Human nephrosclerosis triggers a hypoxia-related glomerulopathy.

In the kidney, hypoxia contributes to tubulointerstitial fibrosis, but little is known about its implications for glomerular damage and glomerulosclerosis. Chronic hypoxia was hypothesized to be involved in nephrosclerosis (NSC) or "hypertensive nephropathy." In the present study genome-wide expression data from microdissected glomeruli were studied to examine the role of hypoxia in glomerulosclerosis of human NSC. Functional annotation analysis revealed prominent regulation of hypoxia-associated biological processes in NSC, including angiogenesis, fibrosis, and inflammation. Glomerular expression levels of a majority of genes regulated by the hypoxia-inducible factors (HIFs) were significantly altered in NSC. Among these HIF targets, chemokine C-X-C motif receptor 4 (CXCR4) was prominently induced. Glomerular CXCR4 mRNA induction was confirmed by quantitative RT-PCR in an independent cohort with NSC but not in those with other glomerulopathies. By immunohistological analysis, CXCR4 showed enhanced positivity in podocytes in NSC biopsy specimens. This CXCR4 positivity was associated with nuclear localization of HIF1alpha only in podocytes of NSC, indicating transcriptional activity of HIF. As the CXCR4 ligand CXCL12/SDF-1 is constitutively expressed in podocytes, autocrine signaling may contribute to NSC. In addition, a blocking CXCR4 antibody caused significant inhibition of wound closure by podocytes in an in vitro scratch assay. These data support a role for CXCR4/CXCL12 in human NSC and indicate that hypoxia not only is involved in tubulointerstitial fibrosis but also contributes to glomerular damage in NSC.

Viral RNA and DNA trigger common antiviral responses in mesangial cells.

Extrarenal viral infections commonly trigger glomerulonephritis, usually in association with immune complex disease. The Ig component of immune complexes can activate glomerular cell Fc receptors, but whether complexed viral nucleic acids contribute to glomerular inflammation remains unknown. Because of the types of Toll-like receptors (Tlrs) expressed by glomerular mesangial cells, we hypothesized that viral single-stranded RNA and DNA would activate mesangial cells via Tlr-independent pathways and trigger overlapping antiviral immune responses. Consistent with this hypothesis, 5'-triphosphate RNA (3P-RNA) and non-CpG DNA activated murine primary glomerular mesangial cells to secrete Cxcl10 and Il-6 even in cells derived from mice deficient in the Tlr adaptor proteins Myd88 and Trif. Transcriptome analysis revealed that 3P-RNA and non-CpG-DNA triggered almost identical gene expression programs, especially the proinflammatory cytokine Il-6, several chemokines, and genes related to type I IFN. We observed similar findings in glomerular preparations after injecting 3P-RNA and non-CpG-DNA in vivo. These effects depended on the formation of complexes with cationic lipids, which enhanced nucleic acid uptake into the cytosol of mesangial cells. Small interfering RNA studies revealed that 3P-RNA recognition involves Rig-1, whereas non-CpG-DNA did not require Rig-1 or Dai to activate glomerular mesangial cells. We conclude that 3P-RNA and double-stranded DNA trigger a common, TLR-independent, antiviral response in glomerular mesangial cells, which may promote glomerulonephritis in the setting of viral infection.

Culture-induced changes in blood-brain barrier transcriptome: implications for amino-acid transporters in vivo.

Tight homeostatic control of brain amino acids (AA) depends on transport by solute carrier family proteins expressed by the blood-brain barrier (BBB) microvascular endothelial cells (BMEC). To characterize the mouse BMEC transcriptome and probe culture-induced changes, microarray analyses of platelet endothelial cell adhesion molecule-1-positive (PECAM1(+)) endothelial cells (ppMBMECs) were compared with primary MBMECs (pMBMEC) cultured in the presence or absence of glial cells and with b.End5 endothelioma cell line. Selected cell marker and AA transporter mRNA levels were further verified by reverse transcription real-time PCR. Regardless of glial coculture, expression of a large subset of genes was strongly altered by a brief culture step. This is consistent with the known dependence of BMECs on in vivo interactions to maintain physiologic functions, for example, tight barrier formation, and their consequent dedifferentiation in culture. Seven (4F2hc, Lat1, Taut, Snat3, Snat5, Xpct, and Cat1) of nine AA transporter mRNAs highly expressed in freshly isolated ppMBMECs were strongly downregulated for all cultures and two (Snat2 and Eaat3) were variably regulated. In contrast, five AA transporter mRNAs with low expression in ppMBMECs, including y(+)Lat2, xCT, and Snat1, were upregulated by culture. We hypothesized that the AA transporters highly expressed in ppMBMECs and downregulated in culture have a major in vivo function for BBB transendothelial transport.

Transcript-specific expression profiles derived from sequence-based analysis of standard microarrays.

BACKGROUND: Alternative mRNA processing mechanisms lead to multiple transcripts (i.e. splice isoforms) of a given gene which may have distinct biological functions. Microarrays like Affymetrix GeneChips measure mRNA expression of genes using sets of nucleotide probes. Until recently probe sets were not designed for transcript specificity. Nevertheless, the re-analysis of established microarray data using newly defined transcript-specific probe sets may provide information about expression levels of specific transcripts. METHODOLOGY/PRINCIPAL FINDINGS: In the present study alignment of probe sequences of the Affymetrix microarray HG-U133A with Ensembl transcript sequences was performed to define transcript-specific probe sets. Out of a total of 247,965 perfect match probes, 95,008 were designated "transcript-specific", i.e. showing complete sequence alignment, no cross-hybridization, and transcript-, not only gene-specificity. These probes were grouped into 7,941 transcript-specific probe sets and 15,619 gene-specific probe sets, respectively. The former were used to differentiate 445 alternative transcripts of 215 genes. For selected transcripts, predicted by this analysis to be differentially expressed in the human kidney, confirmatory real-time RT-PCR experiments were performed. First, the expression of two specific transcripts of the genes PPM1A (PP2CA_HUMAN and P35813) and PLG (PLMN_HUMAN and Q5TEH5) in human kidneys was determined by the transcript-specific array analysis and confirmed by real-time RT-PCR. Secondly, disease-specific differential expression of single transcripts of PLG and ABCA1 (ABCA1_HUMAN and Q5VYS0_HUMAN) was computed from the available array data sets and confirmed by transcript-specific real-time RT-PCR. CONCLUSIONS: Transcript-specific analysis of microarray experiments can be employed to study gene-regulation on the transcript level using conventional microarray data. In this study, predictions based on sufficient probe set size and fold-change are confirmed by independent means.

Improved elucidation of biological processes linked to diabetic nephropathy by single probe-based microarray data analysis.

BACKGROUND: Diabetic nephropathy (DN) is a complex and chronic metabolic disease that evolves into a progressive fibrosing renal disorder. Effective transcriptomic profiling of slowly evolving disease processes such as DN can be problematic. The changes that occur are often subtle and can escape detection by conventional oligonucleotide DNA array analyses. METHODOLOGY/PRINCIPAL FINDINGS: We examined microdissected human renal tissue with or without DN using Affymetrix oligonucleotide microarrays (HG-U133A) by standard Robust Multi-array Analysis (RMA). Subsequent gene ontology analysis by Database for Annotation, Visualization and Integrated Discovery (DAVID) showed limited detection of biological processes previously identified as central mechanisms in the development of DN (e.g. inflammation and angiogenesis). This apparent lack of sensitivity may be associated with the gene-oriented averaging of oligonucleotide probe signals, as this includes signals from cross-hybridizing probes and gene annotation that is based on out of date genomic data. We then examined the same CEL file data using a different methodology to determine how well it could correlate transcriptomic data with observed biology. ChipInspector (CI) is based on single probe analysis and de novo gene annotation that bypasses probe set definitions. Both methods, RMA and CI, used at default settings yielded comparable numbers of differentially regulated genes. However, when verified by RT-PCR, the single probe based analysis demonstrated reduced background noise with enhanced sensitivity and fewer false positives. CONCLUSIONS/SIGNIFICANCE: Using a single probe based analysis approach with de novo gene annotation allowed an improved representation of the biological processes linked to the development and progression of DN. The improved analysis was exemplified by the detection of Wnt signaling pathway activation in DN, a process not previously reported to be involved in this disease.