Poonam Sansanwal

Expression of Complement Components Differs Between Kidney Allografts from Living and Deceased Donors

A disparity remains between graft survival of renal allografts from deceased donors and from living donors. A better understanding of the molecular mechanisms that underlie this disparity may allow the development of targeted therapies to enhance graft survival. Here, we used microarrays to examine whole genome expression profiles using tissue from 53 human renal allograft protocol biopsies obtained both at implantation and after transplantation. The gene expression profiles of living-donor kidneys and pristine deceased-donor kidneys (normal histology, young age) were significantly different before reperfusion at implantation. Deceased-donor kidneys exhibited a significant increase in renal expression of complement genes; posttransplantation biopsies from well-functioning, nonrejecting kidneys, regardless of donor source, also demonstrated a significant increase in complement expression. Peritransplantation phenomena, such as donor death and possibly cold ischemia time, contributed to differences in complement pathway gene expression. In addition, complement gene expression at the time of implantation was associated with both early and late graft function. These data suggest that complement-modulating therapy may improve graft outcomes in renal transplantation.

Mitochondrial Autophagy Promotes Cellular Injury in Nephropathic Cystinosis

The molecular and cellular mechanisms underlying nephropathic cystinosis, which exhibits generalized proximal tubular dysfunction and progressive renal failure, remain largely unknown. Renal biopsies from patients with this disorder can reveal abnormally large mitochondria, but the relevance of this and other ultrastructural abnormalities is unclear. We studied the ultrastructure of fibroblasts and renal proximal tubular epithelial cells from patients with three clinical variants of cystinosis: Nephropathic, intermediate, and ocular. Electron microscopy revealed the presence of morphologically abnormal mitochondria and abnormal patterns of mitochondrial autophagy (mitophagy) with a high number of autophagic vacuoles and fewer mitochondria (P < 0.02) in nephropathic cystinosis. In addition, we observed increased apoptosis in renal proximal tubular epithelial cells, greater expression of LC3-II/LC3-I (microtubule-associated protein 1 light chain 3), and significantly more autophagosomes in the nephropathic variant. The autophagy inhibitor 3-methyl adenine rescued cell death in cystinotic cells. Cystinotic cells had increased levels of beclin-1 and aberrant mitochondrial function with a significant decrease in ATP generation and an increase in reactive oxygen species. This study provides ultrastructural and functional evidence of abnormal mitophagy in nephropathic cystinosis, which may contribute to the renal Fanconi syndrome and progressive renal injury.

A Peripheral Blood Diagnostic Test for Acute Rejection in Renal Transplantation

Monitoring of renal graft status through peripheral blood (PB) rather than invasive biopsy is important as it will lessen the risk of infection and other stresses, while reducing the costs of rejection diagnosis. Blood gene biomarker panels were discovered by microarrays at a single center and subsequently validated and cross‐validated by QPCR in the NIH SNSO1 randomized study from 12 US pediatric transplant programs. A total of 367 unique human PB samples, each paired with a graft biopsy for centralized, blinded phenotype classification, were analyzed (115 acute rejection (AR), 180 stable and 72 other causes of graft injury). Of the differentially expressed genes by microarray, Q‐PCR analysis of a five gene‐set (DUSP1, PBEF1, PSEN1, MAPK9 and NKTR) classified AR with high accuracy. A logistic regression model was built on independent training‐set (n = 47) and validated on independent test‐set (n = 198)samples, discriminating AR from STA with 91% sensitivity and 94% specificity and AR from all other non‐AR phenotypes with 91% sensitivity and 90% specificity. The 5‐gene set can diagnose AR potentially avoiding the need for invasive renal biopsy. These data support the conduct of a prospective study to validate the clinical predictive utility of this diagnostic tool.

Non-HLA antibodies to immunogenic epitopes predict the evolution of chronic renal allograft injury

Chronic allograft injury (CAI) results from a humoral response to mismatches in immunogenic epitopes between the donor and recipient. Although alloantibodies against HLA antigens contribute to the pathogenesis of CAI, alloantibodies against non-HLA antigens likely contribute as well. Here, we used high-density protein arrays to identify non-HLA antibodies in CAI and subsequently validated a subset in a cohort of 172 serum samples collected serially post-transplantation. There were 38 de novo non-HLA antibodies that significantly associated with the development of CAI (P<0.01) on protocol post-transplant biopsies, with enrichment of their corresponding antigens in the renal cortex. Baseline levels of preformed antibodies to MIG (also called CXCL9), ITAC (also called CXCL11), IFN-γ, and glial-derived neurotrophic factor positively correlated with histologic injury at 24 months. Measuring levels of these four antibodies could help clinicians predict the development of CAI with >80% sensitivity and 100% specificity. In conclusion, pretransplant serum levels of a defined panel of alloantibodies targeting non-HLA immunogenic antigens associate with histologic CAI in the post-transplant period. Validation in a larger, prospective transplant cohort may lead to a noninvasive method to predict and monitor for CAI.

Caspase-4 may play a role in loss of proximal tubules and renal injury in nephropathic cystinosis

Nephropathic cystinosis is characterized clinically by generalized proximal renal tubular dysfunction, renal Fanconi Syndrome and progressive renal failure. Glomerular–proximal tubule disconnection has been noted in renal biopsies from patients with nephropathic cystinosis. In vitro studies performed in cystinotic fibroblasts and renal proximal tubular cells support a role for apoptosis of the glomerulotubular junction, and we have further extended these studies to human native cystinotic kidney specimens. We performed semi-quantitative analysis of tubular density in kidney biopsies from patients with nephropathic cystinosis and demonstrated a significant reduction (p = 0.0003) in the number of proximal tubules in the kidney tissue of patients with cystinosis compared to normal kidneys and kidneys with other causes of renal injury; this reduction appears to be associated with the over-expression of caspase-4. This study provides the first quantitative evidence of a loss of proximal tubules in nephropathic cystinosis and suggests a possible role of caspase-4 in the apoptotic loss of proximal tubular cells. Further work is needed to elucidate if this injury mechanism may be causative for the progression of renal functional decline in nephropathic cystinosis.

Inhibition of Intracellular Clusterin Attenuates Cell Death in Nephropathic Cystinosis

Nephropathic cystinosis, characterized by accumulation of cystine in the lysosomes, is caused by mutations in CTNS. The molecular and cellular mechanisms underlying proximal tubular dysfunction and progressive renal failure in nephropathic cystinosis are largely unclear, and increasing evidence supports the notion that cystine accumulation alone is not responsible for the end organ injury in cystinosis. We previously identified clusterin as potentially involved in nephropathic cystinosis. Here, we studied the expression of clusterin in renal proximal tubular epithelial cells obtained from patients with nephropathic cystinosis. The cytoprotective secretory form of clusterin, as evaluated by Western blot analysis, was low or absent in cystinosis cells compared with normal primary cells. Confocal microscopy revealed elevated levels of intracellular clusterin in cystinosis cells. Clusterin in cystinosis cells localized to the nucleus and cytoplasm and showed a filamentous and punctate aggresome-like pattern compared with diffuse cytoplasmic staining in normal cells. In kidney biopsy samples from patients with nephropathic cystinosis, clusterin protein expression was mainly limited to the proximal tubular cells. Furthermore, expression of clusterin overlapped with the expression of apoptotic proteins (apoptosis-inducing factor and cleaved caspase-3) and autophagy proteins (LC3 II and p62). Silencing of the clusterin gene resulted in a significant increase in cell viability and attenuation of apoptosis in cystinosis cells. Results of this study identify clusterin as a pivotal factor in the cell injury mechanism of nephropathic cystinosis and provide evidence linking cellular stress and injury to Fanconi syndrome and progressive renal injury in nephropathic cystinosis.

Abnormal mitochondrial autophagy in nephropathic cystinosis

Cystinosis, which is characterized by lysosomal accumulation of cystine in many tissues, was the first known storage disorder caused by defective metabolite export from the lysosome. The molecular and cellular mechanisms underlying nephropathic cystinosis, the most severe form, which exhibits generalized proximal tubular dysfunction and progressive renal failure, remain largely unknown. We used renal proximal tubular epithelial (RPTE) cells and fibroblasts from patients with 3 clinical variants of cystinosis: nephropathic, intermediate, and ocular to explore the specific injury mechanism in nephropathic cystinosis. We demonstrate enhanced autophagy of mitochondria, increase in apoptosis and mitochondrial dysfunction in the nephropathic cystinosis phenotype. Furthermore, specific inhibition of autophagy results in significant attenuation of cell death in nephropathic cystinosis. This study provides ultrastructural and functional evidence of abnormal mitochondrial autophagy in nephropathic cystinosis, which may contribute to the renal Fanconi syndrome and progressive renal injury.

Insights into novel cellular injury mechanisms by gene expression profiling in nephropathic cystinosis.

Nephropathic cystinosis is a rare, inherited metabolic disease caused by functional defects of cystinosin associated with mutations in the CTNS gene. The mechanisms underlying the phenotypic alterations associated with this disease are not well known. In this study, gene expression profiles in peripheral blood of nephropathic cystinosis patients (N = 7) were compared with controls (N = 7) using microarray technology. In unsupervised hierarchical clustering analysis, cystinosis samples co-clustered, and 1,604 genes were significantly differentially expressed between both groups. Gene ontology analysis revealed that differentially expressed genes in cystinosis were enriched in cell organelles such as mitochondria, lysosomes, and endoplasmic reticulum (p ≤ 0.030). The majority of the differentially regulated genes were involved in oxidative phosphorylation, apoptosis, mitochondrial dysfunction, endoplasmic reticulum stress, antigen processing and presentation, B-cell-receptor signaling, and oxidative stress (p ≤ 0.003). Validation of selected genes involved in apoptosis and oxidative phosphorylation was performed by quantitative real-time polymerase chain reaction (PCR). Electron microscopy and confocal imaging of cystinotic renal proximal tubular epithelial cells further confirmed anomalies in the cellular organelles and pathways identified by microarray analysis. Further analysis of these genes and pathways may offer critical insights into the clinical spectrum of cystinosis patients and ultimately lead to novel links for targeted therapy.

p62/SQSTM1 prominently accumulates in renal proximal tubules in nephropathic cystinosis

BackgroundNephropathic cystinosis, a lysosomal storage disorder, is associated with generalized proximal tubular dysfunction and progressive renal failure. The underlying molecular and cellular mechanisms leading to renal tubular injury remain largely unknown. Abnormal induction of autophagy has been shown in cystinosis. We have studied the autophagic flux in cystinosis by evaluating autophagy-specific substrates.MethodsLC3 and p62 expression was evaluated by (1) immunohistochemistry performed on kidney biopsies obtained from four nephropathic cystinosis patients, four patients with renal injury due to causes other than cystinosis, and four normal kidney tissues and (2) fluorescence imaging in cultured renal proximal tubular epithelial (RPTE) cells obtained from four nephropathic cystinosis patients and two lots of normal primary RPTE cells, both in basal and starvation conditions. p62 expression was also corroborated by western blot analysis in RPTE cells.ResultsThere was a significant buildup of p62 protein in patients with nephropathic cystinosis, specifically in the proximal tubules in kidney biopsies and RPTE cells (p = 0.0004), and the accumulation was further enhanced upon starvation. Cystinotic RPTE cells exhibited a significant co-localization of p62 with LC3.ConclusionsOur findings indicate a potential block in the autophagic flux in cystinosis, thus providing key insights into the underlying mechanisms of tubular injury in cystinosis.

A Five-Gene Peripheral Blood Diagnostic Test for Acute Rejection in Renal Transplantation

Monitoring of renal graft status through peripheral blood (PB) rather than invasive biopsy is important as it will lessen the risk of infection and other stresses, while reducing the costs of rejection diagnosis. Blood gene biomarker panels were discovered by microarrays at a single center and subsequently validated and cross‐validated by QPCR in the NIH SNSO1 randomized study from 12 US pediatric transplant programs. A total of 367 unique human PB samples, each paired with a graft biopsy for centralized, blinded phenotype classification, were analyzed (115 acute rejection (AR), 180 stable and 72 other causes of graft injury). Of the differentially expressed genes by microarray, Q‐PCR analysis of a five gene‐set (DUSP1, PBEF1, PSEN1, MAPK9 and NKTR) classified AR with high accuracy. A logistic regression model was built on independent training‐set (n = 47) and validated on independent test‐set (n = 198)samples, discriminating AR from STA with 91% sensitivity and 94% specificity and AR from all other non‐AR phenotypes with 91% sensitivity and 90% specificity. The 5‐gene set can diagnose AR potentially avoiding the need for invasive renal biopsy. These data support the conduct of a prospective study to validate the clinical predictive utility of this diagnostic tool.