Susanne Eder

Neuroendocrine Secretory Protein 55

Abstract: The chromogranins constitute a class of acidic proteins comprising the structurally related chromogranins A and B and secretogranin II. These proteins are widely distributed in endocrine and nervous tissues; they are localized to the large dense core vesicles and released from them after stimulation of cells. In all the tissues examined chromogranins are proteolytically processed into small peptides, some of which have defined physiological activities. Chromogranin A plays a key role in large dense core vesicle biogenesis and can induce the formation of the regulated pathway. We have recently cloned neuroendocrine secretory protein 55 (NESP55), a protein that shares several features with the class of chromogranins. NESP55 is a soluble, acidic, heat‐stable secretory protein that is expressed exclusively in endocrine and nervous tissues, although less widely than chromogranins. NESP55 is genomically imprinted and transcribed only from the maternal allele. It is proteolytically processed in some tissues into the small octapeptide GAIPIRRH located at the C terminus of NESP55. In the brain NESP55 is found in cell bodies and axons but not in terminals. At the subcellular level NESP55 is localized to a large vesicle, which is anterogradely transported by the fast axonal flow in neurons. From this vesicle NESP55 is constitutively released. However, in some tissues like the adrenal, medulla, and bovine splenic nerve, NESP55 is also found in the large dense transmitter storage organelles. Thus, NESP55 represents a novel peptidergic marker for a large constitutively secreting vesicle pool found in the central and peripheral nervous system.

Neuroendocrine Secretory Protein 55 (NESP55): Alternative Splicing onto Transcripts of the GNAS Gene and Posttranslational Processing of a Maternally Expressed Protein

Recent studies established a novel genomically imprinted gene located 45 kb upstream of the human GNAS1 locus. This locus encoded for the Neuroendocrine Secretory Protein with an apparent molecular weight of 55,000 (NESP55), which is transcribed exclusively from the maternal allele. We sequenced rat and human NESP55 and investigated tissue-specific splicing of its mRNA and posttranslational modifications of the protein in various tissues. Alternative mRNA splicing of NESP55 was analyzed by sequencing of cDNA clones, RT-PCR and Northern blotting. Two main splice variants, which were generated in a tissue-specific manner, were identified: The open reading frame encoding NESP55 was spliced onto exons 2–13 of Gsα in the adrenal medulla, pituitary and the brain. In addition, in the pituitary a second shorter, prominent mRNA transcript was generated by splicing of NESP55 onto exons 2, 3 and N1 of Gsα. Several of the cDNA clones isolated contained inverted repeats of 50–150 bp at their 5′ or 3′ termini, which might form hairpin stems and thus alter mRNA stability. The NESP55 open reading frame encoded a hydrophilic protein of 28,018 Da (human) and 29,218 Da (rat), respectively, which resembled the class of acidic, neuroendocrine secretory proteins collectively called chromogranins. NESP55 is highly conserved among mammalian species. It is posttranslationally acidified by the addition of keratan sulfate glycosaminoglycan chains and differentially processed by endopeptidases in various endocrine and neuronal tissues.

Secretion and Molecular Forms of NESP55, a Novel Genomically Imprinted Neuroendocrine-Specific Protein from AtT-20 Cells

NESP55 (neuroendocrine secretory protein of Mr 55,000) is a paternally imprinted proteoglycan, expressed specifically in endocrine cells and the nervous system. We investigated the subcellular localization and secretion of NESP55 in AtT-20 cells. NESP55 accumulated in the medium linearly over 24 h exceeding its intracellular content 3.7-fold by that time. Incubation of cells at 16°C, to block protein export, inhibited basal secretion by 79%. Stimulation of AtT-20 cells with 8-Br-cAMP increased secretion of NESP55 by only 45%. The NESP55 secretory vesicles sedimented at a density of 1.2–1.4 M, which is slightly lighter than that of the large dense core vesicles. Immunofluorescence studies revealed immunoreactivity in the Golgi apparatus and a punctuate staining of processes or neurites. Our data demonstrate that NESP55 is mainly sorted to and released from a population of constitutive secretory vesicles, which are transported out of the perikarya into processes or axons. In addition, some NESP55 is also routed to the regulated pathway. The signal peptide of NESP55, as determined with peptide antisera, is 46 amino acids long and represents the best conserved region of this molecule suggesting that the signal peptide may have a function of its own. The subcellular localization and export of NESP55 from cells are reminiscent of neuronal proteoglycans forming the extracellular matrix, which are implicated in the development and maintenance of neuronal circuits and mechanisms of axonal guidance.

Increased Renal Versican Expression Is Associated with Progression of Chronic Kidney Disease

Novel prognostic markers for progression of kidney disease are needed to distinguish patients who might benefit from a more aggressive nephroprotective therapy. Expression of the proteoglycan versican was evaluated in renal transcriptomics profiles and in an independent set of 74 renal biopsies. Versican levels were correlated to histologic damage scores and to renal outcome, and versican expression and regulation was evaluated in vitro. In transcriptomics profiles of renal tissue versican was positively correlated with (i) histological parameters in kidney biopsies, (ii) progressive decline of renal function in proteinuric kidney diseases, and (iii) impaired renal function and histology scores in diabetic nephropathy. In an independent cohort of 74 biopsies of glomerular diseases renal RNA levels of versican isoforms V0 and V1, but not V2 and V3 correlated significantly with creatinine after a mean follow up time of 53 months. Versican isoforms V0 and V1 together with serum creatinine at time of biopsy and the degree of glomerulosclerosis predicted 20% and 24% of the variability of creatinine at follow up, which was significantly more than serum creatinine and histological parameters alone (16%). However, when patients with acute kidney failure at time of biopsy (n = 5) were excluded, the additive predictive value of versican V1 was only marginally higher (35%) than creatinine and glomerulosclerosis alone (34%). Versican isoforms V0 and V1 were primarily expressed in vitro in proximal tubule cells and in fibroblasts. The results in humans were confirmed in three rodent models of kidney disease, in which renal versican expression was significantly upregulated as compared to corresponding controls. These data show for the first time an association of renal versican isoform V0 and V1 expression with progressive renal disease.

Metallothioneins and renal ageing

BACKGROUND Human lifespan is increasing continuously and about one-third of the population >70 years of age suffers from chronic kidney disease. The pathophysiology of the loss of renal function with ageing is unclear. METHODS We determined age-associated gene expression changes in zero-hour biopsies of deceased donor kidneys without laboratory signs of impaired renal function, defined as a last serum creatinine >0.96 mg/dL in females and >1.18 mg/dL in males, using microarray technology and the Significance Analysis of Microarrays routine. Expression changes of selected genes were confirmed by quantitative polymerase chain reaction and in situ hybridization and immunohistochemistry for localization of respective mRNA and protein. Functional aspects were examined in vitro. RESULTS Donors were classified into three age groups (<40, 40-59 and >59 years; Groups 1, 2 and 3, respectively). In Group 3 especially, genes encoding for metallothionein (MT) isoforms were more significantly expressed when compared with Group 1; localization studies revealed predominant staining in renal proximal tubular cells. RPTEC/TERT1 cells overexpressing MT2A were less susceptible towards cadmium chloride-induced cytotoxicity and hypoxia-induced apoptosis, both models for increased generation of reactive oxygen species. CONCLUSIONS Increased expression of MTs in the kidney with ageing might be a protective mechanism against increased oxidative stress, which is closely related to the ageing process. Our findings indicate that MTs are functionally involved in the pathophysiology of ageing-related processes.

Guidelines and clinical practice at the primary level of healthcare in patients with type 2 diabetes mellitus with and without kidney disease in five European countries

Background: The number of patients with type 2 diabetes mellitus and diabetes mellitus–associated chronic kidney disease varies considerably between countries. Next to differences in genetic as well as life style risk factors, varying practices in medical care delivery might cause this diversity. Method: The PROVALID study recruited 4000 patients with type 2 diabetes mellitus at the primary level of healthcare in five European countries (Austria, Hungary, The Netherlands, Poland and Scotland). Baseline data were used to describe patient characteristics and compare the adherence to ADA (American Diabetes Association) and KDIGO (Kidney Disease: Improving Global Outcomes) guidelines with respect to metabolic and blood pressure control, use of renin–angiotensin system–blocking agents, statins and acetylsalicylic acid between the countries. Results: About 34.8% of the population had evidence of diabetes mellitus–associated chronic kidney disease. The median HbA1c level of the cohort was 6.8% (ranging from 6.5 in Poland to 7.0% in Scotland). Mean blood pressure was 136/79 (±17/10) and significantly higher in subjects with elevated albuminuria. These individuals also were more often treated with renin–angiotensin system–blocking agents (74.1% vs 84.6%), whereas the use of statins was driven by cardiovascular comorbidity. Acetylsalicylic acid was used in only 28.9% subjects. Despite similar cardiovascular comorbidities and renal function, the use of renin–angiotensin system–blocking agents varied significantly between the countries from 66.7% to 87.4%. An even higher variability was observed for patients >40 years of age using statins (39.8%–82.7%) and administration of acetylsalicylic acid in patients older than 50 years (5.2%–43.8%). Conclusion: Our study shows that medical practice in type 2 diabetes mellitus patients with and without renal disease is different in European countries. Longitudinal follow-up will reveal if this diversity affects clinical endpoints.