Uroscopy in the 21st century: high-field NMR spectroscopy

From the experiments described, it can be seen that there are different research approaches that can be taken and these are summarized in Table 1. Whereas much scientific research is principally hypothesis led, there remains, nevertheless, an important place for exploratory research. High resolution NMR can measure, directly and simultaneously, a wide range of endogenous metabolites in biological fluids and has the unique capability of providing structural information on the metabolites detected. It has proved to be a powerful research tool with which to study inherited metabolic diseases, renal disease, drug metabolism, and toxicity, and can be used to monitor the effects of drug therapy. For instance, by using a library of experimental toxins one can map the metabolic profile of site-specific nephron injury. With this approach in man one could eventually take an unknown disease such as Balkan nephropathy and predict the initial site of tubular injury, the mode of injury and therefore the kind of toxin capable of producing that injury. NMR spectroscopic techniques are still advancing rapidly, with ever increasing sensitivity and sophistication of NMR pulse sequences to enhance structural elucidation in complex mixtures. Given the advances in directly coupled HPLC-NMR and even HPLC-NMR-mass spectroscopy it is likely that these technologies in conjunction with pattern recognition will make major contribution to our understanding of renal processes and provide new diagnostic insights in the 21st century.      

Activation of guinea pig eosinophil respiratory burst by leukotriene B4: role of protein kinase C

Leukotriene B4 (LTB4) and the protein kinase C activator, 4-beta-phorbol dibutyrate (PDBu), both induced a pronounced and concentration-dependent stimulation of hydrogen peroxide (H2O2) generation by purified guinea pig peritoneal eosinophils in the concentration range 1 nM-1 microM. The LTB4 response was inhibited competitively by the specific LTB4 receptor antagonist, U-75302, with a KB of 25 nM, while the concentration-response curves for both stimuli were shifted rightwards (3.8-fold and 2.8-fold for LTB4 and PDBu, respectively) by the competitive protein kinase C inhibitor, 1-O-hexadecyl-2-O-methylglycerol at a concentration of 300 microM. LTB4 appears, therefore, to induce respiratory burst in eosinophils via a receptor-mediated mechanism involving protein kinase C.     

TAP genes and immunity

The transporter associated with antigen processing (TAP) is a member of the ATP-binding cassette transporter family that specializes in delivering cytosolic peptides to class I molecules in the endoplasmic reticulum. The TAP is a major target of genetic alteration in tumours and disruption by viral inhibitors. In some species, TAP genes have co-evolved with MHC class I molecules to deliver peptides that are customised for particular alleles. In humans, MHC class I polymorphism determines the level of tapasin-mediated association with TAP and subsequent peptide optimisation within the peptide-loading complex (PLC). MHC class I molecules that still load peptides without complexing to the TAP might be more resistant to viral interference of the PLC and less sensitive to competition for TAP by other class I allotypes.     

Localization of a gene for otosclerosis to chromosome 15q25-q26

Among white adults otosclerosis is the single most common cause of hearing impairment. Although the genetics of this disease are controversial, the majority of studies indicate autosomal dominant inheritance with reduced penetrance. We studied a large multi- generational family in which otosclerosis has been inherited in an autosomal dominant pattern. Five of16 affected persons have surgically confirmed otosclerosis; the remaining nine have a conductive hearing loss but have not undergone corrective surgery. To locate the disease- causing gene we completed genetic linkage analysis using short tandem repeat polymorphisms (STRPs) distributed over the entire genome. Multipoint linkage analysis showed that only one genomic region, on chromosome 15q, generated a lod score >2.0. Additional STRPs were typed in this area, resulting in a lod score of 3.4. STRPs FES (centromeric) and D15S657 (telomeric) flank the 14. 5 cM region that contains an otosclerosis gene.      

X-linked liver glycogenosis type II (XLG II) is caused by mutations in PHKA2, the gene encoding the liver alpha subunit of phosphorylase kinase

X-linked liver glycogenosis type II (XLG II) is a recently described X- linked liver glycogen storage disease, mainly characterized by enlarged liver and growth retardation. These clinical symptoms are very similar to those of XLG I. In contrast to XLG I patients, however, XLG II patients do not show an in vitro enzymatic deficiency of phosphorylase kinase (PHK). Recently, mutations were identified in the gene encoding the liver alpha subunit of PHK (PHKA2) in XLG I patients. We have now studied the PHKA2 gene of four unrelated XLG II patients and identified four different mutations in the open reading frame, including a deletion of three nucleotides, an insertion of six nucleotides and two missense mutations. These results indicate that XLG II is due to mutations in PHKA2. In contrast to XLG I, XLG II is caused by mutations that lead to minor structural abnormalities in the primary structure of the liver alpha subunit of PHK. These mutations are found in a conserved RXX(X)T motif, resembling known phosphorylation sites that might be involved in the regulation of PHK. These findings might explain why the in vitro PHK enzymatic activity is not deficient in XLG II, whereas it is in XLG I.      

A distinctive glomerular lesion complicating placental site trophoblastic tumor: report of two cases

Renal disease with distinctive pathologic features developed in two young women who had placental site trophoblastic tumors. The renal abnormalities were manifested by proteinuria in both cases and by hematuria in one case; blood pressure was elevated in one of the patients. Pathologic examination of the kidneys showed distinctive glomerular abnormalities, characterized mainly by the presence of occlusive eosinophilic deposits in many of the glomerular capillary lumina, most of which stained for fibrinogen-related antigens and IgM by immunohistochemical techniques. Ultrastructural examination showed the deposits to consist mainly of granular material that contained packets of fibrillar material with the appearance of fibrin. The uterine tumors were composed of mononucleated and multinucleated cells with abundant cytoplasm that infiltrated between the muscle bundles of the myometrium; in both tumors there was prominent deposition of eosinophilic material that had the tinctorial properties of fibrin and that stained for fibrinogen and IgM in immunoperoxidase studies. The renal abnormalities disappeared after hysterectomy in one case; the other patient, who was receiving chemotherapy and had disseminated intravascular coagulation, died with leukopenia and sepsis. The clinical and pathologic features in these cases suggest that the renal abnormalities were related to the uterine tumors and that the production of immune complexes and/or the activation of intravascular coagulation by the tumors were pathogenetic mechanisms.     

Detection of two forms of GP330. Their role in Heymann nephritis.

Heymann nephritis is characterized by glomerular immune deposits that contain a glycoprotein called gp330. The deposits are believed to result from shedding of immune complexes formed on podocytes. Complexes are also shed from proximal tubule cells, when antibodies combine with gp330 on the cell surface. We performed the present study to investigate what portion of the gp330 molecule is shed, using a rabbit antiserum against a peptide deduced to be in the cytoplasmic domain of gp330, as well as a rabbit antiserum and two monoclonal antibodies that recognize extracellular epitopes of gp330. The anti-cytoplasmic peptide antiserum precipitated from Fx1A (a crude renal cortical membrane preparation), a protein with a mass of about 440 kd that was reactive with two monoclonal anti-gp330 antibodies. (In our experiments, the protein called gp330 generally has a mass estimated to be about 440 kd.) The anti-cytoplasmic peptide antiserum also reacted with a truncated gp330 protein produced in transfected COS cells. Immunohistochemical studies showed that all the antibodies recognized the same group of epithelial cells. However, as seen in immunoultrastructural studies of proximal tubules, the anti-cytoplasmic peptide antiserum reacted only with components at the base of microvilli, whereas the anti-gp330 ectodomain antibodies identified material not only at the base, but over the surface of microvilli as well. In rats with Heymann nephritis, glomerular deposits and material shed into tubule lumens reacted with antibodies against extracellular epitopes of gp330, but not with the anti-cytoplasmic peptide antiserum. We propose that there are two forms of gp330 on the cell surface of proximal renal tubules. One form is restricted to coated pit regions at the base of microvilli and has a cytoplasmic domain containing a sequence deduced from a partial complementary DNA encoding gp330. The other form is present over microvilli (and possibly at the base of microvilli as well) and lacks the cytoplasmic domain deduced from the complementary DNA. The complexes that are shed in Heymann nephritis contain either a portion of gp330 cleaved from the full-length molecule or a form of gp330 that lacks the cytoplasmic domain.