http://www.kidney-international.org basic research & 2015 International Society of Nephrology

Glycosylation patterns of kidney 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 . This study insulin deficiency (type 1 diabetes, ‘juvenile’) or insulin aims to establish the link between 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 was which, in a substantial segment of patients, leads to end-stage used in the screening of 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 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

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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 .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 PHA-L Phaseolus vulgaris Tri- and tetra-antennary complex oligosaccharides SJA Sophora japonica b-GalNAc; b-Gal ConA Concanavalia ensiformis a-Man branched and terminal WGA Triticum vulgaris GlcNAc; GalNAc; Neu5Ac sWGA (succinylated WGA) Triticum vulgaris GlcNAc PNA Arachis hypogea Galb1–3GalNAca-Thr/Ser Jacalin Artocarpus integrifolia Galb1–3GalNAca-Thr/Ser; GalNAc-a-Thr/Ser GSI Griffonia simplicifolia a-GalNAc; GalNAca-Thr/Ser; a-Gal RCA120 Ricinus communis LacNAc Abbreviations: Fuc, fucose; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; Gal, (34); LacNAc, N-acetyllactosamine; Neu5Ac, N-acetylneuraminic acid.

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STZCTRL STZ CTRL STZ CTRL STZ CTRL STZ CTRL STZ CTRL STZ CTRL STZ CTRL STZ CTRL STZ CTRL STZ CTRL STZ CTRL 2w 2w 5w 5w 8w 8w 2w 2w 5w 5w8w 8w 2w 2w 5w 5w8w 8w 250 2w 2w 5w 5w 8w 8w 250 250 150 150 150 250 100 100 100 75 75 75 150 50 50 50 100 32 75 32 32 25 25 25 20 20 20 50 15 15 15 MW MW MW MW MW MW 32 UEA/Actin PHA-E/Actin GSI/Actin

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Normalized I.I count 100 Normalized I.I count 5 1 0 0 0 2w 5w 8w 2w 5w 8w 2w 5w 8w Figure 1 | Lectins efficiently detect streptozotocin (STZ)-induced differential glycosylation. Coomassie staining (a) and western blots (b–d) of pools of five rat kidney cortex lysates per treatment and time point: STZ 2 w; CTRL 2 w; STZ 5 w; CTRL 5 w; STZ 8 w; CTRL 8 w. Double staining was performed in the western blots (b–d) probing the same membrane with biotinylated lectin (red) and anti-actin (green). Differentially expressed glycoproteins are indicated with arrows. Equal amounts of total protein were loaded in each lane. In panels e–g, actin-normalized expression levels of selected protein bands from each lectin blot are displayed. Data represent one of three independent experiments. 2 w, 2 weeks; 5 w, 5 weeks; 8 w, 8 weeks; CTRL, citrate control; MW, molecular weight; PHA-E, Phaseolus vulgaris; UEA, Ulex europaeus. controls than in the STZ-treated samples at all time points. In SDS-PAGE and subsequently stained by colloidal Coomassie, Figure 1d, the preferentially O-glycan-binding lectin GSI the protein patterns of both bound fractions were remarkably binds to the 250- and 150-kDa protein bands in the control more complex than what appeared in the lectin blots but not in the treated samples. (Figure 2f). The major 180-kDa protein band of the PNA- The actin-normalized quantification of the protein bound fraction together with the 100- and 150-kDa RCA- domains mentioned is presented in panels E–G of Figure 1. bound protein bands were excised from the Coomassie- stained gel and proteins identified using MS.

RCA120 and PNA lectins reveal remarkable disease-associated changes in the glycoprofiles of the kidney cortex and MS identification of selected glycoproteins allow the isolation of differentially expressed glycoproteins Tryptic peptides derived from the overnight in-gel digestion by LAC of the excised gel plugs were separated by reversed-phase In the RCA blot (Figure 2a), a lower degree of general 120 high-performance liquid chromatography and analyzed by glycosylation in STZ-treated mice is visibly observed in linear trap quadrupole MS. Protein identification was protein bands of approximately 150, 100, and 85 kDa, conducted by searching the tandem MS data against the respectively. Disease-associated differences in the expression UniProtKB/Swissprot protein database (Rattus species) using of the 150-kDa band are more evident in 2 w and 8 w MASCOT search engine. The full list of proteins identified samples. In the case of lower-molecular-weight bands, a from the bands excised from the Coomassie-stained gels is general decrease in signature glycosylation pattern appeared reported in Table 2. Despite the identification of several to progress with the onset of the disease. Similarly, in panel b potential marker candidates, DPP4 was shortlisted for further (Figure 2), the O-glycan-specific PNA also reveals a investigations owing to its strong implications in diabetes remarkable change in expression of the 180-kDa protein and known localization in the kidney.25 band, strongly expressed in 5 w and 8 w control samples (CTRL). As previously described, the quantification of the actin-normalized IR intensity of the described protein bands RCA120 binds to tubular glycoproteins and clusters is presented in Figure 2c, d. As DPP4 was isolated by RCA120 affinity chromatography, we To isolate and identify the proteins yielding the charac- further investigated the localization of RCA120-binding teristic reduction of RCA120 and PNA binding to diabetic epitopes in the kidney by lectin fluorescence. This was kidney cortex samples, LAC was performed using PNA- and performed on frozen sections of 2 w and 5 w kidney cortex RCA120-conjugated agarose beads. When analyzed by lectin probed with fluorescein-labeled RCA120 (green) and with blots, the PNA-bound fraction appeared to be principally DAPI (4’,6-diamidino-2-phenylindole) for nuclear staining composed by an approximately 180-kDa protein band, (blue, Figure 3). RCA120 appears to detect mostly the luminal whereas the RCA120 blot presented two major bands of and basolateral aspects of tubuli. No apparent differences in approximately 150 and 100 kDa (Figure 2e), respectively. the tissue-binding pattern of the lactose-specific lectin can be When a greater amount of total protein was loaded on the observed between STZ-treated and control samples.

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Figure 2 | Ricinus communis agglutinin I (RCA120) and peanut agglutinin (PNA) lectins reveal remarkable disease-associated changes in the glycoprofiles of the kidney cortex and allow the isolation of differentially expressed glycoproteins by lectin affinity chromatography. RCA (a) and PNA (b) western blots of kidney cortex lysates of streptozotocin (STZ)-treated and control rats at 2-, 5-, and 8-week post-injection time points. Differentially expressed glycoproteins are indicated with arrows. Actin-normalized expression levels of selected protein bands from each lectin blot are displayed in panels c and d. Affinity chromatography of 5 and 2 w CTRL samples was performed with PNA- and RCA-agarose beads, respectively. The efficiency of the chromatographic separation was assessed by lectin blot (e) and Coomassie staining of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) of bound fractions (f). Double staining was performed in all western blots (a, b and e) probing the same membrane with biotinylated lectin (red) and anti-actin (green). 2 w, 2 weeks; 5 w, 5 weeks; 8 w, 8 weeks; CTRL, citrate control; MW, molecular weight.

DPP4 expression is upregulated in STZ-treated rat kidney time points DPP4 was more evenly distributed between cortex glomeruli and tubuli. DPP4 expression levels were validated by western blotting. Figure 4 depicts DPP4 blots of single animal kidney cortex DPP4 is glyco-decorated with RCA120-binding epitopes, lysates at 2, 5, and 8 w after induction of diabetes. DPP4 whose expression levels are reduced in STZ diabetes in 2 w protein expression levels (Figure 4d) appeared to be kidney cortex lysates upregulated in STZ-treated animals. Interestingly, the most The glycosylation status of the target protein DPP4 was significant difference in DPP4 expression between STZ- assessed by immunoprecipitation and lectin/western blots treated and control animals is observed at the 2 w time point (Figures 6 and 7). The immunoprecipitations carried out on (Po0.05), with a higher inter-animal variability being pooled 2 w STZ and CTRL kidney cortex lysates using either observed in later stages of the disease. At the 5 w time point, a commercial anti-DPP4 or RCA120 confirmed (1) the an appreciable but not statistically significant (P ¼ 0.054) validity of the proprietary anti-DPP4 (gift of prof H. upregulation of DPP4 is observed in diabetic samples. Kawachi; Figure 6c); (2) that DPP4 carries RCA120-binding epitopes (Figure 6d); and (3) that the main 100-kDa band DPP4 is localized in the glomeruli and at the brush border of recognized and affinity-purified by RCA120 in Figures 2e and proximal convoluted tubuli f is indeed DPP4 (Figures 6e and f). With these studies, it was Frozen kidney cortex sections were probed with anti-DPP4, impossible to quantitatively compare the levels of DPP4 whose fluorescence signal was obtained by the use of TRITC- glycosylation in the immunoprecipitation-bound fractions of conjugated secondary antibody (in red in Figure 5), whereas STZ vs. CTRL. To overcome this issue, western/lectin blots nuclear staining was visualized (in blue) by DAPI. No evident from single animals at the 2 w time point were probed differences in DPP4 distribution were observed. DPP4 was simultaneously with RCA120 and DPP4 (Figure 7). Actin- less predominant in glomeruli at the 2 w time point in both normalized IR intensities of DPP4 and RCA120 expression treated and control samples (panels a and d), whereas in later levels (Figure 7d) were plotted as ratio in Figure SM3,

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Table 2 | List and bibliographic analysis of selected protein bands from PNA and RCA120 affinity chromatography––bound fractions and identified by MS analysis using MASCOT search engine Excised SwissProt Implication in Glycosylation band accession Gene MW MASCOT diabetes studies LAC (kDa) Protein name number name (kDa) Score (Reference) (Reference) PNA 180 Low-density lipoprotein receptor–related protein 2 precursor (Megalin; P98158 Lrp2 519 2061 61 62 gp330) Aminopeptidase N (Kidney Zn peptidase; CD13 antigen) P15684 Anpep 109 362 63 64 Mucin and cadherin–like protein precursor (Mu-protocadherin; GP100) Q9JIK1 Cdhr5 91 331 — 65 Glutamyl aminopeptidase (Aminopeptidase A) P50123 Enpep 108 247 66 67 Sodium/potassium-transporting ATPase alpha-1 chain precursor P06685 Atp1a1 113 146 68 — (Sodium pump 1) (Na( þ )/K( þ )ATPase1) RCA 150 Aminopeptidase N ((Kidney Zn peptidase; CD13 antigen) P15684 Anpep 109 2480 63 64 Glutamyl aminopeptidase (Aminopeptidase A) P50123 Enpep 108 905 66 — Dipeptidyl peptidase 4 (T-cell activation antigen CD26; GP110 glycoprotein) P14740 Dpp4 88 330 25 48–52 Neutral and basic amino acid transport protein rBAT Q64319 Slc3a1 79 138 — — Sodium/potassium-transporting ATPase alpha-1 chain precursor (Sodium P06685 Atp1a1 113 128 68 — pump 1) (Na( þ )/K( þ )ATPase1) 100 Dipeptidyl peptidase 4; T-cell activation antigen CD26; GP110 glycoprotein P14740 Dpp4 88 2109 25 48–52 Glutamate carboxypeptidase 2 P70627 Folh1 85 276 69 70 Neprilysin; Neutral endopeptidase; CD10 antigen P07861 Mme 86 239 54,71 72,73 Neutral and basic amino acid transport protein rBAT Q64319 Slc3a1 79 198 — — Alpha-1-inhibitor 3 precursor P14046 A1i3 164 198 74 75 Murinoglobulin-2 precursor Q6IE52 Mug2 162 172 — — Aminopeptidase N; Kidney Zn peptidase; CD13 antigen P15684 Anpep 109 156 63 64 Meprin A subunit alpha precursor Endopeptidase-2; MEP-1 Q64230 Mep1a 85 217 76 77,78

Abbreviations: LAC, lectin affinity chromatography; MS, mass spectrometric analysis; MW, molecular weight; PNA, peanut agglutinin; RCA, Ricinus communis;RCA120, Ricinus communis agglutinin I.

DPP4 enzymatic activity is suppressed by STZ treatment Enzymatic activity was assessed by measuring the fluorescent intensity of the hydrolysis products of the DPP4 sub- strate H-Gly-Pro-b-naphthylamide. DPP4 activity appears to increase in an age-dependent manner both in STZ-treated and control animals, with the highest activity levels registered in 8 w samples (Figure 8a). STZ treatment resulted in a statistically significant suppression of enzymatic activity at CTRL 2 W CTRL 5 W the 2-week time point (P ¼ 0.018).

Neprilysin enzymatic activity is reduced in STZ-treated animals To verify whether reduced enzymatic activity is specific to DPP4 or is observed also for other membrane peptidases, neprilysin (NEP) enzymatic activity was assessed. As for DPP4, the fluorescent intensity of the hydrolysis products of the NEP substrate N-Dansyl-D-Ala-Gly-pNOH2-Phe-Gly STZ 2 W STZ 5 W ([D]AG(pN)PG) was measured. STZ-treated samples exhib- ited lower activity levels than age-matched healthy controls at Figure 3 | Ricinus communis agglutinin I (RCA120) binds to all time points (Figure 8b). In the diabetic kidney cortex, glomerular and tubular glycoproteins. Lectin fluorescence micrographs of frozen kidney cortex sections of streptozotocin NEP activity levels increased in an age-dependent manner (STZ)-treated and control rats at 2 and 5 w post infection probed with despite remaining significantly lower than the healthy fluorescein-labeled RCA (green) and 4’,6-diamidino-2-phenylindole controls at all time points. (DAPI) nuclear staining (blue). Bar ¼ 100 mm. 2 w, 2 weeks; 5 w, 5 weeks; CTRL, citrate control. DISCUSSION Glycosylation is one of the most common post-translational modifications of proteins, found on more than half of revealing a statistically significant trend. This analysis con- the proteins encoded in eukaryotic genomes.26 Despite the firmed the original hypothesis—i.e., changes in the glycosy- fact that recent investigations have generously reinforced lation of DPP4 do occur in the early onset of STZ diabetes the role of glycosylation as a post-translational modification in rat kidney cortices. for healthy kidney function,27–34 there have been limited

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Figure 4 | Dipeptidyl peptidase-IV (DPP4) expression is upregulated in streptozotocin (STZ)-treated rat kidney cortex. DPP4 (red) and actin (green) western blots of rat kidney cortex lysates of individual cortex samples (a–c). Equal amounts of total protein were loaded in each lane. Infrared (IR)-intensity analysis of DPP4 expression levels were normalized against actin (d). Data are represented as the mean±s.e.m. of one of two independent experiments. ***Pp0.0001 compared with the STZ-treated group. 2 w, 2 weeks; 5 w, 5 weeks; 8 w, 8 weeks; CTRL, citrate control; MW, molecular weight; NS, non significant. systematic studies on the potential of glycan changes in Interestingly, enhanced fucosylation levels of alpha-1-acid diabetic kidney disease progression. glycoprotein and other serum glycoproteins have been In this study, 5 out of 12 lectins used in the initial blot previously observed in type 1 diabetes patients35 and in db/ screening revealed remarkable alterations of the samples’ db mice and humans with type 2 diabetes.36 glycoprofile during disease progression (Figures 1 and 2). It LAC fractionation, followed by MS analysis of the RCA120- has to be noted that in this study whole kidney cortex lysates bound 100- and 150-kDa and PNA-bound 180-kDa protein were screened. Isolation of glomerular- and tubular-enriched bands revealed an intriguing list of glycoproteins with, in fractions may reveal additional profile alterations even with most cases, known implications in diabetes and a documen- lectins that, in this study, proved to be inefficient. In contrast ted link between protein activity and glycosylation (see to our expectations, diabetes onset appeared to result in a Table 2). lower binding signature, with the exception of UEAI. PHA-E, Interestingly, some of the identified glycoproteins, namely RCA, GSI, and PNA lectins bind preferentially to terminal aminopeptidase N, aminopeptidase A, and Meprin A are glucose/N-acetylglucosamine (Glc/GlcNAc) and galactose/N- involved in angiotensin signaling, tubule sodium handling, acetylgalactosamine (Gal/GalNAc) residues on N- and O- and in matrix degradation, all processes notoriously impli- linked glycans. The decrease in the lectin binding to these cated in diabetic complications of the kidney, thus provi- glycoepitopes can be attributed either to a decrease in the ding scope for further investigations. DPP4 was identified protein backbone expression or to specific glycan modifica- from the RCA120 100-kDa band with 36% sequence coverage tion such as abnormal capping of the glycan chains with and 20 unique peptides. Because of its established implica- terminal sialic acids or branching monosaccharides such as tion in diabetes, DPP4 was shortlisted as the most interesting Fuc. The latter suggestion is supported by the binding profile candidate for further investigations.37,38 obtained with the Fuc-binding UEA-I. This lectin exhibits DPP4 is a homodimeric serine peptidase that is widely high-affinity binding toward 2’-fucosyl-N-acetyllactosamine expressed on the surface of a variety of epithelial, endo- structures,24 and in our blots a notable increase in fucosylated thelial, and lymphoid cells, and that circulates as secreted glycoprotein binding is observed for high-molecular-weight form in plasma.39 In recent years, DPP4 inhibitors including protein bands in the 5- and 8-week STZ samples (Figure 1). sitagliptin and vildagliptin have been approved for

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2W 5W 8W STZ CTRL

Proximal CT Collecting Treatment Week Glomeruli Tubuli Brush border/ Distal CT duct lumen Basolateral

CTRL 2 ** ** ** – – – CTRL 5 *** ** ** – – NA CTRL 8 *** ** ** – – – STZ 2 ** ** ** * * – STZ 5 *** ** ** – – NA STZ 8 *** ** ** – – NA Figure 5 | Dipeptidyl peptidase-IV (DPP4) is localized mainly in the glomeruli and at the brush border of the proximal convoluted tubuli. Immunofluorescence micrographs of streptozotocin (STZ)-treated (a–c) and control (d–f) rat kidney cortex sections probed with DPP4 (red) and 4’,6-diamidino-2-phenylindole (DAPI) nuclear staining (blue). Bar ¼ 50 mm. (g) Visual scoring of 10 slides per treatment and time point. 2 w, 2 weeks; 5 w, 5 weeks; 8 w, 8 weeks; CTRL, citrate control; NA, not available.

therapeutic use for the treatment of diabetes type 2. They protein backbone are crucial in the maintenance of podocyte exert their glucose-regulatory action by inhibiting the slit pore integrity and functional glomerular filtration.45,46 degradation of the incretine hormone, glucagon-like- Removal of sialic acid leads to rapid podocyte foot efface- peptide, by DPP4.40 ment and proteinuria in mice.47 Both RCA120 and DPP4 immunofluorescence analyses The absence of RCA120-binding epitopes in the glomeruli (Figures 3 and 5), however, failed for an accurate topological reveals a potential limitation for the sole use of this particular identification of tissue sites more strongly affected by the lectin in future studies if glomerular proteins have also to be disease onset. Notably, despite the presence of DPP4 on both targeted. As glomerular RCA120-binding glycans could be tubular and glomerular aspects of the kidney, RCA120 masked by the presence of terminal sialic acid, fluorescent micrographs reveal a binding predominance for digestion of microscopy slides before RCA120 fluorescent tubular regions. A majority of all glomerular proteins are staining or parallel use of sialic acid–binding lectins could be glycosylated, and pioneering studies have shown that their used to unveil glomerular epitopes. glycosylation is crucial for the functionality at this site. In this study, semiquantitative western blot analysis of Accordingly, nephrin, the key structural-functional molecule DPP4 expression levels revealed an increase in STZ-diabetic of the glomerular filtration barrier, is heavily glycosylated,41 rats with the early stages of disease, revealing the most and experimental impairment of the podocyte glycosylation significant differences between diabetic and control samples machinery has proven to have a marked effect on the cellular (Figure 4). This is in agreement with the data of Kirino et al. localization of this protein.28 Interestingly, N-glycosylation showing Dpp4 mRNA expression to be elevated in the kidneys has a pivotal role for the tight regulation of also other of STZ-treated rats when compared with healthy controls.25 slit diaphragm components including the transient receptor Double-staining blots performed with anti-DPP4 and RCA120 potential channel 6, which mediates uptake of cations in (Figure 7) cleared the discrepancy between the trend observed 42,43 podocytes. The most explored glomerular molecule in the RCA120 and DPP4 blots, confirming a diabetes- studied for glycosylation and function is podocalyxin.44 associated alteration of DPP4 glycosylation at the early stages The strong negative charges owing to abundant sialic acid of the disease onset. Notably, DPP4 is heavily glycosylated with and sulfate moieties on the N- and O-glycans decorating the nine potential N-glycosylation sites in humans, mouse, and

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Ponceau Anti-DPP4(1) Figure 6 | Dipeptidyl peptidase-IV (DPP4) immunoprecipitation and Ricinus communis (RCA) pull-down of 2 w kidney cortex lysates confirms the presence of Ricinus communis agglutinin I (RCA120)-binding epitopes on DPP4. Pools of three rat kidney cortex lysates per treatment at the 2 w time point were either immunoprecipitated (IP) using a commercial anti-DPP4 antibody, anti-DPP4(1) (a–d), or affinity- purified by RCA chromatography (e and f). Starting material (streptozotocin (STZ)/citrate control (CTRL)), bound (STZ B/CTRL B), and flow-through (FT) fractions were analyzed by western/lectin blots. To assess the quality of the pull-downs, membranes were probed with Ponceau staining (a and e). IP efficiency was validated by probing the membranes with commercial and proprietary anti-DPP4 (red; B and C, respectively). Double staining was performed by probing membrane C with biotinylated RCA120lectin (green). RCA120 pull-down fractions were probed with commercial anti-DPP4 (green, f). Equal volumes of elution fractions were loaded; in all other lanes, 5 mg of total protein was loaded. Data represent one of two independent experiments.

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50 50 50 Anti-DPP4 37 37 25 25 50 20 20 37

RCAAnti-actin RCA/DPP4 merge

Figure 7 | Streptozotocin (STZ) diabetes induces a reduction in the expression of Ricinus communis agglutinin I (RCA120)-binding epitopes on dipeptidyl peptidase-IV (DPP4) in 2 w kidney cortex lysates. RCA120 (green; a and d), commercial anti-DPP4 (red; b and d), or anti-actin (red; c) western blots of rat kidney cortex lysates of individual cortex samples from 2 w STZ-diabetic and control rats. Equal amounts of total protein were loaded in each lane. Data are represented as the mean±s.e.m. of two independent experiments. CTRL, citrate control.

DPP4 activity previous reports.25 As a form of control, enzymatic activity of NS NEP was also assessed in the same experimental settings. 200 NS STZ NEP is responsible for the degradation and inactivation * CTRL 150 of a number of bioactive peptides, such as endothelins, angiotensins I and II, bradykinin, atrial natriuretic peptide, 100 and enkephalins.53 In this study, NEP activity in the kidney 50 cortex was found to be significantly decreased at all time points in diabetic rats compared with healthy controls, in 0 agreement with previous studies by us and others.23,54 2W 5W 8W In conclusion, in this study, plant lectins were shown to detect diabetes-induced dysregulation of protein glyco- NEP activity 40 sylation and in diabetic rat kidney cortex. This STZ ** represents a strong alternative to scouting of new markers **** ** CTRL 30 arising from costly genomics and proteomics approaches. 20 MATERIALS AND METHODS

pmol/min/mg10 pmol/min/mg Animals and DN model Diabetes was induced to 9-week-old male Sprague–Dawley rats 0 (Charles River, Wilmington, MA) by STZ administration, as de- 2W 5W 8W scribed previously.55 Rats were killed at three different time points Figure 8 | Dipeptidyl peptidase-IV (DPP4) and neprilysin (NEP) (2, 5, and 8 weeks) from the end of the pharmacological treatment. enzymatic activities are suppressed by streptozotocin (STZ) Clinical parameters of the rat study are reported in Supplementary treatment. Enzymatic activities of DPP4 (a) and NEP (b) in kidney Material Figure 1. Tissues were immediately dissected, cut to pieces, cortex lysates of STZ-treated and control rats were assessed by snap-frozen in liquid nitrogen, and stored at 80 1C. Kidney cortex measuring the fluorescent intensity of the hydrolysis products of samples were analyzed in this study. H-Gly-Pro-b-naphthylamide and N-Dansyl-D-Ala-Gly-pNOH2-Phe-Gly ([D]AG(pN)PG), respectively. Data are represented as the mean±s.e.m. of three biological replicates in one of three independent experiments. *Pp0.05; **Pp0.01; ****Pp0.0001. 2 w, Quantitative lectin and western blots 2 weeks; 5 w, 5 weeks; 8 w, 8 weeks; NS, non significant. Antibodies used in this study were mouse monoclonal anti-actin antibody (Sigma-Aldrich, St Louis, MO), IRDye 800 CW Goat anti- rat48 and 3 potential O-glycosylation sites.49 Over the years, Mouse IgG and IRDye 680 goat anti-rabbit IgG (Li-COR contrasting reports have addressed the functional link between Biosciences, Lincoln, MA), rabbit polyclonal anti-DPP4 (courtesy of Prof Hiroshi Kawachi, Niigata University, Japan), and mouse DPP4 glycosylation and its enzymatic activity.50–52 Notably, an anti-rat DPP4 antibody (BioLegend, San Diego, CA). Biotinylated interesting parallel between the loss of RCA120-binding lectins used in this study (ConA, GSL 1, Jacalin, LCA, PHA(E), epitopes and decrease in DPP4 enzymatic activity was PHA(L), PNA, Pisum sativum agglutinin, RCA120, Sophora japonica observed in this study at the early stages of the disease onset agglutinin, UEA-1, WGA, succinylated WGA) were all purchased (Figure 8). Despite the enhanced protein levels, a reduced renal from Vector Laboratories (Peterborough, UK). IRDye 680 Strepta- DPP4 enzymatic activity in STZ rats is in agreement with vidin was purchased from Li-COR.

Kidney International 9 basic research A Ravida` et al.: Glycosylation patterns in diabetic nephropathy

Lectin- and immunofluorescence microscopy Science, London, UK). Searches were carried out with trypsin Cryosections (6 mm) of kidney tissues were fixed with ice-cold specificity (one missed cleavage allowed), 0.5 Da for MS and 0.5 Da acetone for 5 min. A minimum of four nonconsecutive slides from for MSMS (oxidations of Methionine and Propionamide Cys were each biological replicate (n ¼ 3) per treatment and time point were set as variable modifications). A MASCOT score 461 was used for both RCA120 and DPP4 staining. Glycan epitopes were considered significant. probed with 10 mg/ml fluorescein-labeled RCA120 (Vector Labora- 2 þ 2 þ tories) solution (in 1% bovine serum albumin in Mg and Ca - DPP4 immunoprecipitation enriched Tris buffer) for 20 min at RT in the presence and absence of Protein-A sepharose (Invitrogen, Camarillo, Ca) beads were inhibiting galactose (0.5 M in enriched Tris buffer). In immuno- incubated with 50 mg of monoclonal mouse anti-rat DPP4 antibody fluorescence experiments, the following antibodies were used: rabbit (BioLegend) in phosphate-buffered saline in ice for 3 h. After polyclonal anti-DPP4 (courtesy of Prof Hiroshi Kawachi) and binding of the antibodies, beads were washed with phosphate- TRITC-conjugated anti-rabbit antibody (Jackson Laboratory, buffered saline. Antibodies were cross-linked to protein-A Sepharose Sacramento, CA). After being mounted with Vectashield anti-fading by the addition of 25 mmol/l dimethyl pimelimidate (Sigma- solution with DAPI (Vector Laboratories), all the specimens were Aldrich) resuspended in 0.2 M triethanolamine, pH 8.2. The viewed with Leica DM IL LED microscope using 10x, 20x, and 40x reaction was performed twice in end-over-end rotation for 30 min HI PLAN I objectives (Leica Microsystems, Wetzlar, Germany) at room temperature in the dark. The remaining reactive amino equipped with an epifluorescent source and a filter system for FITC groups were quenched by the addition of 50 mmol/l ethanolamine and TRITC fluorescence. Images were processed with Adobe in phosphate-buffered saline for 60 min at room temperature. The Photoshop CS4 software (Adobe System, San Jose, CA). antibodies cross-linked to beads were abundantly washed in phosphate-buffered saline and in incubation buffer (10 mmol/l LAC Hepes, pH7.4, 150 mmol/l NaCl, 1 mmol/l CaCl2, 1 mmol/l MgCl2, Kidney cortex lysates from 5 w CTRL and 2 w CTRL were incubated and 1 mmol/l MnCl2). Pools of three rat kidney cortex lysates per 1 with PNA-agarose and RCA120-Agarose slurry (Vector Laboratories), treatment at the 2 w time point were incubated overnight at 4 Cin respectively, in end-over-end agitation overnight at þ 4 1C. end-over-end rotation. After extensive washes with binding buffer, The unbound fraction was collected and, following extensive bound proteins were eluted by Laemmli buffer. washing, columns were incubated for 3 h at room temperature with 2 column volumes of 0.5 M galactose (Sigma-Aldrich), Enzymatic assay (quantification of DPP4 and NEP peptidase and bound fractions were eluted. High concentrations of salts activity) and galactose were removed from both unbound and bound Sample preparation. Frozen kidney cortex samples of three fractions by diafiltration in Amicon Ultra centrifugal filters with rats per treatment and time point were homogenized according to a a 3-KDa cutoff (Millipore Corporation, Billerica, MA, USA). The previously described protocol.58 To enhance sample solubility, 3-[(3- chromatography efficiency was assessed by lectin blot, as described cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) above. was added to the homogenates to a final volume of 1% (v/v) and The bound fraction was separated by SDS-PAGE and visualized samples were vortexed for 24 h at þ 4 1C. After incubation and full 56 by Colloidal Coomassie staining. The Coomassie-stained gel was sample solubilization, CHAPS was removed by dialysis (MWCO acquired by AlphaImager (Alpha Innotech, Santa Clara, CA). From 3.5 kDa) against water. the Coomassie-stained gels, the protein bands at 150, 100 kDa Enzymatic assays. As previously described,59,60 enzymatic (RCA120), and 180 kDa (PNA) were excised and MS analysis was activities were assessed by measuring the fluorescent intensity performed as described below. of the hydrolysis products of H-Gly-Pro-b-naphthylamide and N-Dansyl-D-Ala-Gly-pNOH2-Phe-Gly ([D]AG(pN)PG) (both from MS analysis Sigma-Aldrich), substrates for DPP4 and NEP, respectively. The gel plugs were digested with trypsin according to standard protocols.57 Digested samples were resuspended in 20 ml of 0.1% Statistics TFA in 2% ACN solution and analyzed by nano LC-system All data were analyzed for normality before statistical testing by (Ultimate 3000 system; Dionex, Sunnyvale, CA) coupled to an Prism 6.0 (GraphPad Software, La Jolla, CA) software. In cases in linear trap quadrupole mass spectrometer (Thermo Fisher Scientific, which multiple group comparisons were made, data were analyzed Dublin, Ireland); peptides were eluted with the following binary using one-way analysis of variance. For comparisons between two gradients: solvent A (2% ACN and 0.1% formic acid in LC-MS- groups, the Student’s t test was used. In all tests, Po0.05 was grade water) and 0–65% solvent B (80% ACN and 0.08% formic acid deemed significant. in LC-MS-grade water) for 60 min using a nanoRPC column (PepMap C18, 75 mmid250 mm, 3 mm particle and 100 A˚ pore size DISCLOSURE (Dionex)). The linear trap quadrupole was operated in a data- The authors disclose no conflict of interest. dependent acquisition mode with the Xcalibur software (Thermo Scientific, Dublin, Ireland). Full mass spectra were recorded in ACKNOWLEDGMENTS profile mode over a mass range of 300–2000m/z and tandem mass We thank Dr Finbarr O’Sullivan (NICB) for granting access and spectra recorded in profile mode. Dynamic exclusion was enabled assistance at the cryotome and Ms Louise Sewell (CBAS) for her with an exclusion duration of 30 s. Protein identification searches technical assistance, and Prof Hiroshi Kawachi, Niigata University, were performed using the information in the tandem mass spectra Japan for the kind gift of anti-DPP4. AR is supported by DCU Research by searching against the UniProtKB/Swissprot protein database Fellowship award. This study was supported by The Irish Health (Rattus species) using MASCOT search engine (Version 2.3, Matrix Research Board Grant HRA/09/62.

10 Kidney International A Ravida` et al.: Glycosylation patterns in diabetic nephropathy basic research

SUPPLEMENTARY MATERIAL 25. Kirino Y, Sato Y, Kamimoto T et al. Interrelationship of dipeptidyl Figure S1. Clinical parameters of the rat study. peptidase IV (DPP4) with the development of diabetes, dyslipidaemia Figure S2. PSA, PHA-L, SJA, ConA, WGA, sWGA, and Jacalin do not and nephropathy: a streptozotocin-induced model using wild-type and DPP4-deficient rats. J Endocrinol 2009; 200: 53–61. reveal appreciable changes in glycosylation during STZ-diabetes 26. Apweiler R, Hermjakob H, Sharon N. On the frequency of protein progression in rat kidney cortices. glycosylation, as deduced from analysis of the SWISS-PROT database. Figure S3. STZ diabetes induces a reduction in the RCA/DPP4 ratio in Biochim Biophys Acta 1999; 1473:4–8. 2 w kidney cortex lysates. 27. Tamura K, Manabe N, Uchio K et al. Characteristic changes in Supplementary material is linked to the online version of the paper at carbohydrate profile in the kidneys of hereditary nephrotic mice (ICGN http://www.nature.com/ki Strain). J Vet Med Sci 2000; 62: 379–390. 28. Yan K, Khoshnoodi J, Ruotsalainen V et al. N-linked glycosylation is critical for the plasma membrane localization of nephrin. J Am Soc Nephrol 2002; REFERENCES 13: 1385–1389. 29. Vogtla¨nder NP, Tamboer WP, Bakker MA et al. Reactive oxygen species 1. World Health Organisation fact sheet N 312. August 2011. deglycosilate glomerular alpha-dystroglycan. Kidney Int 2006; 69: 2. Potenza MA, Nacci C, Gagliardi S et al. Cardiovascular complications in diabetes: lessons from animal models. Curr Med Chem 2011; 18: 1526–1534. 1806–1819. 30. Nicolaou N, Margadant C, Kevelarn SH et al. Gain of glycosylation in 3. Bentata Y, Haddiya I, Latrech H et al. Progression of diabetic nephropathy, integrin a3 causes lung disease and nephrotic syndrome. 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