks. Various important underexpressed genes, such as SMAD6, were associated with aberrantly methylated, intronic enhancers, and these changes were validated in an independent cohort. MOTIF analysis of aberrantly hypermethylated regions revealed enrichment for binding sites of AP2a, AHR, HAIRY, ARNT, and HIF1 transcription factors, reflecting contributions of dysregulated hypoxia signaling pathways in RCC. The functional importance of this aberrant hypermethylation was demonstrated by selective sensitivity of RCC cells to low levels of decitabine. Most importantly, methylation of enhancers was predictive of adverse prognosis in 405 cases of RCC in multivariate analysis. In addition, parallel copy-number analysis from MspI representations demonstrated novel copy-number variations that were validated in an independent cohort of patients. Conclusions: Our study is the first high-resolution methylome analysis of RCC, demonstrates that many kidney-specific enhancers are targeted by aberrant hypermethylation, and reveals the prognostic importance of these epigenetic changes in an independent cohort. Clin Cancer Res; 20(16); 4349–60. ©2014 AACR.