Altered Chaperone and Protein Turnover Regulators Expression in Cultured Skin Fibroblasts from Type 1 Diabetes Mellitus with Nephropathy Paolo Tessari,*,† Lucia Puricelli,† Elisabetta Iori,† Giorgio Arrigoni,‡,§ Monica Vedovato,† Peter James,‡ Anna Coracina,† and Renato Millioni† Department of Clinical and Experimental Medicine, Chair of Metabolism, University of Padova, Padova, Italy, ProteinTechnology Wallenberglab, Lund, Sweden, and Department of Biological Chemistry, University of Padova, Padova, Italy Received August 30, 2006
In type-1 diabetes mellitus (T1DM) with diabetic nephropathy (DN), accumulation of abnormal proteins in the kidney and other tissues may derive from constitutive alterations of intracellular protein recognition, assembly, and turnover. We characterized the proteins involved in these functions in cultured skin fibroblasts from long-term T1DM patients with [DN+] or without [DN-] nephropathy but similar metabolic control, and from matched healthy subjects. 2-D gel electrophoresis and MS-MALDI analysis were employed. The [DN+] T1DM patients, compared with the two other groups, exhibited increased abundance of a high-molecular weight isoform of protein disulphide-isomerase A3 and a decrease of two low-molecular weight isoforms. They also had increased levels of heat shock protein (HSP) 60 kDa isoform #A4, of HSP71 kDa isoform #A30, and of HSP27 kDa isoform #6, whereas the HSP27 kDa isoforms #A90 and #A71 were decreased. Cathepsin beta-2 (#40), the cation-independent mannose 6-phosphate receptor binding protein 1 (CIMPR) (#A27), and annexin 2 (#A9) were also decreased in the [DN+] T1DM patients, whereas the RNA-binding protein regulatory subunity (#38) and the translationally-controlled tumor protein (TCTP) (#A45) were increased. These changes of chaperone-like proteins in fibroblasts may highlight those of the kidney and be patho-physiologically related to the development of nephropathy in T1DM. Keywords: proteomics • fibroblasts • diabetic nephropathy • protein folding • chaperones
Introduction Diabetic nephropathy (DN) is a chronic microvascular complication of type 1 diabetes mellitus (T1DM) occurring in ≈40% of patients during their lifetime.1 Not only environmental but also genetic factors are involved in DN, because patients with similar disease duration, metabolic control, and exposure to other environmental factors may or may not develop DN.2,3 Nevertheless, the causative gene(s) of DN remain elusive. Should DN be genetically determined,4,5 such a predisposition will likely be expressed as specific phenotypes detectable only in cells and tissues of T1DM subjects with DN. Onset, progression, and chronicization of DN are associated with molecular and metabolic phenomena and with wellcharacterized renal structural and functional changes.6,7 In advanced stages of the disease, thickening of the glomerular basal membranes, mesangial matrix accumulation, increase in the size of proximal tubuli, and alterations of the podocytes are commonly found in the kidney.6 Proteinuria and/or de* To whom correspondence should be addressed. Prof. Paolo Tessari, Dipartimento di Medicina Clinica e Sperimentale, Universita` di Padova, via Giustiniani 2, 35128 Padova, Italy. Phone, +39- 0498211748; Fax, +390498754179; E-mail,
[email protected]. † Department of Clinical and Experimental Medicine. ‡ ProteinTechnology Wallenberglab. § Department of Biological Chemistry.
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Journal of Proteome Research 2007, 6, 976-986
Published on Web 01/18/2007
creased glomerular kidney filtration can develop.8 These changes are likely the result of altered protein expression and/or turnover, which could be themselves genetically determined. The expression of altered proteins in cells may be controlled and/or limited by systems that can recognize and remove abnormal molecules, regulate protein assembly, spatial arrangement, and folding.9,10 Thus, an abnormal protein profile may result not only from an altered protein expression per se but also from the failure by regulatory proteins to remove and/ or reassemble proteins with altered structure and/or folding. These regulatory proteins are generically named chaperones,11 and they include heat-shock proteins (HSP), as well as proteins that play a role in intramolecular thiol linkages, RNA expression, proteolysis, signal transduction, and cell communication.12-14 Skin fibroblasts are widely used as an in vitro model to study alterations constitutively expressed also in other body cells; therefore, they represent a useful model to study genetically linked phenotypes associated to the pathogenesis of a disease, such as the development of chronic diabetic complications.2,3,15 Therefore, the aim of this study was to characterize, in cultured skin fibroblasts from long-term T1DM patients without 10.1021/pr060443n CCC: $37.00
2007 American Chemical Society
research articles
Chaperone-Like Protein Changes in Diabetic Nephropathy
Table 1. Clinical Features of T1DM Patients With and Without Nephropathy and Normal Control Subjectsa
no. (male/female) age (yr) body mass index (kg/m2) mean blood pressure (mmHg) serum creatinine (µmol/L) (range) duration of diabetes (yr) glycated hemeoglobin (%) albumin excretion rate (µg/min) (range) a
normal control subjects
T1DM patients without nephropathy
T1DM patients with nephropathy
2/3 37 ( 4.6 22.9 ( 1.2 93.3 ( 10 86 ( 9 (79-94) -
2/3 39.4 ( 5 24.1 ( 0.4 95.2 ( 2.7 85 ( 6 (70-92) 22.8 ( 3.2 9.3 ( 0.8 9.9 (0-11.3)
2/3 35.6 ( 3.8 22.2 ( 1.2 99.1 ( 6.8 347 ( 160 (73-860) 21.8 ( 4.5 10.5 ( 1.3 662.5 (625-922)
Values are means ( SEM, except for albumin excretion rate, which is given as median and range.
and with established DN, the abundance of proteins involved in the regulation of intracellular protein recognition, assembly, and turnover, using a proteomic approach.
Methods Subjects. Five long-term T1DM patients with diabetic nephropathy, defined as a urinary albumin excretion rate (AER) of >200 µg/min in sterile urine, five T1DM patients without nephropathy (AER:
