Relevance of Protein Content within the Renal Scaffold for Kidney Bioengineering and Regeneration

Chronic kidney disease is currently a major public health problem around the world. Although hemodialysis increases survival of patients with end-stage renal disease, kidney transplantation remains the only potentially curative treatment. However, transplantation as a therapeutic option is limited by availability of suitable donor organs. This situation highlights the urgent need to find new and potentially inexhaustible sources of transplantable organs. Perfusion decellularizarion of whole organs is a novel approach to organ engineering and regeneration. In the present research, we used a continuous perfusion decellularization protocol to eliminate cellular componet of kidney and evaluated residual scaffold components after decellularizarion process by proteomics analysis. Our proteomic data show that this protocol results in incomplete removal of cellular proteins. However, unlike other authors, we assume that proteins retained within decellularized kidney scaffold could be the basis for specific homing and celular differentation in the recellularization process.


INTRODUCTION
Chronic kidney disease (CKD) is currently a major public health worldwide problem [1,2].CKD affects a significant percentage of the population; mainly due to that their leading causes are highly prevalent disorders such as aging, hypertension, diabetes and vascular disease [3].Although hemodialysis increases survival of patients with end-stage renal disease (ESRD), the kidney transplantation remains the only po-

Harvesting of Rat Kidneys
All experimental protocols were reviewed and approved by the Ethics Committee for Animal Research of the University of Cordoba, and all rats received humane care in compliance with the guiding principles in the Guide for the Care and Use of Laboratory Animals.
The animals were sacrificed by exsanguination under general anaesthesia (i.p. sodium thiopental).After a median laparotomy and systemic heparinization through the infrahepatic inferior vena cava for anticoagulation, renal artery, renal vein and ureter were complety isolated.Subsequently, the whole kidney was harvested and transferred to a cell culture dish.The whole kidney was maintained hydrated with cooled PBS while the main vasculature of the organ was cannulated.

Whole-Organ Perfusion Decellularization
We performed a decellularization protocol following the method used by Guyette J et al. [11], with minor modifications.Briefly, renal artery was cannulated with a 24-G cannula and fixed with 3/0 silk suture.The kidney was manually perfused with 10 ml of diluted heparin (Sanofi, Gentilly, France) solution to remove residual blood.Then, the cannulated kidney was mounted into the decellularization chamber and was connected to the peristaltic pump (Fresenius-Kabi, Bad Homburg, Germany).The perfusión rate for each step was set at 0.4 mL/min.The decellularization process was initiated by perfusion with PBS for 30 minutes, followed by 0.66% SDS (Thermo Fisher Scientific, Waltham, MA, USA) in deionized water for 18 hours until organ translucency was confirmed.Finally, the kidney was perfused with 1% Triton X-100 (Acros Organics, Fisher Scientific) in deionized water for 30 minutes.Following decellularization, we washed the kidney scaffolds with PBS containing 10,000 U/mL penicillin G, 10 mg/mL streptomycin, and 25 μg/mL amphotericin-B (Sigma-Aldrich, St. Louis, MO, USA) at 0.4 mL/min constant arterial perfusion for 96 hours.Wash solution was changed every 12 hours.
All used solutions were buffered at ph 7.4 and prepared under sterile conditions.The buffered solutions were kept to 4˚C until used.

Visualization of the Vascular System
To visualize the three-dimensional microvasculature in the scaffolds after decellularization, a blue colored solution was injected throught cannulated renal artery of the decellularized kidney.To performed the solution was used Commasie Brilliant Blue R (Sigma-Aldrich).

Quantification of Residual Detergent
Removal of SDS detergent of the kidney perfusate solution was quantified using the Stains All Dye reagent (Sigma-Aldrich) by absorbance readings at 438 nm as previously described by Rusconi F et al. [16].

Basic Histology Assessment of the Kidney Scaffolds
To assess cell and nuclear clearance as well as preservation of extracellular matrix, hematoxylin and eosin (H&E) and silver methamine staining were performed in decellularized and native kidneys.Briefly, decellularized and native kidneys transverse sections were fixed in 4% formalin, dehydrated through a graded series of ethanol, and embedded in paraffin.Three-micrometer paraffin sections were stained with H&E and methenamine silver and examined by normal light microscopy.

Protein Extraction and Digestion
Protein lysates from native and decellularized kidney were prepared by homogenizing tissue in lysis buffer (7 M urea, 4% CHAPS (w/v), 0.5 M DTT) containing protease inhibitor cocktail (Sigma-Aldrich) using a rotating blade homogenizator.Samples were cleaned by protein precipitation with TCA/acetone and solubilised in 50 µL of 0.2% RapiGest SF (Waters, Milford, MA, USA) in 50 mM ammonium bicarbonate.The total protein content was measured using the Qubit Protein Assay Kit (Thermo Fisher Scientific); 50 µg of protein was subjected to trypsin digestion following a protocol adapted from Vowinckel et al. [17].Briefly, protein samples were incubated with 5 mM DTT at 60˚C for 30 min and then with 10 mM iodoacetamide at room temperature and in darkness for 30 min.Sequencing Grade Modified Trypsin (Promega, Madison, WI, USA) was added at a 1:40 trypsin:protein ratio and incubated at 37˚C for 2 hours; the same amount of trypsin was again added and incubated at 37˚C for another 15 hours.RapiGest was then precipitated by centrifugation after incubating with 0.5% TFA at 37˚C for 1 hour.

Mass Spectrometry Analysis
The peptide solutions were analysed by a shotgun data-dependent acquisition (DDA) approach using nano-LC-MS/MS.One μg of each pooled sample (2 μL) was separated into a nano-LC system Ekspert nLC415 (Eksigent, Dublin, CA, USA) using an Acclaim PepMap C18 column (75 μm × 25 cm, 3 µm, 100 Å) (Thermo Fisher Scientific) at a flow rate of 300 nl/min.Water and ACN, both containing 0.1% formic acid, were used as solvents A and B, respectively.The gradient run consisted of 5% to 30% B for 120 min.
Eluting peptides were directly injected into a hybrid quadrupole-TOF mass spectrometer Triple TOF 5600+ (Sciex, Redwood City, CA, USA) operated with a data-dependent acquisition system in positive ion mode.A NanoSpray III ESI source (Sciex) was used for the interface between nLC and MS, with an application of 2600 V voltage.The acquisition mode consisted of a 250 ms survey MS scan from 350 to 1250 m/z followed by an MS/MS scan from 230 to 1700 m/z (60 ms acquisition time, rolling collision energy) of the top 65 precursor ions from the survey scan, for a total cycle time of 4.2 s.The fragmented precursors were then added to a dynamic exclusion list for 15 s; any singly charged ions were excluded from the MS/MS analysis.
The peptide and protein identifications were performed using Protein Pilot software (version 5.0.1,Sciex) with a Rattus Swiss-Prot concatenated target-reverse decoy database (downloaded in May 2015), containing 7927 target protein sequences, specifying iodoacetamide as Cys alkylation.The false discovery J. Biomedical Science and Engineering rate (FDR) was set to 0.01 for both peptides and proteins.

GO Analysis and Bioinformatic Tools
The proteins identified in native and kidney scaffold were described using Uniprot knowledgebase (http://www.uniprot.org/).The list of gene names from each dataset was used to generate a Venn diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/).Network analysis protein-protein interactions of the identified proteins were performed using STRING (https://string-db.org/).The confidence score for selection was 0.7.Gene ontology (GO) terms enrichment was calculated using Gene Ontology enRIchment anaLysis and visuaLizAtion (GOrilla) tool (http://cbl-gorilla.cs.technion.ac.il/).Significance for each GO term was assessed with p-values that were corrected using FDR q-value according to the Benjamini and Hochberg (1995) method.

Kidney Decellularization
In our laboratory, the continuous perfusion decellularization protocol was based on the combination of two detergents (SDS and Triton X-100).Detergent perfusion and washing of rat kidney was maintained with low and constant physiological perfusion pressures to allow gradual adaptation to changes in vascular resistance.As shown in the Figure 1 retained overall shape and the macroscopic structure, and became white and translucent due to the removal of the cellular contents.Furthermore, the three dimensional structure of the vasculature was preserved and intact (Figure 1

(C)).
According to previous studies, SDS perfusion shows severe cellular toxicity in subsequent recellularization processes [11,18].Analysis of several samples of the perfusate solution during decellularization process confirmed removal of SDS detergent.After perfusing Triton-X100, we observed a marked decrease in the levels of SDS becoming undetectable in the washing process, suggesting complete removal of residual SDS (Figure 1(D)).

Histological Characterization of the Rat Renal ECM Scaffold
The microscopic examination revealed the renal scaffold microarchitecture preserved and the integrity of the glomerular, tubular and vascular structures.Histologic evaluation with H&E staining showed pink eosinophilic structures typical of collagen fibrils, whereas no basophilic staining typical of cellular nuclear material was observed (Figure 2(E)), as compared to normal rat kidney staining (Figure 2(B)).
Moreover, methenamine silver staining was performed to assess preservation of basement membranes.Figure 2(F) showed the characteristic pattern (in black) of the glomerular and tubular basement membranes intact after the decellularization process and confirming successfully eliminate the cellular component compared to normal rat kidney staining (Figure 2(C)).

Proteomic Analysis of the Kidney Scaffold
The proteins identified in native and kidney scaffold were described using Uniprot knowledgebase (Data Supplementary).As expected, fewer distinct proteins were detected after decellularization process, process, 296 proteins were conserved to both native and kidney scaffold and 128 were exclusively represented in renal scaffold (Data Supplementary).Figure 3(B) shows the interaction network of the proteins with high confidence.The interactions among themselves indicates that the proteins are biologically connected.
GOrilla tool using a single ranked list of genes was performed to analyze the cellular component ontology in the decellularized rat kidney.The system recognized 421 genes out of 424 gene terms entered, only 419 of these genes were associated with a GO term.The results revealed 20 GO terms enriched for cellular component (Figure 4, Table 1).Some highly enriched cellular component terms were associated to protein complex and ECM.Associated genes for extracellular matrix term (GO:0031012) are shown in the Table 2.Each gene name is specified by gene symbol followed by a short description of the gene.Proteins were subsequently categorized into their functional and biological processes.GOrilla tool using two unranked lists of genes (scaffold as target list and native as background list) was performed.The system recognized 1404 genes out of 1414 gene terms entered, only 1092 of these genes were associated with a GO term.The results revealed 2 GO terms enriched for molecular function (Figure 5, Table 3) and 3 GO terms enriched for biological process (Figure 6, Table 4).
The biological process terms identified were related to metabolic process including organic acid metabolic process (GO:0006082), oxoacid metabolic process (GO:0043436) and carboxylic acid metabolic process (GO:0019752).Several enrichment molecular function terms were related to cofactor binding (GO:0048037) and secondary active transmembrane transporter activity (GO:0015291).

DISCUSSION
Renal transplantation is currently the most effective treatment for ESRD that represents one of the primary mortality reason globally.Because of this, the demand for transplantable organs continues to grow and the number of available organs seems to have plateaued in recent years.Consequently, there is a urgent need to identify new, potentially inexhaustible sources of clinically applicable and transplantable kidneys.
Perfusion decellularization of whole organs is a novel approach to organ engineering and regeneration.The use of a decellularized kidney scaffold is based on the mechanical and biological property of the ECM, which can maintain natural cellular architecture and some residual molecules that may advantage recellularization, differentiation and proliferation of the host cells.Kidney decellularization has been successfully achieved using a variety of approaches and protocols.Recently, Destefani et al. showed a comprehensive investigation about different protocols for rat kidney decellularization [19].Although, to date no specific protocol has demonstrated to be superior to the others.
In this study, the continuous perfusion decellularization protocol was based on the combination of two detergents (SDS and Triton X-100).We perfused kidneys using a peristaltic pump to ensure monitoring of perfusion pressure.Perfusion pressure was maintained in physiological range (up to 120   mmHg) during the entire duration of the decellularization process.This approach differs from the gravity-based perfusion system adopted by Guyette J et al. [11].Moreover, we decided to use a lower concentration of SDS (0.66%), because high concentrations of SDS can lead to the destruction of the ECM [20][21][22].Histological data showed that our decellularization procedure was effective in maintaining kidney microarchitecture.Furthermore, even the integrity of the basal membrane is preserved, which is a basic requirement for successful endothelialization of the vessels [23].Recellularization process requires not only the preservation of the overall architectural integrity of the organ but the maintenance of a vascular network intact, both are indispensable for ensuring adequate oxygenation of the organ.Taking this into consideration, we demostrated that the vascular tree was preserved after the decellularization process, which was evidenced by perfusion through renal artery of blue colored solution.
In this research, we employed a proteomics approach (by LC-MS/MS) to generate a comprehensive protein expression profile of kidney scaffold.As expected, fewer distinct proteins were detected after decellularization process.We identified 424 proteins in the kidney scaffold of which 296 proteins were conserved to both native and kidney scaffold.In the decellularized kidney scaffold, we identified a large number of both remnant cellular and ECM proteins.During creation of a decellularized scaffold, it is crucial to preserve the organ-specific ECM "zip codes" that support site appropriate cell attachment and differentiation [15,24].The identification of acellular matrix composition by LC-MS/MS confirms the expression of the main components of ECM including collagen type I, laminin, fibronectin, vimentin and Currently, the few published reports of proteome composition of decellularized biological scaffolds focus primarily on ECM proteins [15,[25][26][27].In this work, along with ECM proteins, we identified numerous cellular proteins mainly related to metabolic processes, in binding and transporter activity.Interestingly, we identified proteins involved in kidney development (Slc34a1, Aqp1, Mme, Podxl, Sdc4, Nphs2, Hspa4, Cat, Hrsp12, Cyp4a8, Asl, Aldh1a1, Aldh9a1, Acat1, Ass1).Particularly, Podxl, Aqp1 and Nphs2 are involved in kidney epithelium development and glomerulus development.Specifically, Podxl play an essential role in the formation and maintenance of podocyte foot processes [28] and Podxl-deficiency prevents renal tubule formation [29].

CONCLUSION
Collectively, our data reveal that decellularized kidney scaffold retain characteristic cues from local microenvironment that could be the basis for specific homing and celular differentation in the recellularization process.Thus, we assume that the goal of decellularization is to identify the crucial proteins that must remain within the biological scaffolds in order to complete a successful recellularization process.

Figure 1 .
Figure 1.Representative pictures of kidney (A) before and (B) after decellularization process.Mean length of native and scaffold kidney was 1.8 cm.(C) Visualizarion fo the vascular network.(D) SDS quantification in kidney perfusate solutions.

Figure 4 .
Figure 4. GO classification of enriched cellular component in renal scaffold.

Figure 5 .
Figure 5. GO classification of enriched molecular function in renal scaffold.

Figure 6 .
Figure 6.GO classification of enriched biological process in renal scaffold.

Table 1 .
GO classification of enriched cellular component.

Table 3 .
GO classification of enriched molecular function.

Table 4 .
GO classification of enriched biological process.