SELDI-TOF mass spectrometry-based protein profiling of kidney tissue.

Protein profiling has numerous applications in renal research including the detection of protein biomarkers with aberrant expression levels during disease development. Such information is essential for early diagnosis and will aid the improvement of patient management and minimise the progression of disease. Further to this, data generated from these studies will assist the elucidation of the precise mechanisms of disease development and can lead to the discovery of potential drug targets. Surface enhanced laser desorption/ionisation time of flight mass spectrometry (SELDI-TOF MS), is emerging as a popular profiling tool for such studies. It incorporates the methods of solid-phase chromatography and TOF-MS in a single platform. This chapter provides a guide for establishing kidney profiling experiments using SELDI-TOF MS and will cover the following topics: 1) preparation of tissue extracts; 2) array processing, including optimisation of conditions for biomarker discovery; and 3) data acquisition/analysis.

Recombinant expression, purification and characterisation of the HMG domain of human SRY.

The HMG domain is a DNA binding and bending 'architectural' motif involved in chromatin re-modelling during transcription. Recombinant SRY HMG domain protein, 88 amino acids in length, has been produced in E. coli. Using FPLC and a stirred ultra-filtration cell, this domain has been purified to homogeneity and concentrated to yield milligram quantities. Functional characterisation studies of the pure, concentrated SRY HMG domain show the recombinantly expressed protein to be active in terms of DNA binding and calmodulin binding activities.

Evaluation of relaxin's antifibrotic action by SELDI-TOF mass spectrometry-based profiling of relaxin knockout mice, a model of progressive fibrosis.

Surface-enhanced laser desorption ionization-time-of-flight mass spectrometry was employed to identify potential biomarkers for early-onset fibrosis. These biomarkers were then used to evaluate the efficacy of relaxin to reverse or ameliorate the development of the condition.

Aberrant protein expression in plasma and kidney tissue during experimental obstructive nephropathy.

Kidney failure is a major health problem worldwide. Patients with end-stage renal disease require intensive medical support by dialysis or kidney transplantation. Current methods for diagnosis of kidney disease are either invasive or insensitive, and renal function may decline by as much as 50% before it can be detected using current techniques. The goal of this study was, therefore, to identify biomarkers of kidney disease (associated with renal fibrosis) that can be used for the development of a non-invasive clinical test for early disease detection. We utilized two protein-profiling technologies (SELDI-TOF MS and 2-D) to screen the plasma and kidney proteome for aberrantly expressed proteins in an experimental mouse model of unilateral uretric obstruction, which mimics the pathology of human renal disease. Several differentially regulated proteins were detected at the plasma level of day-3-obstructed animals, which included serum amyloid A1, fibrinogen α, haptoglobin precursor protein, haptoglobin and major urinary proteins 11 and 8. Differentially expressed proteins detected at the tissue level included ras-like activator protein 2, haptoglobin precursor protein, malate dehydrogenase, α enolase and murine urinary protein (all p<0.05 versus controls). Immunohistochemistry was used to confirm the up-regulation of fibrinogen. Interestingly, these proteins are largely separated into four major classes: (i) acute-phase reactants (ii) cell-signaling molecules (iii) molecules involved in cell growth and metabolism and (iv) urinary proteins. These results provide new insights into the pathology of obstructive nephropathy and may facilitate the development of specific assay(s) to detect and monitor renal fibrosis.

Restricted, but abundant, expression of the novel rat gene-3 (R3) relaxin in the dorsal tegmental region of brain.

Relaxin is a peptide hormone with known actions associated with female reproductive physiology, but it has also been identified in the brain. Only one relaxin gene had been characterized in rodents until recently when a novel human relaxin gene, human gene-3 (H3) and its mouse equivalent (M3) were identified. The current study reports the identification of a rat homologue, rat gene-3 (R3) relaxin that is highly expressed in a discrete region of the adult brain. The full R3 relaxin cDNA was generated using RT-PCR and 3' and 5' RACE protocols. The derived amino acid sequence of R3 relaxin retains all the characteristic features of a relaxin peptide and has a high degree of homology with H3 and M3 relaxin. The distribution of R3 relaxin mRNA in adult rat brain was determined and highly abundant expression was only detected in neurons of the ventromedial dorsal tegmental nucleus (vmDTg) in the pons, whereas all other brain areas were unlabelled or contained much lower mRNA levels. Relaxin binding sites and relaxin immunoreactivity were also detected in the vmDTg. These together with earlier findings provide strong evidence for a role(s) for multiple relaxin peptides as neurotransmitters and/or modulators in the rat CNS.

Purification and characterization of relaxin from the tammar wallaby (Macropus eugenii): bioactivity and expression in the corpus luteum.

The objective of this study was to isolate and purify prorelaxin or mature relaxin from the tammar wallaby corpus luteum (CL), determine their structure and bioactivity, and test the hypothesis that enzymatic cleavage of prorelaxin occurs in late gestation. Tammar relaxin peptides were extracted from pooled corpora lutea of late pregnant tammars using a combination of HPLC methods, and they were identified using Western blotting with a human (H2) relaxin antisera and matrix-assisted laser desorption ionization time of flight mass spectrometry. Although no prorelaxin was identified, multiple 6-kDa peptides were detected, which corresponded to the predicted mature tammar relaxin amino acid sequence, with an A chain of 24 amino acids, and different B chain lengths of 28, 29, 30, and 32 amino acids. Tammar relaxin bound with high affinity to rat cortical relaxin receptors and stimulated cAMP production in the human monocytic cell line, THP-1, which expresses the relaxin receptor. Analysis of individual CL indicated that equivalent amounts of mature relaxin peptides were present throughout gestation and also in unmated tammars at equivalent stages of the luteal phase in the nonpregnant cycle. Immunoreactive relaxin was localized specifically to the luteal cells of the CL and the intensity of immunostaining did not vary between gestational stages. These data show that the CL of both pregnant and unmated tammar wallabies produces mature relaxin and suggests that relaxin expression in this species is not influenced by the conceptus. Moreover, the presence of mature relaxin throughout gestation implies that prohormone cleavage is not limited to the later stages of pregnancy