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Pre-clinical alterations in the serum of COL(IV)A3-/mice as early biomarkers of Alport syndrome Petra Muckova, Sindy Wendler, Diana Rubel, Rita Büchler, Mandy Alert, Oliver Gross, and Heidrun Rhode J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00814 • Publication Date (Web): 20 Oct 2015 Downloaded from http://pubs.acs.org on October 30, 2015

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Journal of Proteome Research

For TOC only 254x190mm (96 x 96 DPI)

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• birth • genotyping • sample collection until week 9.5 • pooling of serum samples

Fig. 2

• pre-fractionation of proteins • digestion of sub-fractions by trypsin • mass spectrometry • data analysis by Proteome Discoverer

• comparison of corresponding subfractions by Sieve 2.0 and 2.1 Fig. 3 ACS Paragon Plus Environment

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Pre-clinical alterations in the serum of COL(IV)A3-/- mice as early biomarkers of Alport syndrome 1,2

Petra Muckova, 1Sindy Wendler, 3Diana Rubel, 1Rita Büchler, 1Mandy Alert, 3Oliver Gross,

1

Heidrun Rhode*

1

Institute of Biochemistry I, University Hospital Jena, Nonnenplan 2-4, 07740 Jena, Germany

2

Clinic of Neurology, University Hospital Jena, Erlanger Allee 101, 07740 Jena, Germany

3

Department of Nephrology and Rheumatology, University Medicine Göttingen,

Robert-Koch Str. 40, 37075 Göttingen, Germany

Corresponding author: *E-Mail: [email protected] Tel.: +49-3641-938620 Fax: +49-3641-9396302



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Abstract The efficiency of the inhibition of the angiotensin converting enzyme, the most widely used therapy for the Alport syndrome, depends on the onset of the therapy – the earlier the better. Hence, early progressive biomarkers are urgently required to allow for pre-clinical diagnosis, an early start of possible therapy as well as the monitoring of this therapy. In the present study, an improved comprehensive and precise proteomic approach has been applied to the serum of juvenile Alport-mice, non-treated and treated, and wild-type controls of various ages to search for biomarkers. With a total of 2542 stringently altered proteins, the serum composition clearly shows a dependency on age, i.e., stage, and therapy. Initially, the serum constituents indicate an enhanced extracellular matrix remodeling, cell damage and the production of particular acute phase proteins. A panel of 15 potential biomarker candidates has been identified. In later stages, renal filtration failure and systemic acute phase reaction determine the composition of the serum; an effect which is well known for manifested human Alport syndrome. With a small number of mouse urine samples, as example the proteomic results for gelsolin could be verified using ELISA. Once verified in man, these early biomarkers would allow for a sensitive and specific diagnosis of the Alport syndrome in children, as well as facilitate the monitoring of a possible therapy.

Keywords: non-invasive biomarkers, serum proteome, pre-clinical diagnosis, therapy monitoring


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Introduction The Alport syndrome (AS) is a hereditary nephropathy characterized by hematuria and proteinuria, often leading to end-stage kidney disease, hearing loss, and ocular changes 1-6. In principal, all mutations in α3, α4, or α5 of collagen type IV lead to the disease. The majority of cases are X-linked inherited due to mutations in the α5 chain of collagen (IV) (COL(IV)A5) 7, 8. Female carriers of X-linked AS may be seriously affected 9, 10. Despite significant efforts to develop treatments, there is still no known cure 11. Currently, the inhibition of the renin angiotensin aldosterone system is the most effective and widely used therapy, as inhibiting the angiotensin converting enzyme (ACE) has been shown to delay renal failure in a time-dependent manner 12-15. This also means that the earlier treatment commences, the greater the benefit will be13. In principle, ACE-inhibition should begin early in childhood and requires long-term administration as well as reliable monitoring. Thus, diagnostic parameters for both the initiation of the treatment as well as its monitoring are urgently required. Genetic analyses verify the disease; however, in previously undiagnosed families with AS, this tool is unfeasible for early diagnostics or to monitor therapy. Histological analysis is highly invasive, risky, and only reveals rather late events of progression. In general, it can be said that common kidney parameters are neither suitable nor practical for monitoring early events and their susceptibility to ACE-inhibition. Currently, there is a lack of knowledge as to whether or not treatment is effective in the very early stages of AS and about the optimum point in time to initiate or change therapy. Therefore, the goal of our study is to determine biomarkers (BMs) for the reliable identification of early signs of glomerular alteration due to AS. These BMs should be identifiable in non-invasive samples (blood and urine). In young children, a comprehensive search for such biomarkers in a pre-clinical state is not possible for both ethical and practical reasons. Instead, COL4A3-/- mice can be used as a rapid mouse model of AS 16. In this mouse model, the initiation of ACE-inhibitor therapy prior to the onset of proteinuria delayed fibrosis and doubled the survival time 17,18. A significantly smaller improvement in outcome was achieved when the inhibition of ACE was started after the onset of proteinuria 17. These experimental data were confirmed with the help of retrospective analysis of human registry data ACS Paragon Plus Environment


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13,19,20

, suggesting that ACE-inhibition delays the end stage renal disease and increases the life

expectancy in AS patients (and heterozygous carriers) in a time-dependent manner 13. This observation highlights the importance of an early, accurate diagnosis of AS, as suggested in expert recommendations 12,21. Prior to our study, a panel of BM candidates has been identified using our proteomic workflow and samples from patients with manifested AS 22. Here, our improved approach 23 is applied to the serum of juvenile AS-mice (COL(IV)A3-/-) to search for BMs.


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Experimental Section Workflow: An overview of the workflow applied is given in Fig. 1. The sample preparation process and the data analysis are presented in Figs. 2 and 3, respectively. Samples: Cross-breeding of heterozygous COL(IV)A3+/- mice by Cosgrove et al. 16 (The Jackson Laboratory, Maine, USA) on a 129X1/SvJ genetic background for more than 20 generations was performed in a pathogen-free environment. PCR-based genotyping was performed as described previously17. The animal diet R/MH (V153x; Ssniff, Soest, Germany) contained 19% protein, 3.3% fat, 4.9% fiber. All animal experiments complied with the German animal care and ethics legislation, were approved by the local authorities, and supervised by veterinarians. ACE-therapy with Ramipril started pre-emptively in week (wk) 4 at the maximum tolerated dosage of 10 mg/kg/day in drinking water. Samples were collected at wks 4.5, 6.0, 7.5, and 9.5. For urine samples, the mice were allowed to urinate on a glass plate. The voided urine was aspirated into microcentrifuge tubes; 15-300 µl pure urine could be obtained without invasive intervention. Blood samples were taken by intracardial puncture immediately after killing. All samples were stored at -80 °C until shipping, and thereafter in liquid nitrogen until use. Proteinuria was measured using a 4-12 % Novex Tris/Glycine polyacrylamide gradient gel (Life Technologies, Carlsbad, USA) stained with Coomassie Blue and analyzed using densitometry, a semiquantitative method described previously 17. Urea was measured in serum aliquots on a Cobas8000, Modular Analyzer Series (Roche Diagnostics, Mannheim, Germany). COL(IV)A3-/- (AS) mice at wk 4.5 did not show any significant differences in comparison to wild-type (wt) controls in terms of proteinuria or renal function (beyond microhematuria) 17, similar to humans at stage 0 of AS with isolated macrohematuria 13. AS mice at 6 wks of age, similar to humans in stage I or early stage II, develop proteinuria; however, renal function tests were still within the wt control range. Groups: Wt control group mice, C-0, were administered with placebo and AS groups comprising AS-0 and AS-R were administered with placebo and Ramipril (R). Just before sample preparation, samples of equal volumes were pooled from three or four individuals of control or AS mice, respectively. Thus, altogether 12 pooled serum samples were prepared: four from controls and eight from ASACS Paragon Plus Environment


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mice. Pools of control groups 4.5C-0, 6C-0, 7.5C-0, 9.5C-0 refer to samples from wk 4.5, 6, 7.5, and 9.5, respectively, and accordingly for AS mice treated with placebo (4.5AS-0, 6AS-0, 7.5AS-0, 9.5AS0), or Ramipril (4.5AS-R, 6AS-R, 7.5AS-R, 9.5AS-R). Sample preparation: According to Wendler et al. 23, a two dimensional native chromatographic fractionation has been applied. Briefly, 100 µL of the pooled serum samples were 1D-separated by size exclusion chromatography (SEC) on a Superdex™ 200 GL column (10/300) controlled by an Äkta purifier™ system (both GE Healthcare) in 10 mM Tris/HCl containing 150 mM NaCl (pH 7.4) at 20 °C. Thus, 48 fractions of 250 µL were collected into chilled 96-well deep well plates (flow rate: 350 µL/min). Automated anion exchange chromatographic 2D-fractionation was simultaneously performed for up to six samples 23. This validated and quality-checked workflow was characterized by coefficients of variation of lower than 10% for protein separation and 30% for mass spectrometric results 23 24. Mass spectrometric analysis: In total, 1176 2D-fractions (>0.03 mg/ml) were analyzed using LCMS/MS. Corresponding 2D-fractions from sample groups of the same age (C-0, AS-0, AS-R) were submitted to MS, using the microplate equipped autosampler (Accela, Thermo Fisher Scientific, USA). A liquid chromatographic separation of peptides was performed on a Hypersil Gold UHPLC column (1.9 µm, 50×1.0 mm) with an Accela 1250 UHPLC system (both Thermo Fisher Scientific, USA) prior to mass spectrometry; using a binary mobile phase, 0.1% formic acid in (A) water and in (B) acetonitrile at a flow rate of 150 µL/min, in a binary gradient of 0-1 min 5% B, 20 min 30% B, 23 min 40% B, 23.125 min 95% B, 25.1-26.5 min 5%-95% B, 26.5-28 min 95% B, 28.1-32 5% B. Tandem mass spectrometry (MS/MS) measurements were carried out on a LTQ Orbitrap Discovery (Thermo Fisher Scientific, USA) by using positive mode heated electrospray ionization (H-ESI) at a vaporizer temperature of 200 °C. Sheath gas flow (30.0) and auxiliary gas flow (10.0) were used to dry the ion spray (both nitrogen gas flows in arbitrary units). The ionization voltage and the temperature of the ion transfer tube were set to 4.5 kV and to 275 °C. The MS/MS system operated in data-dependent TOP10 mode using 1 microscan. For this purpose, ions have been monitored in the LTQ ion trap in full scan centroid mode at m/z 350-1700. The ten most intensive ions were run through collisionACS Paragon Plus Environment

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induced dissociation (CID) for further orbitrap high resolution (30,000) analysis (profile data type). Wideband activation was used. The automatic gain control (AGC) target value for the orbitrap mass analyzer in full scan mode was 1.0 × 106. The LC-MS/MS operated via graphical interface of the Xcalibur software 2.1. All fractions were run in duplicate. After each duplicate a blank (injection of LCMS grade water) was applied to avoid carryover. For quality control and stability check, a digest of human holotransferrin (SERVA Electrophoresis GmbH, 36756) was analyzed four times per microplate 23. Protein identification and data analysis: Analyses of MS/MS spectra were done with Proteome Discoverer 1.3 and Sequest database search (Mouse Fasta database from 12.11.2013 downloaded from www.uniprot.org) algorithm (false discovery rate of 0.01). To detect differences between the three groups, AS-0 vs. C-0 and AS-R vs. AS-0, were analyzed with Sieve 2.0 and 2.1 (Thermo Fisher Scientific) for corresponding fractions in parallel (Control Compare Trend, Fig. 3). 2D-fractions are considered to be corresponding fractions originating from two different samples of animals of the same age and exhibiting the same fraction locations (1D_2D). The Sieve 2.0 results were filtered with the charge-dependent Xcorr (Xcorr 1.5, 2.0, 2.25 and 2.5 for charge 1, 2, 3, and >3, respectively) and with a p-value of 0.05. The Sieve 2.1 results were filtered with Percolator (0.050) and Frames Table Filter (PRElement=0, PRSize>1 and goodID>0); the calculation type used was “relative variance weighting”. Since deviant but similar results were obtained with both versions of Sieve, we combined the results. The resulting data regarding protein ID, peptide counts, frames, hits, ratios, standard deviation, and p-values were labeled with the fraction locations (1D_2D) (Table S1, Supporting Information). Thus, we were able to identify chromatographic clusters of altered proteins (≥2 connected locations). Subsequently, these protein clusters were subdivided into three categories: proteins with exclusively up- (ratio >2) or down-regulation (ratio 300kDa-63kDa) and charge (2D-fractions: 01-18). Transferrin no longer exhibits a clear global up-regulation, but rather a differential alteration of subportions. Only a small fraction of non-charged transferrin is significantly increased and is susceptible to Ramipril (Table S2), whereas the main part remains unchanged or even decreased in AS in comparison to controls. Similarly, the majority of peptides related to A1AT cover a wide range of MW (>400kDa-70kDa) and charge (2D-fraction: 11-24). Here, in contrast to wk 4.5, the majority of fractions of A1AT show increased ratios in AS vs. controls. Fibronectin, inter-alpha-trypsin inhibitor, and leucine rich HEV glycoprotein show a greater up-regulation than in wk 4.5. Again, vomeronasal type 2 receptor 67 is remarkably increased but not reversed by Ramipril. Several proteins are lower in AS-0 than in C-0, among them several fractions of the corticosteroid binding globulin (similar to wk 4.5), carbonic anhydrase 14, and protein Ces1b. The highest percentage of hemopexin (derived from peptide counts) is lower in AS-0 and AS-R than in C-0 than in all identified serpines A3 (F, K, C, M). BM candidates at later stages of AS (wk 7.5, AS stage II) In AS, 43 proteins were identified as stringently up-regulated and decreased in concentration after Ramipril therapy (Table S4, Supporting information). The highest increase was found with haptoglobin, Apo B, and β2-glycoprotein 1. Again, peptides related to transferrin were found in several clusters of an even higher heterogeneity than in wk 6.0. Fractions of transferrin with a higher MW and charge are increased in AS-0, whereas those proteins whose percentage had decreased exhibited a lower MW and charge. In principle, the same and inverse holds true for the complement factor C3 and A1AT, respectively. In AS-0, Apo AIV is increased and decreased at the MW of lipoproteins and the free protein, respectively, and in general down-regulated by Ramipril. Apo AI, AII and CIII are also altered differentially in a variety of fractions. In summary, the pattern of lipoprotein composition and size seems to be altered heavily in later stages of AS. Serpines are also changed:



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whereas serpine A3N is increased in AS-0 (Table S3), all other forms (K, C, F, M) are decreased to at least one fourth. Several proteins are clearly down-regulated in AS-0 compared to C-0, among them murinoglobulins 1 and 2, antithrombin, transthyretin, carboxylesterase 1C, and the transferrin-like protein Gm20425.

3. Evaluation of results Since some BM can be expected to enter the urine compartment (Fig. 6) and no further serum samples were available for evaluation, urine samples were used for the preliminary verification of the proteomic results. Moreover, due to restricted sample amounts and volumes, and the limited availability of suitable test kits from all BM candidates identified, so far only two – gelsolin and COL(I)A1 – could be tested as examples with ELISA. For the measurements of several other promising BM candidates we could not find any test kit or suitable antibodies. In urine, the concentration ranges of gelsolin seem to reflect the concentration ratios of comparative samples in the proteomic study. At 4.5 wks, higher concentrations of gelsolin were found in AS-0 compared to C-0 and AS-R (Fig. 5) in accordance with the proteomic ratios determined by Sieve. At 6 wks, the medians of the gelsolin concentrations in AS-0 and C-0 are similar, verifying the proteomic ratios around 1.0. However, in urine, levels are increased compared to the situation at 4.5 wks, while AS-R show much lower concentrations compared to to wk 4.5. With the few samples at hand at 7.5 wks, similar low concentrations of gelsolin were found in AS and controls. With the available test kit for COL(I)A1, no comparable result could be found. In urine, the very low concentrations of COL(I)A1 did not show any significant differences among all groups (results not shown). All values show a high variability within the groups. It should be noted that we have not been able to normalize our results to corresponding creatinin concentrations due to the limited sample volumes.

Discussion The goal of our present study was to investigate markers for the very early diagnosis in the AS mouse model, including those that appear prior to ultrastructural changes. ACS Paragon Plus Environment

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The α3, 4, and 5-type collagen IV chains are evolutionary highly conserved, and all mammals, including mice, can develop AS. Alport pathogenesis has been very well studied in the COL4A3-/mouse model 16, 25. Our experiments demonstrated that Alport mice exhibit early alterations in blood and urine as probable markers of preclinical renal disease even prior to early ultrastructural changes in the GBM. Even though these biomarkers might not specifically point to a definitive diagnosis of AS and even if the results from Alport mice might not be easily transferable to humans, if confirmed in humans with AS, the early warning signs of a defective GBM should stimulate doctors to send young patients to a pediatric nephrologist for a thorough evaluation including genetic diagnostics. In general, the search method we applied identifies only substantial alterations due to the low protein input and the averaging that comes with pooling. Moreover, the results are confirmed by the fact that the changes in BMs found could also be detected in several adjacent sub-fractions. Although only exemplarily verified with ELISA, our mass spectrometric results were confirmed by follow-up samples during growth and reversibility under ACE-inhibition in independent sets of animals. Summary of serum proteome alterations during progression of AS: At wk 4.5, there are signs of only a moderate APR, indicated by the slight increase of parts of positive APPs. Moreover, some extracellular matrix (ECM) and cellular components might indicate initial fibrosis and tissue damage. The possible origins of these components are shown in Fig. 6. At wk 6.0, signs of typical APR become prominent, indicated by the striking increase of a set of positive APPs and the decline of most subsets of typical negative APPs. Some APPs and other proteins peak in wk 6.0; this might indicate a high disease activity at this age. Nevertheless, some negative APPs or fractions thereof show an untypical alteration, i.e., increase, at wks 4.5 and 6.0. The decline of several LMW portions of various proteins could be compatible with urinary loss of these entities during early failure of glomerular barrier function and, hence, their possible appearance in urine (not yet analyzed by us). At wk 7.5, ECM leakage components are no longer visible. Signs of a typical APR persist, and the decline of LMW protein fractions becomes prominent – probably due to the developing urinary loss. Moreover, some



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plasma proteins are found to be increased only at this stage, e.g., kallikrein, afamin, and ApoB as well as some special cellular proteins. Possible impact of selected proteins that interact with ECM: Isoform 2 of collagen alpha 1(I) chain (COL(I)A1 (2)) is used in ECM collagen networks forming fibrils of collagen I trimers. In the human GBM, together with collagen IV 26, collagen I has been identified 27. Moreover, there is a strong link between collagen I deposition and glomerulosclerosis and fibrosis both as source and implication of cellular impairment (e.g. 28, 29). The identified peptides represent a fragment rather than the entire chain; this could be explained by enhanced proteolysis in AS or the already developing fibrosis in this early stage. Interestingly, the ACE seems to be directly involved in fibrosis (e.g. 30, 31). Its inhibition is reno-protective and diminishes collagen expression. This might be indicated by the reduced serum concentrations of COL(I)A1 (2) and other ECM constituents upon Ramipril therapy. Recently, isoform 3 of sulfhydryl oxidase 1 was identified as an essential disulfide-forming extracellular component which controls matrix composition and function during organ development 32

. The enzyme was localized in the kidney among several rat tissues 33 and thus might be released

into the circulation upon enhanced matrix remodeling. Leucine rich HEV glycoprotein is recognized as an important regulator of angiogenesis during tissue development and neo-angiogenesis mediated by modulating TGF-β signaling 34. Thus it may be involved in renal development and increased and released into the circulation in the case of mismatches. Moreover, TGF-β is known as the central mediator of glomerulosclerosis 35 and might link leucine rich HEV glycoprotein with collagen expression. This protein is continuously up-regulated in AS and can be efficiently reversed by Ramipril at all ages analyzed. Possible significance of selected proteins that derive from cellular compartments: Since the identified peptides could not define the exact origin, these proteins may be damage markers of either renal or extra-renal cells. Although the most strongly altered protein, vomeronasal type 2 receptor 67 (Table 1), belongs to a receptor family mainly expressed in the olfactory system 36, 37, it has also been detected in the kidney and other tissues 37. Expression of betaine homocysteine S methyltransferase 1 was shown in several organs, among them the rodent kidney 38. Gelsolin is a ACS Paragon Plus Environment

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cytosolic calcium-binding protein which interacts with several cellular proteins, such as F-actin 39, 40 and is known to be released from several injured cell types. Changes in this protein could be verified in urine (with ELISA) to be similar to those detected in blood samples (analyzed with mass spectrometry). Possible impact of selected plasma proteins: Some of these proteins reflect alterations in lipoprotein metabolism, and some of them are known as APPs. Remarkably, several APPs are altered contrarily to the usual APR and/or show an interesting relation to the known pathomechanism in AS. These proteins are highlighted here. The identified type of inter-alpha-trypsin inhibitor belongs to a heterogeneous protein family. These proteins are released by the liver as well as by extra-hepatic tissues. In blood, all variants of interalpha-trypsin inhibitor consist of a light chain (lc, bikunin) and one or two heavy chains (hc) 41. Since several forms of inter-alpha-trypsin inhibitor are considered as BM in several diseases (e.g. 42-44), the protein family seems not to be specific for AS. However, the components identified by us (hc1 and hc2) form one special type since they co-elute within our pre-fractionation matrix. Moreover, all three components decline in a synergistic way upon Ramipril therapy in identical sub-fractions. Interestingly, these particular chains are known to form a complex, which has structural and catalytic activities that may reflect kidney injury. This type of inter-alpha-trypsin inhibitor is known to interact by a transesterification reaction with hyaluronan in the ECM of several tissues 41, thereby forming the serum-derived hyaluronan-associated protein complex 45. Kidney proximal tubular cells are surrounded by hyaluronan cables associated with several ECM proteins, among them the same type of inter-alpha-trypsin inhibitor 46 within a functional network. Moreover, this type of inter-alphatrypsin inhibitor is related to fibrosis 47. Additionally, it shows antiproteolytic activity since it interacts with several complement factors, resulting in a suppression of, e.g., C5 activation 41. The identified alteration might be compatible with both, an increased expression during the APR or an increased release from altered ECM in the injured renal cortex. Hc3 and 4 are well-known positive APPs in several species, including mice (e.g. 42, 44, 48-51) and are found here to be increased at later stages. In



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contrast, both the lc and hc2 identified by us are known to be negative APPs in rats and man 43, 52. Thus, the increase of these chains may reflect their release from renal ECM upon remodeling. Transferrin is a well-known negative hepatic APP of several species which declines moderately 53. Moreover, constitutive expression of transferrin mRNA was shown in several extra-hepatic tissues including mouse kidney 54. In 4.5 wk old AS mice, the main part of this protein rises considerably by up to 40-fold (Table S1) and is down-regulated by Ramipril application. However, later alterations in transferrin merely reflect different posttranslational modifications that will be analyzed in more depth in future studies. Thus, like inter-alpha-trypsin inhibitor, the increase of transferrin cannot be considered as APR. Some positive APPs are up-regulated in AS. In mice, haptoglobin and serum amyloid P are described as the major APPs 55, usually with fold-changes of more than ten during acute disturbances. In early AS (wk 4.5) we identified only moderate alterations of these proteins, as well as of several complement factors, angiotensinogen, and plasminogen. The moderate rise of haptoglobin is in accordance with results in mice on the mRNA level 54 and may reflect low-grade inflammation. Later, haptoglobin is increased much further and only partially reversed by Ramipril. There are vast data on the role of the liver-derived retinol binding protein 4, which is also known to be adipose tissue-secreted and responsible for insulin resistance (e.g. 56, 57). This protein has been recognized to be a negative APP for some time 58. Currently, at least in chicken, the protein was shown to be expressed in several extra-hepatic tissues, among them the kidney 59. Additionally, the protein was shown to link chronic renal failure to metabolic alterations in humans (e.g. 60). Taken together, since the rise in AS could not be explained by the acute phase, we hypothesize that the protein is released from the kidney during kidney alteration and might be responsible for the wellknown alteration of lipoprotein metabolism. Known to bind phospholipids and lipopolysaccharides and as a negative APP 61, also β2-glycoprotein 1 is raised in AS. Complement factor H is a plasma protein constitutively produced in multiple forms by the liver and is one of the most important complement control proteins in fluid-phase and on surfaces. It has several ACS Paragon Plus Environment

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roles including targeting damaged cells and debris, preventing complement activation in fluid phase and on host surfaces, as well as controlling the positive feedback loop of complement activation. Complement factor H strongly interacts with CRP 62, C3b and various surface structures 63. Moreover, the protein is known to be expressed in various extra-hepatic tissues with focal regulatory roles, among them are mesangial cells 64 and podocytes 65. It was shown to be up-regulated in glomerulonephritis 66 and thus might be released from these origins. Additionally, complement factor H may be involved in signaling – providing a link to metabolic dysregulation 62. In AS mice, it is increased in blood, first moderately and peaking at 6.0 wks – likely indicating a progression of glomerular damage. Similar kinetics can be seen with complement factor I (Table 1). Hemopexin is a known positive APP 67. Hemopexin probably fulfills many other functions besides heme and metal ion binding. In spite of caution 68, hemopexin seems to act in immune regulation 69, 70

. In the kidney, hemopexin seems to be involved in signaling in nephrin-dependent remodeling of

the actin cytoskeleton in podocytes 71. Human mesangial cells are shown to produce and secrete hemopexin after TNF-α stimulation 72 in vitro. These cells are also able to activate the protease activity of (or linked to) this protein 73. Moreover, hemopexin is accumulated focally in renal proximal tubules upon acute kidney injury in mice. A higher fold-increase of hemopexin protein and mRNA was found after LPS stimulation of cultured renal cells than after heme challenge 74. On the protein level, nephrotoxic challenges like cisplatin and ischemia/reperfusion cause an even higher rise. Notably, since some matrix metalloproteases contain several so-called hemopexin domains, the peptides identified by us could not be attributed to matrix metalloproteinases. In accordance with the results described here, an increase of hemopexin, haptoglobin, fibronectin, complement factor H, and a fraction of transferrin was previously identified proteomically in the serum of children with manifested AS 22. Of these proteins, at least fibronectin has been positively evaluated (in urine) in combination with a variety of BM candidates in immunoassays 75. The identified carboanhydrase isoenzyme 14 is a membrane-bound enzyme found in various cell types and, in mice, mostly expressed in the kidney 76.



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Conclusions All up-regulated proteins that are down-regulated by Ramipril particularly or exclusively in the earliest stage (wk 4.5) may serve as BM candidates for AS diagnosis, identification of progression, as well as monitoring of therapy. These BM candidates preceded the light-microscopically visible changes in the kidney of Alport-mice 17. These 15 proteins are: sub-sets of both transferrin and hemopexin (which are still to be characterized), HMW kininogen II, retinol binding protein 4, serpins A3K, A3C, A3M, and A3F, β2-glycoprotein 1, COL(I)A1 (2), isoform 3 of sulfhydryl oxidase 1, betaine homocysteine S methyltransferase 1, superoxide dismutase [Cu-Zn], vomeronasal type 2 receptor 67, and isoform 2 of gelsolin. Other increased proteins that persist over a longer period might also be useful, such as fibronectin and the ECM-related heavy chains 1 and 2 of inter-alpha-trypsin inhibitor. These candidate BMs have to be evaluated in larger sample counts, either alone or in combination. Some of these serum proteins may enter the urine compartment and may thus be monitored in urine. Once validated, some of the proteins released into the circulation could verify the new insights into the pathogenesis of AS as already discussed above.

Acknowledgements: Financial support: Thuringia Ministry for Education, Science and Culture (2010 FE 9001, 2013 FE 9075) and the European Regional Development Fund (2010 FE 9001, 2013 FE 9075), Federal Ministry for Education and Research (01GM1304). Language editing: Laura McMillan, Stephanie Luther.

Disclosure: The authors declare no conflict of interest.


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Supporting information: Table S1 Mass spectrometric data obtained by Sieve 2.0 and 2.1: Protein ID, Description, Peptides, Frames, Hits, Ratios, Standard Deviation (StDev), pValue, Fraction number (1D_2D) inclusive prefixes indicating sample pairs of A, 4.5 weeks; B, 6.0 weeks; and C, 7.5 weeks.

Table S2 Serum proteome alterations in AS-mice at week 4.5 - that are reversed by Ramipril.





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Figures: Figure 1: Flow-chart of the methodology



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Figure 2: Diagram of the pre-fractionation process and the preparation for mass spectrometry Experimental details are described in the Experimental Section and in 23. After a serial 1D-SEC of the samples, all subsequent steps (2D-AEC, all preparation steps of tryptic digestion, and mass spectrometry) are performed in parallel and mainly automated. The location, i.e., fraction number(s), of a protein identified with mass spectrometry preserves information on both the native molecular weight and the negative net charge.


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Figure 3: Diagram of the analysis of mass spectrometric data In corresponding sub-fractions, i.e. sub-fractions of the same 1D- and 2D-number, from samples of the same age, the ratios of each identified protein obtained with Sieve 2.0 and 2.1 were compared as indicated in Figure 3, AS-0 vs. C-0 and AS-R vs. AS-0. This was done separately with samples obtained at identical points in time, 4.5 wks, 6.0 wks, and 7.5 wks. All fold-changes above two, i.e., reliably above the experimental error, are considered as confident deviation. Peptide counts have been used to identify those chromatographic sub-fractions containing the main part of the respective proteins.



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Figure 4: Chromatogram of serum proteins of mice after native SEC

All plasma proteins co-elute in four main peaks under the native conditions used. The molecular weight according to column calibration and the dominant constituents of these peaks are indicated in A.

A: Untreated AS-mice (AS-0) of four ages (wk 4.5-9.5); B: healthy control mice (C-0) and C: AS mice treated with Ramipril (AS-R) at ages of 4.5 and 9.5 wks.


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Figure 5: Boxplots of gelsolin concentrations in mouse urine measured with ELISA

AS-0: untreated AS mice; C-0: healthy control mice; AS-R: Ramipril treated AS mice; n: number of samples measured.



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Figure 6: Essential structures and hypothetical protein flows in both the healthy glomerulus and the altered glomerulus in AS

The following figure summarizes the hypothetical origins and flows of the BM candidates we identified. The expression of these proteins was approved at least in the kidney, sometimes in special glomerular cell types according to literature data as indicated in the Discussion. P, podocyte (green, healthy; pink, disturbed in AS); M, mesangial cell (blue, healthy; diamonds, disturbed in AS); E, endothelial cell; GBM, glomerular basement membrane (green, healthy, red, mismatched in AS); SD, slit diaphragm; Arrows indicate the expected flow of proteins derived from these structures, their color reflecting the protein’s probable origin. Thickness and number of arrows correlate to the amount of protein. Grey arrows indicate the flow of blood plasma proteins. Hypothetical origin of the following biomarker candidates are indicated by symbols: * COL(I)A1, isoform 3 of sulfhydryl oxidase 1; ** leucine rich HEV glycoprotein; ♦ betaine homocysteine S methyltransferase 1, gelsolin; vomeronasal type 2 receptor 67, complement factor H; ∇ inter-alpha-trypsin inhibitor; transferrin, retinol binding protein 4, hemopexin. The additional, well-known alterations following proteinuria and tubular failure are not depicted here.


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