Genomewide expression profiles of rat model renal isografts from brain dead donors.

BACKGROUND: It has been well documented that two factors, brain death (BD) and ischemia/reperfusion (I/R) injury, have distinct but overlapping adverse influences on the clinical outcome of renal transplantation. METHOD: We previously established a rat model of renal isografting from brain dead donors. In the present study, we performed genomic expression profiling with a high-density oligonucleotide microarray to identify genes that were upregulated or downregulated by BD and/or I/R injury. RESULTS: Among a total of 20,550 genes, most of those upregulated by BD were genes for adhesion molecules and cytokines or for chemokines such as Gro1 and IP-10. When overexpression of these genes was assessed by real-time reverse transcriptase-polymerase chain reaction, it was only observed one hr after the engraftment of kidneys from BD donors and returned to baseline thereafter, indicating the presence of an acute systemic inflammatory response to BD. Analysis of biologic networks demonstrated the activation of specific pathways that were clearly different for BD and I/R injury. The p53 and NFkappaB pathway was involved in the acute response to BD, whereas the Myc, Jun, and c-fos pathway was involved in I/R injury. Investigation of secretory protein genes identified LCN2 and SPP1 as candidate genes for biologic markers. CONCLUSION: Because our experimental system is a good model of renal transplantation from brain dead or living human donors, our data may be useful for elucidating the pathologic processes involved and for identification of novel markers for graft dysfunction of renal transplantation.

Expression analysis of a mouse orthologue of HSFY, a candidate for the azoospermic factor on the human Y chromosome.

Heat shock transcription factor on Y (HSFY) is located in one of three candidate regions for azoospermic factor (AZF), AZFb on the Y chromosome. We and others have already revealed that some azoospermic males are missing the regions of the Y chromosome including HSFY. Previously, we showed that murine HSFY-like sequence [mHSFYL (Riken cDNA 4933413G11Rik)], which is the mouse orthologue of HSFY, is exclusively expressed in testis. The sequences encoding the presumed DNA-binding domain in HSFY and mHSFYL were found in other mammals such as dogs, cows and chickens. To elucidate mHSFYL expression in the testes in detail, we carried out in situ hybridization. mHSFYL was predominantly expressed in round spermatids. Furthermore, we clarified the intracellular distribution of mHSFYL in COS1 cells with HA- or GFP-tagged proteins. Both HA-mHSFYL and GFP-mHSFYL were located in the nucleus. Our results suggest that HSFY/mHSFYL may have evolutionarily conserved functions for spermatogenesis.

Palindromic AT-rich repeat in the NF1 gene is hypervariable in humans and evolutionarily conserved in primates.

Palindromic sequences are dispersed in the human genome and may cause chromosomal translocations in humans. They constitute unsequenced gaps in the human genome because of their resistance to PCR amplification, cloning into vectors, and sequencing. We have overcome these difficulties by using a combination of optimized PCR conditions, cloning in a recombination-deficient E. coli strain, and RNA polymerases in sequencing. Using these methods, we analyzed a palindromic AT-rich repeat (PATRR) in the neurofibromatosis type 1 (NF1) gene on chromosome 17 (17PATRR). The 17PATRR manifests a size polymorphism due to a highly variable length of (AT)(n) dinucleotide repeats within the PATRR. 17PATRRs can be categorized into two types: a longer one that comprises a nearly or completely perfect palindrome, and a shorter one that represents its deleted asymmetric derivative. In vitro analysis shows that the longer 17PATRR is more likely to form a cruciform structure than the shorter one. Two reported t(17;22)(q11;q11) patients with NF1, whose breakpoints were identified within the 17PATRR, have translocations that are derived from perfect or nearly perfect palindromic alleles. This implies that the symmetric structure of a PATRR can induce a translocation. We identified conserved PATRRs within the NF1 gene in great apes and similar inverted repeats in two Old World monkeys, but not in New World monkeys or other mammals. This indicates that the palindromic region appeared approximately 25 million years ago and elongated during primate evolution. Although such palindromic regions are usually unstable and disappear rapidly due to deletion, the 17PATRR in the NF1 gene was stably conserved during evolution for reasons that are still unknown.

Global gene expression profiling of renal scarring in a rat model of pyelonephritis.

Renal scarring is a serious complication of chronic pyelonephritis that occurs due to vesicoureteral reflux. In our study, we performed global expression profiling of the kidney during renal scarring formation in a rat pyelonephritis model. An inoculum of Escherichia coli was injected directly into the renal cortex. Histologically, renal scarring developed during the 3-to-4 week period after injection. The time-course expression profile of 18,442 genes was then analyzed using microarrays, followed by validation with real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Most of the genes found to be up-regulated during renal scarring are associated with immune and defense responses, including cytokines, chemokines and their receptors, complement factors, adhesion molecules and extracellular matrix proteins. These genes were up-regulated as early as 1 week after injection, when no fibrotic changes were yet evident, peaked at 2 weeks, and gradually decreased thereafter. However, a subset of cytokine genes was found to be persistently activated even at 6 weeks after injection, including interleukin (IL)-1beta, transforming growth factor (TGF)-beta, and IL-3. Further statistical analysis indicated that the pathways mediated by these cytokines are activated concomitantly with renal scarring formation. The products of these genes may thus potentially be novel non-invasive diagnostic or prognostic biomarkers of renal scarring.

Genetic variation affects de novo translocation frequency.

Translocation is one of the most frequently occurring human chromosomal aberrations. The constitutional t(11;22)(q23;q11), which is the only known recurrent non-Robertsonian translocation, represents a good model for studying translocations in humans. Here we demonstrate polymorphisms of the palindromic sequence at the t(11;22) breakpoint that affect the frequency of de novo translocations in sperm from normal males. A typical allele consists of a perfect palindrome, producing ~10-5 de novo t(11;22) translocations. Alleles with an asymmetric center do not form the t(11;22). Our data show the importance of genome sequence on chromosomal rearrangements, a class of human mutation that is thought to be random.