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Glycosylation Patterns of Kidney Proteins Differ in Rat Diabetic

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Glycosylation Patterns of Kidney Proteins Differ in Rat Diabetic http://www.kidney-international.org basic research & 2015 International Society of Nephrology Glycosylation patterns of kidney proteins differ in rat diabetic nephropathy Alessandra Ravida`1, Luca Musante1, Marjut Kreivi1, Ilkka Miinalainen1, Barry Byrne1, Mayank Saraswat1, Michael Henry2, Paula Meleady2, Martin Clynes2 and Harry Holthofer1 1Centre for BioAnalytical Sciences, Dublin City University, Dublin, Ireland and 2National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland Diabetic nephropathy often progresses to end-stage kidney Diabetes mellitus is one of the leading noncommunicable disease and, ultimately, to renal replacement therapy. chronic diseases, with more than 340 million people affected Hyperglycemia per se is expected to have a direct impact on worldwide.1 The high level of blood glucose caused by either the biosynthesis of N- and O-linked glycoproteins. This study insulin deficiency (type 1 diabetes, ‘juvenile’) or insulin aims to establish the link between protein glycosylation and resistance (type 2 diabetes, ‘adult onset’) may lead, over time, progression of experimental diabetic kidney disease using to an array of complications, especially of the cardiovascular orthogonal methods. Kidneys of streptozotocin-diabetic and system.2 Diabetic kidney damage (diabetic nephropathy, DN) control rats were harvested at three different time points is one of the most burdensome of diabetic complications, post streptozotocin injection. A panel of 12 plant lectins was which, in a substantial segment of patients, leads to end-stage used in the screening of lectin blots. The lectins UEAI, PHA-E, kidney disease with consequent renal replacement, i.e., GSI, PNA, and RCA identified remarkable disease-associated dialysis and kidney transplantation, as the only therapeutic differences in glycoprotein expression. Lectin affinity options.3 The presence of minute amounts of albumin in the chromatography followed by mass spectrometric analyses urine (microalbuminuria) is regarded as an early sign of led to the identification of several glycoproteins involved declining filtration function of the kidneys,4 but in many in salt-handling, angiogenesis, and extracellular matrix regards it is neither a specific nor a predictive marker of DN.5 degradation. Our data confirm a substantial link between Leakage of proteins, such as albumin, into the urine glycosylation signature and diabetes progression. (proteinuria) implies an ongoing impairment of the kidney Furthermore, as suggested by our findings on dipeptidyl filtration function. The gaps in understanding the peptidase-IV, altered protein glycosylation may reflect pathophysiology of worsening kidney function associated changes in biochemical properties such as enzymatic activity. with diabetes calls for new perspectives in discovery research Thus, our study demonstrates the unexplored potential of to provide novel diagnostic markers and therapy targets. protein glycosylation analysis in the discovery of molecules From a biochemical point of view, high circulating levels linked to diabetic kidney disease. of glucose, inaccessible to many cell types because of insulin Kidney International advance online publication, 14 January 2015; deficiency or resistance, are bound to alter the physiological doi:10.1038/ki.2014.387 equilibrium of many enzyme-driven, as well as direct, KEYWORDS: chronic kidney disease; diabetes; proteomic analysis chemical reactions. Thus, excess of circulating reducing sugars appears as the driving force for the generation of advanced glycation end products via the nonenzymatic Maillard reaction.6 Furthermore, in diabetes, high levels of glucose enter the enzymatically driven hexosamine bio- synthetic pathway, leading to excess of circulating N-acetyl- glucosamine (GlcNAc) and subsequent protein O-GlcNAc modifications.7 A great amount of work has been dedicated to unravel the role of proteoglycans of the extracellular matrix, such as heparan sulfate (HS), in charge-selective permeability of the glomerular basement membrane.8 In particular, alterations of HS side chains of agrin, the major Correspondence: Harry Holthofer, Centre for BioAnalytical Sciences, Faculty HSPG in the glomerular basement membrane, have been of Science and Health, Dublin City University, Glasnevin, Dublin 9, Ireland. reported in a number of renal pathologies. Notably, a E-mail: [email protected] decrease of HS (but not agrin) and inverse correlation Received 3 February 2014; revised 2 October 2014; accepted 9 October between HS staining in the glomerular basement membrane 2014 and proteinuria has been reported in DN.9 Decrease of HS Kidney International 1 basic research A Ravida` et al.: Glycosylation patterns in diabetic nephropathy was found to be because of depolymerization by reactive further biochemical investigations, suggesting a potential oxygen species, hyperglycemia-driven downregulation of HS functional link between changes in protein glycosylation, synthesis,10 and, mostly, by increased glomerular expression biochemical properties, and enzymatic activity. of the HS-degrading enzyme heparanase.11 Interestingly, a In the light of the findings, this study introduces the use of number of reports highlight that alteration of HS expression, simple lectin assays as a new approach to biomarker mining sulphation, and/or structural changes are not found as initial and therapeutic target discovery. events in DN, but rather correlate with proteinuria in 12,13 advanced stages of the disease. RESULTS Much less is known of the direct impact of hyperglycemia Screening of rat kidney cortex lysates by lectin blots: lectins on glycan biosynthetic processes such as N- and O-linked efficiently detect STZ-induced differential glycosylation glyco-decoration of cellular proteins during the early onset of The focus of this work was to study the initial stages of the disease. diabetic complications of the kidney at three different time Lectins are ‘carbohydrate-binding and cell-agglutinating points (2, 5, and 8 weeks post STZ injection) with a specific proteins of nonimmune origin’, with binding specificity for interest in the cortical kidney region. Clinical parameters of terminal or subterminal carbohydrate residues of glycan side the rats used in this study are reported in Supplementary chains of both glycoproteins and glycolipids.14 The use of Material Figure S1. Lectin blots were carried out on pools of lectins and, in particular, the widely available plant lectins to cortex lysates from three to five animals per treatment and explore glycoconjugate profiles of clinical specimens and time point. A total of 12 commercially available biotinylated experimental models has been developed in the 1970s and plant lectins were used and detected with infrared (IR)- perfected throughout the following decades.15 In the infancy labeled streptavidin. The glycan-binding specificities of the of glycobiology, they have been used by us and others to lectins used have been recently investigated by the Con- reveal altered glycosylation profiles associated with diabetic sortium for Functional Glycomics using glycan microarrays24 damage of the kidney in human subjects and in experimental and are summarized in Table 1. models of diabetes.16–19 The Coomassie-stained sodium dodecyl sulfate (SDS)- Streptozotocin (STZ) is a selective toxin that targets polyacrylamide gel electrophoresis (PAGE) in Figure 1a fails the insulin-producing pancreatic b-cells in the rat and to reveal appreciable differences in the protein profile is a well-established method to induce type 1 diabetes between treated and control animals, with the exception of in rodent models.20 Recently, several proteomics-based an apparent 14-kDa protein band highly expressed in the STZ studies have investigated the differential expression of 5-week (5 w) kidney. The lectins Pisum sativum agglutinin, proteins in the kidney of rats with STZ-induced diabetes.21–23 PHA-L, Sophora japonica agglutinin, wheat-germ agglutinin In this study, kidney cortex samples of STZ-diabetic rats (WGA), and succinylated WGA (sWGA) showed unremark- were analyzed to establish a connection between disease able differences in the specific banding patterns (Supplemen- progression and altered protein glycosylation. Notably, Ulex tary Material Figure S2), suggesting that the glycoepitopes europaeus agglutinin (UEAI), Phaseolus vulgaris agglutinin E detected by these lectins are not appreciably altered in the (PHA-E), Griffonia simplicifolia (GSI), Ricinus communis kidney cortex during early disease progression. agglutinin I (RCA120), and peanut agglutinin (PNA) Instead, the fucose (Fuc)-binding UEAI, interestingly, identified remarkable disease-associated differences in glyco- reveals a distinct banding at approximately 140 kDa, which is protein expression. Lectin affinity chromatography (LAC) strongly present in 5 w and 8-week (8 w) samples (Figure 1b). followed by mass spectrometric (MS) analyses led to the In Figure 1c, the lectin PHA-E, with binding specificity to identification of a panel of target glycoproteins. Among biantennary N-glycans with bisecting GlcNAc, recognizes these, dipeptidyl peptidase-IV (DPP4) was selected for preferentially the high-molecular-weight proteins in the Table 1 | Lectin origin and nominal glycan-binding specificities according to CFG glycan array public data Abbreviation Origin Nominal binding specificity PSA Pisum sativum GlcNAc; LacNAc UEA Ulex europaeus Fuca1–2LacNAc PHA-E Phaseolus vulgaris Biantennary complex N-glycans
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