Application of Modular Therapy for Renoprotection in Experimental Chronic Kidney Disease.

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1 APPLICATION OF MODULAR THERAPY FOR RENOPROTECTION IN EXPERIMENTAL

Tissue Engineering Part A Downloaded from online.liebertpub.com by Ucsf Library University of California San Francisco on 03/13/15. For personal use only.

CHRONIC KIDNEY DISEASE David M. Kepecs1 M.Sc., Yanling Zhang1 MD Ph.D., Kerri Thai1, Suzanne L. Advani1 B.Sc., Darren A. Yuen1 MD Ph.D., Kim A. Connelly1 MD Ph.D., Hari Kosanam2 Ph.D., Eleftherios Diamandis2 MD Ph.D., Michael V. Sefton3 Sc.D., and Richard E. Gilbert1 MD Ph.D.

1

Keenan Research Centre for Biomedical Science of St. Michael’s Hospital, 209 Victoria

Street, Toronto, Ontario, Canada, M5B 1T8

2

Department of Pathology & Laboratory Medicine, Mt. Sinai Hospital, 60 Murray Street,

Toronto, Ontario, Canada, M5T 3L9

3

Donnelly Centre for Cellular and Biomedical Research, University of Toronto, 164 College

Street, Toronto, Ontario, Canada, M5S 3G9

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2 ABSTRACT


Cell-based regenerative therapies, offer a new, alternative approach to the treatment of chronic disease. Specifically, studies by our laboratory and others have shown that a subpopulation of cells derived from the bone marrow, known as early outgrowth cells (EOCs), are able to attenuate the progression of chronic kidney disease (CKD). Here we examined the efficacy of a tissue engineering system, in which EOCs were embedded into sub-millimeter sized collagen cylinders. These small individual units are referred to as modules and together form a functional microtissue. Due to their resemblance to endothelial cells, late outgrowth cells (LOCs) were used to coat the module surface, hypothesizing that as such they would promote vascularization and enhance engraftment of the encapsulated EOCs. These coated modules were transplanted subcutaneously into the subtotally nephrectomized rat model of CKD. While coated module therapy significantly improved both renal structure and function, non-coated modules with embedded EOCs were unable to reproduce these salutary effects on the kidney. Nevertheless, in both treatments, the embedded EOCs quickly degraded the modular environment and were seen to migrate to the liver, spleen and bone marrow as early as 6 days after transplantation. With the efflux of EOCs and, unexpectedly, no evidence of vascularization, we hypothesized that the LOCs did not enhance EOC engraftment, but rather augmented the renoprotection provided by EOCs by secretion of their own soluble and potent anti-fibrotic factors. To the best of our knowledge, this is the first study to document an effective subcutaneous approach for renoprotection.

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3 INTRODUCTION


The prevalence of chronic kidney disease (CKD) is expected to increase dramatically in the face of an aging population and the pandemic of diabetes. Unfortunately, despite current treatments, the majority of patients still experience progressive renal dysfunction and continue on to end stage disease.1 In the face of these therapeutic limitations, new and affordable therapies are urgently needed. The use of cell-based regenerative therapies is promising for the treatment of many chronic diseases, including CKD. Although organ-specific progenitor cells have been examined, most clinical and animal model-based studies have examined cells derived from the bone marrow. Among the various types of bone marrow derived cells (BMDCs), a sub-population defined by its culture characteristic as early outgrowth cells (EOCs), has been shown to be renoprotective in experimental CKD models.2-4 Notably, however, despite dramatic improvements in kidney structure and function, the administered EOCs were largely absent from the kidney. Instead, the EOCs were largely found in the liver, spleen and bone marrow,2 suggesting that EOCs exert their beneficial effects in an endocrine manner, facilitating tissue repair by secreting soluble anti-fibrotic factors from remote locations.

In contrast to this endocrine hypothesis, current therapeutic approaches to cell therapy are based on the paracrine mechanism of action that requires cells to be administered as close as possible to the site of injury. This invasive approach has been used in the heart where cells have been administered into coronary arteries or into the myocardium.5,6 While complications are relatively infrequent, a number of important concerns may limit the use of such an approach. Firstly, the invasive nature of cell administration requires specialized 3

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4 imaging facilities and carries with it the risk entailed by invasive procedures.

Secondly,


administration of highly proliferative, relatively undifferentiated cells also carries a risk of neoplastic transformation.7-15 Indeed, the latter was highlighted in a recent case report 15 in which a patient with kidney disease who had received BMDCs, injected into the kidney parenchyma, subsequently presented with masses at the sites of injection and hematuria. Nephrectomy revealed the masses as angiomyeloproliferative lesions that were derived from the administered cells. To obviate the need for direct injection into the site of injury, we sought to explore modular tissue engineering and subcutaneous delivery as an alternative therapeutic modality.

A recent “bottom-up” approach to tissue engineering involves the fabrication of small collagen modules that allow for the encapsulation of cells.16 As a consequence of collagen’s strong cell adhesive properties, the surface of collagen modules can be seeded with endothelial cells (ECs) to enhance vascularization.17 Previous reports in rats17,18 and SCID/bg mice19 have demonstrated that this EC coverage leads to the formation of vessel-like structures which can integrate into the host’s vascular system. This vasculature is expected not only to ensure the adequate oxygenation of the cells within the tissue construct but in this context to also facilitate the entry of the EOC-derived anti-fibrotic factors into the host’s circulation.

While EC coating of modules enhances vascularization, their use is limited by the difficulties in preparing primary, autologous EC cultures or the requirement for immunosuppressive treatment if allogeneic ECs are used.

To provide a feasible alternative, we considered

autologous/syngeneic bone marrow-derived, endothelial-like cells.

These cells are

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5 phenotypically and functionally similar to those of mature endothelium, arising after prolonged


incubation of the circulating mononuclear cells from which EOCs are derived. After 14-21 days of cell culture, endothelial-like cells emerge as rapidly proliferating colonies20 that given their long period of cell culture are commonly referred to as late outgrowth cells (LOCs).21

We hypothesized that a collagen module that contained EOCs within and LOCs on its surface would have the advantage of: (i) enabling EOC secretions to easily permeate and enter the systemic circulation, (ii) require a minimally invasive procedure of administration that would also permit their easy removal in the event of a rationale to terminate therapy (e.g., neoplastic transformation) and (iii) maintain long-term viability as a consequence of LOC-facilitated vascularization. Here, we investigate the renoprotective effects of EOC-containing modules, with or without LOC coverage, comparing these approaches with the intravenous injection of EOCs.

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6 METHODS


Cell isolation and culture EOCs were cultured as previously described.22 Briefly, bone marrow cells were flushed from the femora and tibiae of 3-4 week old male Fischer 344 (F344) rats with sterile phosphatebuffered saline (PBS). The collected cells were plated in differential endothelial cell culture medium (EGM-2, Lonza, Walkersville, MD) on human fibronectin-coated tissue culture flasks and incubated at 37C with 5% CO2 for 10 days to produce EOCs. The culture medium was changed every 2-3 days. To generate LOCs, bone marrow cells were plated at a 1:4 dilution and cultured as described above. After 2 weeks in culture, confluence was reached and the adherent cells were trypsinized and collected by centrifugation at 1400 rpm for 7 minutes. The supernatant was discarded, and the cell pellet was resuspended in EGM-2 and once again plated at a 1:4 dilution. After an additional 10 days in culture, cells reached confluence and were considered LOCs.

Cell Characterization EOCs and LOCs were detached from their flasks using trypsin or Accutase (Stemcell technologies, Vancouver, BC), the latter being used exclusively for those cells immunostained with VE-cadherin. Afterwards, 1x106 LOCs and 1x106 EOCs were transferred into individual 15 mL falcon tubes and centrifuged at 1400 rpm for 5 min. Each individual cell pellet was then suspended in 400 μL of BSA/PBS, followed by the addition of a specific antibody at 0.01μg/μL. For the six cell surface markers assessed in this experiment, the following antibodies were tagged with specific fluorophores: CD34 (APC), VEGFR2 (V450), CD133 and VE-cadherin (PE), CD45 (PE/Cy7) and isolectin (FITC). Each sample was then incubated for 6

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7 20 min at 4°C and individually subjected to flow cytometry with the MACSQuant analyzer


(Miltenyi biotec, Cambridge, MA). The percentage of fluorescently labeled cells was then determined using MACSQuantify software (Miltenyi biotec).

Module fabrication Acidified type-1 collagen (3.1mg collagen/ml; Cohesion technologies, Palo Alto, CA) was mixed with 10xMEM (Invitrogen) and neutralized with 0.8 NaHCO 3, as previously described.23 Neutralized type-1 collagen with or without EOCs (Sigma, Mississauga, ON) was then infused via syringe pump (Razel Scientific Instruments, Inc., Stamford, CT) into the side inlet of a custom-designed T-junction device via polysulfone Masterflex Tygon L/S 13 lab tubing (ColeParmer Instrument Co., Vernon Hills, IL).24 At the same time, compressed air (75.8 kPa) was directed into the upper inlet of the T-junction. With this apparatus, the compressed air meets the collagen fraction at the intersection region of the T-junction whereby the pressurized air generates collagen modules of approximately 2mm in length. The thus-formed collagen modules were then propelled from the T-junction’s lower outlet into three meters of gassterilized polyethylene tubing (PE60, 0.76 mm ID, Becton Dickinson and Company, Franklin Lakes, NJ). The tubing was then removed from the T-junction outlet and placed within a humidified incubator for 1-2 hours at 37ºC and 5% CO2 to allow the collagen to gel. To recover modules, a 10 mL air-filled syringe was connected to the tubing by an 18G needle (Becton Dickinson) to expel the modules into a non-tissue culture treated dish containing EGM-2. Pseudo-endothelialization of the module surface was performed as described previously with ECs.17 Briefly, EOC-containing modules were incubated with 3-5x106 LOCs in a 15mL falcon tube containing 5mL of EGM-2 and gently rocked for 1 hour to ensure maximal

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8 coverage. After rocking, the module suspension was transferred to a non-tissue culture


treated dish and placed in a 37°C, 5% CO2 incubator for 24 hours.

Viability assessment of module encapsulated EOCs After 24 hrs of incubation, the culture medium was aspirated and modules underwent three washes with PBS until their final suspension in 1mL of PBS. A Live/Dead kit (Life Technologies, Burlington, ON) was used to quantify the percentage of viable EOCs within the modules, whereby 1 µL calcein (live cells, excitation/emission: ~495/~515) and 2 µL of ethidium bromide (dead cells, excitation/emission: ~530/~615) were incubated with the module suspension for 30 minutes at 20C. Modules were then washed three times with PBS and imaged using a Zeiss LSM510-META confocal microscope. Fifteen modules were randomly selected and quantified for the presence of both live (green fluorescence) and dead (red fluorescence) EOCs to calculate the proportion that were viable. The LOCs, used in the in vivo studies to facilitate vascularization were not included in this in vitro component.

3

H-proline incorporation

Anti-fibrotic activity was examined by quantifying the incorporation of [3H]-proline as an index of fibroblast collagen production. Following serum starvation, NRK-49F cells (ATCC, Manassas, VA), a renal fibroblastic cell line, were incubated with 0.5 mL of conditioned medium for 4 hours. Unconditioned serum-free endothelial basal medium (EBM-2) served as a negative control. Fibroblasts were stimulated with transforming growth factor-β1 (TGF-β1) at a concentration of 20 ng/mL, and incubated with [ 3H]-proline (1 μCi/well, L-[2,3,4,5-3H]proline; Amersham Biosciences, QC) for 44 hours, as an index of collagen production.

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9 Fibroblasts were harvested, washed four times with PBS, solubilized in 0.75 ml of 1 M NaOH


and then neutralized with 0.5 ml 1 M HCl. The incorporation of exogenous [3H]-proline was measured using a liquid scintillation counter (LS 6000 Beckman Instruments, Inc.).25 Subtotal nephrectomy animal model The subtotally nephrectomized or remnant kidney rat provides a well-established model of CKD with close similarities to what is seen in humans. In brief, F344 rats (Charles River, Montreal, QC) of 12 weeks of age underwent one-step subtotal nephrectomy (SNX, n = 49) or sham surgery (n = 10), as previously described.26 Briefly, animals were anesthetized with inhaled 2.5% isoflurane. The right kidney was removed via subcapsular nephrectomy and infarction of approximately two thirds of the left kidney was achieved via selective ligation of 2 out of the 3 or 4 branches of the renal artery. Sham surgery consisted of laparotomy and manipulation of both kidneys before wound closure. All animal studies were approved by the St. Michael’s Hospital Animal Ethics Committee.

Module and EOC injection Four weeks after SNX surgery, urinary protein excretion was examined to ensure similar distributions of proteinuria among each group prior to the initiation of treatment. Based on these values, animals were randomly allocated into four groups: (1) modules containing 106 EOCs, (2) LOC coated modules containing 106 EOCs, (3) cell free modules, and (4) 106 EOCs in sterilized PBS. After 24 hours of incubation, each plate of modules was washed three times with sterilized PBS to remove any culture medium residuum and suspended in 0.5mL of PBS in a 1mL syringe. Immediately afterwards, modules were injected subcutaneously into the right hindquarter using an 18G needle. The intravenous

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10 administration of 1 x 106 EOCs in sterile PBS, prepared as previously described,2 served as


the positive control.

Kidney function Prior to surgery and 4 and 8 weeks after it, animals were housed in metabolic cages to collect 24-hour urine samples for subsequent determination of urine protein excretion using the benzethonium chloride method. Systolic blood pressure was also measured at these time points in conscious rats using an occlusive tail-cuff plethysmograph attached to a pneumatic pulse transducer (Powerlab, ADInstruments, Colorado Springs, CO) as previously described.27 Prior to termination, glomerular filtration rate (GFR) was assessed using a modified FITC-inulin plasma clearance assay.28 Briefly, 3.74 L/g body weight of FITC-inulin was injected into the tail vein of each rat. Venous blood was then sampled at 3, 7, 10, 15, 35 and 55 minutes after receiving FITC-inulin. The concentration of this agent was then assayed by its fluorescence with a Spectramax M5e microplate reader (Molecular Devices, Sunnyvale, CA) with 485 nm excitation and 527 nm emission settings. GFR was calculated using a two phase, exponential decay curve and non-linear regression method, as previously described28 in which GFR = I/(A/ + B/), where I is the amount of FITC-inulin injected, A and B are the yintercept values for the two decay rates, and  and  are the decay constants for the distribution and elimination phases. Histochemistry and immunohistochemistry At the end of the study, 8 weeks post-surgery, animals were terminated.

Kidneys were

excised as was the subcutaneous tissue containing the modules. Tissues were immersion fixed in 10% neutral buffered formalin, embedded in paraffin or flash frozen in liquid nitrogen 10

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11 and embedded in cryostat matrix (Tissue-Tek, Sakura, Kobe, Japan). Formalin-fixed kidneys


were

sectioned

and

immunohistochemically.

stained

with

Periodic-acid

Schiff

(PAS)

stain

or

examined

Subcutaneous tissue containing modules were fixed in formalin,

embedded in paraffin, and sectioned before staining with Masson’s trichrome and haematoxylin and eosin (H&E).

Glomerulosclerosis was assessed on PAS-stained sections using a semi-quantitative technique in a masked fashion, as described previously.26 The degree of sclerosis was subjectively graded on a scale of 0-4: 0, normal; 1, sclerotic area of 50-75% (moderate to severely sclerotic); 4, sclerotic area of >75% (severely sclerotic). A glomerulosclerosis index was calculated for each animal by averaging scores from all the glomeruli in a kidney section, as also previously reported.26

To visualize ECs in subcutaneous tissue, sections were immunostained with the mouse antirat monoclonal antibody CD31 (Abcam, Toronto, ON) while glomerular ECs were immunostained with the mouse anti-rat monoclonal Aminopeptidase P antibody JG-12 (Bender Medsystems, Atlanta, GA).27 Glomerular capillary density was expressed as the proportional area of JG-12 immunostaining in 30 randomly selected glomeruli from each rat and quantified using computer-assisted image analysis, as previously described.27 Tubulointerstitial fibrosis was assessed by examining the accumulation of collagen type IV in the renal cortex, detected by immunostaining with a goat anti-rat type IV collagen polyclonal antibody (Southern Biotech, Birmingham, AL), and quantified as the proportional area in 9

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12 non-overlapping 20X fields for each animal, using computer-assisted image analysis, as


previously described.29 All analyses were performed with prior masking of the identity of the study group from which the sections had been obtained.

CMTMR and CMFDA Labeling and Tracking To determine the fate of EOCs and LOCs in vivo, cell tracking studies were performed. EOCs were firstly incubated with a 5 mM solution of 5-(and-6)-4-chloromethyl-benzoyl-amino-tetramethylrhodamine (CMTMR, Invitrogen, Carlsbad, CA) for 30 minutes. Immediately afterwards these fluorescently labeled EOCs were encapsulated into collagen modules. LOCs were incubated with a 15mM solution of 5-Chloromethylfluorescein Diacetate (CMFDA, Invitrogen) for 30 min and then used to coat the surface of the EOC-containing modules. These coated modules were then placed in a 37°C, 5% CO2 incubator for 24 hours, and injected subcutaneously into the right hindquarter 4 weeks after surgery. Animals (n=3) were sacrificed 6 days after module injection. The presence of CMTMR and CMFDA positive cells was determined on a Zeiss LSM510-META confocal microscope by counting their presence in 10 randomly selected fields in a 40 mm frozen section embedded in cryostat matrix.30 Sections were counterstained with wheat germ agglutinin 488 (WGA 488, Invitrogen) to better visualize tissue architecture.

Statistical analysis All data are shown as mean  SEM. A minimum number of 3 independent experiments were performed for all in vitro experiments, and analyzed by a one-way ANOVA with a post-hoc Tukey’s test. Differences between in vivo groups were analyzed by a one-way ANOVA with a

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13 post-hoc Fischer’s Least Signficant Difference (LSD) test. Accordingly, for histological


experiments, we also used a one-way ANOVA with a post-hoc LSD test, with the exception of the glomerulosclerosis index, which was analyzed using a Kruskal-Wallis with a post-hoc Dunn’s test. All statistics were performed using GraphPad Prism 6.00 for Mac OS X (GraphPad Software, San Diego, CA). A change was considered statistically significant when p90% of LOCs still expressed endothelial surface markers, they were less likely to express either CD133 or CD45 markers when compared with EOCs (Table 1).

Cell viability and activity in vitro Since the plan for in vivo experiments was to subcutaneously implant these modules 24 hours after fabrication, we assessed the viability of EOCs in the uncoated module at this crucial time point.

Using the Live/Dead assay, the viability of the encapsulated EOCs was >90%.

Furthermore, EOCs that fluoresced red were uniformly distributed throughout the module (Fig. 1A), suggesting the absence of any diffusion constraints of either oxygen or nutrients to the core of the module.

We also examined the module’s permeability to secreted anti-fibrotic factors. As previously demonstrated,2 when treated with conditioned medium from EOCs (EOC-CM) a significant reduction in the extent of 3H-proline incorporation in fibroblasts stimulated with TGF-β was noted. The extent of collagen production was similarly attenuated when fibroblasts were treated with the conditioned medium generated from modules containing EOCs (Fig. 1B), 14

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15 confirming that the modules are, indeed, permeable to the anti-fibrotic factors produced by


EOCs.

EOCs encapsulated in pseudo-endothelialized modules preserve renal function Untreated SNX animals develop heavy proteinuria, hypertension (SBP) and a substantial reduction in GFR. In accordance with earlier reports by our laboratory, this progression of kidney injury was diminished by intravascular EOC infusion. Similar preservation of GFR was noted in animals that had received LOC-coated, EOC-containing modules when compared with non-treated controls (Fig. 2), however not to the same extent as sham-operated animals. Reduction in blood pressure was also evident as was the extent of proteinuria, albeit the latter fell short of our predefined level of statistical significance (Table 2). No significant benefit was noted in SNX rats treated with uncoated EOC containing modules with findings that were intermediate between empty and coated modules.

EOCs encapsulated in coated modules attenuate renal fibrosis and capillary rarefaction When compared with sham-operated animals, SNX rat kidneys display widespread capillary loss along with extensive fibrosis in both the glomerulus (glomerulosclerosis) and the tubulointerstitium (tubulointerstitial fibrosis). As previously reported, the administration of EOCs attenuates the extent of capillary rarefaction, glomerulosclerosis and tubulointerstitial fibrosis in SNX rats.2 Coated module therapy was similarly effective in ameliorating both fibrosis (Fig. 3 and 4) and capillary loss (Fig. 5) while uncoated EOC containing modules were not.

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16


EOCs lead to module degradation and cell migration Coated modular therapy was hypothesized to provide renoprotection by creating a vascularized niche for encapsulated EOCs. However, no augmented vascularization was observable at the site of module implantation. Indeed, neither modules nor the EOCs that they contained on implantation were visible by light microscopy.

Instead, only scattered

remnants of modules were observed at the sites of implantation (Fig. 6). Here, the absence of an inflammatory infiltrate in the region where the cell containing modules had been implanted, suggested that EOC secreted collagenases may have been responsible for module degradation.33

Consistent with the degradation of modules, tracking studies revealed the absence of EOCs within the subcutaneous tissues in which the modules had been implanted.

In contrast,

labeled EOCs were abundantly present in the bone marrow, liver and spleen, albeit less so than when the EOCs were injected intravenously (Fig. 7).

LOCs also demonstrate potent anti-fibrotic activity In contrast to EOCs where tracking studies showed that they had vacated the modules, fluorescently labeled LOCs were notably still present on the module surface 6 days after transplantation. Despite the comparatively fewer labeled EOCs in the liver, spleen and bone marrow of animals that had received coated modules when compared with those treated with intravenous EOCs, both groups showed similar attenuation in fibrosis and preservation of kidney function. Accordingly, we considered that while ostensibly endothelial cell-like, LOCs

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17 might also exert anti-fibrotic effects.

Indeed, when conditioned medium from LOCs was


subjected to the 3H-proline assay of fibrogenesis, it was found to be equally potent as EOCCM in attenuating TGF-β stimulated 3H-proline incorporation (Fig. 8).

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18 DISCUSSION


In the present study, we explored modular tissue engineering with the aim of developing a clinically feasible approach to treating CKD. We hypothesized that vascularized subcutaneous modules would enable EOCs to remain viable in the longterm while secreting their biologically active factors that would ultimately reach the systemic circulation. The present study showed that modular therapy did, indeed, reproduce the benefits seen with intravascular EOC infusion, doing so by subcutaneous delivery. Notably, only modules coated with LOCs were renoprotective, with non-coated modules yielding no significant differences in renal structure and function when compared with empty modules. The enhanced renoprotection seen with coated module therapy was originally hypothesized to be the result of module vascularization, given the similarities between LOCs and mature ECs.

As such, they were expected to

replicate the previously reported superior efficacy of modules coated with allogeneic ECs17,18,34 but without the need for concomitant immunosuppression since cells were syngeneic.

On histological examination, however, not only was there no evidence of

vascularization but only scattered remnants of the modules remained. Empty modules, on the other hand, remained intact.

A recent report by Gupta and Sefton34 demonstrated that islet-encapsulated modules remain intact for at least 60 days after implantation, a considerably longer time than used in our study. Syngeneic islets were used to avoid an immune-mediated inflammatory response that may be harmful to the encapsulated islets or modular device. Specifically for the latter, proinflammatory cytokines, like tumor necrosis factor (TNF-α)35 and Interleukin-10 (IL-10)36 have been shown to induce the expression of collagenases, which in turn have the potential to 18

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19 degrade these modular constructs. In our study, we also employed a syngeneic approach by


isolating BMDCs from healthy, genetically identical donors. Accordingly, as expected, we found no evidence of inflammation at the site of module implantation. In light of these findings, we considered the possibility that rather than an inflammatory infiltrate, the EOCs were responsible for degrading their own modules. Indeed, previous studies have shown that EOCs secrete a range of matrix-degrading enzymes that include the collagenases, matrix metalloproteinases 2,3 and 19,33 that likely contributed to the degradation of the modules’ collagenous meshwork and for the egress of the cells that they contain.

In light of the extensive module degradation, we speculated that the EOCs would migrate to the liver, spleen and bone marrow to exert their renoprotective effects. This premise seemed conceivable given the migratory nature of BMDCs and the embryonic sites of extramedullary hematopoiesis within these organs. While the encapsulated EOCs did egress and migrate to the liver, spleen and bone marrow, we did not observe these cells at nearly the same abundance as seen with intravascular EOC infusion.2 While the precise mechanisms underlying the relative paucity of module-derived EOCs within these organs is unknown, we speculate that it may be a consequence of apoptosis, necrosis or migration following the injection of modules into the relatively avascular subcutaneous tissue. As such, the decreased dosage of EOCs provides a plausible explanation for why non-coated module therapy offers intermediate, yet non-significant renoprotection.

These findings led us to

speculate that LOCs might also contribute to the anti-fibrotic effects of the coated modules.

Since LOCs are derived from the bone marrow like EOCs, only cultured for longer, it seemed reasonable to hypothesize that LOCs may also secrete soluble anti-fibrotic factors capable of 19

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20 mediating renoprotection. Akin to the conditioned medium derived from EOCs, LOC-CM also


potently reduced proline incorporation in renal fibroblasts. This finding suggests that the presence of LOCs on the surface of the module conferred an additional degree of anti-fibrotic activity rather than contributing to a vascular network for the modules. However, unlike EOCs that had almost all migrated to the liver, spleen and bone marrow to exert their anti-fibrotic effect, LOCs seem more likely to have secreted such factors from within the vicinity of the module. Unlike previous reports17,18,34 we did not use mature ECs for the endothelialization of the module surface. Instead we used syngeneic marrow-derived LOCs to prevent the need for immunosuppressive treatment. The difference between these LOCs and mature ECs offers a potential explanation for the minimal vascularization seen at site of transplantation. While LOCs strongly expressed endothelial markers, there was also concomitant expression of hematopoietic markers, albeit at lower levels, suggesting our marrow-derived LOCs are perhaps not fully functional ECs but rather present a more progenitor-like phenotype. The poor vascularization could also be attributed to the fact that ECs are in constant contact with extracellular matrix proteins in vivo37 and with the degradation of collagen these LOCs are left without a scaffold and are unable to properly function as endothelium.

With its low

antigenicity and strong cell adhesion properties, collagen is ideal for tissue engineering. However, its susceptibility to enzymatic degradation, as exemplified in this study suggest that other materials may be more suitable if they are to be used with EOCs. Indeed, alginate, a naturally occurring polysaccharide, might prove superior, after modification to enable LOC/EC attachment, given its previous use in cell encapsulation38,39 where it has been shown not to degrade in the presence of EOCs.39 Alternative strategies, however, would include the 20

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21 continued use of collagen matrices that had been impregnated with collagenase inhibitors


such as marimistat or the use of poloxamine/collagen interpenetrating networks.40

In summary, this is the first study, to the best of our knowledge, to document an effective subcutaneous approach for renoprotection. Coated module therapy was successful in attenuating the functional and structural manifestations of CKD, with efficacy similar to intravascular EOC infusion.

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22 ACKNOWLEDGEMENTS


The authors would like to thank Christine Kuliszewski, Jen Switzer, and Deborah Koh for their technical support of the animal studies. We would also like to express our gratitude to Chuen Lo and Dean Chamberlain for their technical assistance and guidance throughout the operation of these studies. The authors would like to acknowledge the financial support we received from the Canadian Institute of Health Research (CIHR) and the Heart and Stroke Foundation of Ontario. David Kepecs also acknowledges support from the Queen Elizabeth II/ Heart and Stroke Foundation Graduate Scholarship in Science and Technology, as well as an Ontario Graduate Scholarship. Dr. Darren Yuen was previously supported by a KRESCENT postdoctoral fellowship, and currently holds the KRESCENT New Investigator and Canadian Diabetes Association Clinician Scientist Award. Dr. Richard Gilbert is the Canadian Research Chair in Diabetes Complications and this research was supported in part by the Canadian Research Chair Program.

AUTHOR DISCLOSURE STATEMENT No competing financial interests exist.

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Address correspondence to: Richard E. Gilbert, MD Ph.D. Chief, Division of Endocrinology Keenan Research Centre, Li Ka Shing Knowledge Institute 209 Victoria Street Room 508 Toronto, ON Canada M5B 1T8 Email: [email protected]

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FIG. 1. In vitro assessment of EOCs embedded in uncoated modules. (A) Live/Dead assay of embedded EOCs revealed an average of 91% cells were viable (green fluorescence) 24 hours after module fabrication. (B) Conditioned medium generated from non-coated modules with embedded EOCs significantly attenuated 3H proline incorporation in TGF-β stimulated renal fibroblasts. * p

Combination therapy in chronic kidney disease?

A perspective on anti-CCN2 therapy for chronic kidney disease.

Salivary Alterations in Rats with Experimental Chronic Kidney Disease.

Combination therapy with renin-angiotensin-aldosterone system inhibitor telmisartan and serine protease inhibitor camostat mesilate provides further renoprotection in a rat chronic kidney disease model.

Experimental evidence that preexisting chronic kidney disease is a risk factor for acute kidney injury.

Exercise as an Adjunct Therapy In Chronic Kidney Disease.

Iron therapy in anaemic adults without chronic kidney disease.

Screening for Chronic Kidney Disease.

The WISHED Trial: implementation of an interactive health communication application for patients with chronic kidney disease.

Progression from acute kidney injury to chronic kidney disease: clinical and experimental insights and queries.

Role of hypoxia in progressive chronic kidney disease and implications for therapy.

Rhabdomyolysis associated with initiation of febuxostat therapy for hyperuricaemia in a patient with chronic kidney disease.

Chronic anticoagulation in chronic kidney disease.

Experimental validation of the new modular application of the upper bound theorem in indentation.

Chronic kidney disease: considerations for nutrition interventions.

Emerging drugs for chronic kidney disease.

Renal denervation--implications for chronic kidney disease.

Challenges and opportunities for stem cell therapy in patients with chronic kidney disease.

Safety of medical therapy in patients with chronic kidney disease and end-stage renal disease.

Advances in Chronic Kidney Disease.

Anti-TNFα therapy for chronic inflammatory disease in kidney transplant recipients: Clinical outcomes.

Chronic kidney disease in children.

Chronic kidney disease.

Cell-based therapy for kidney disease.