Repeated treatment with bone marrow cell secretory products maintains long-term renoprotection in experimental chronic kidney disease: a placebo-controlled trial

Conditioned medium

Conditioned medium was generated as previously described 8]. In brief, bone marrow cells were flushed from the femora and tibia of 4-week-old
male Fischer F344 rats and plated in endothelial cell growth medium (EGM-2, Lonza,
Walkersville, MD) on human fibronectin-coated tissue culture flasks and incubated
at 37 °C with 5 % CO
₂
for 10 days to give rise to EOCs. EGM-2 consists of serum-free endothelial basal medium-2
(EBM-2, Lonza) supplemented with growth factors such as vascular endothelial growth
factor, epithelial growth factor, and fetal bovine serum. After repeatedly washing
the EOCs with phosphate-buffered saline to remove any residual serum, subconfluent
cells were incubated with serum-free EBM-2 for 24 h to collect EOC-released factors
8]. Culture supernatant was then collected and the resulting cell-free, EOC-conditioned
medium (EOC CM) was concentrated tenfold using a 10-kDa cutoff centrifuge filtration
column (Millipore, Billerica, MA), followed by filtration through a 0.45-?m filter
(Millipore). Unconditioned medium (UCM) consisting of serum-free endothelial basal
medium-2 not incubated with cells was similarly concentrated. Half-milliliter aliquots
of concentrated EOC CM and UCM were stored at ?80 °C until administered as thrice-weekly
tail vein injections.

Animal model

Studies were performed in subtotally nephrectomized rats, a model of non-immune progressive
kidney disease that like chronic kidney disease in humans is characterized by proteinuria,
declining glomerular filtration rate, and hypertension. Twenty-nine, 8-week-old F344
rats underwent one-step subtotal nephrectomy (SNX) according to previously published
protocols 8], 11]. All animal studies were approved by the St. Michael’s Hospital Animal Ethics Committee
(ACC862) and followed the guidelines for animal use and care as described by the Canadian
Council on Animal Care.

Study design

Four weeks following SNX, rats were randomized to receive unconditioned medium (No
Therapy group, n?=?14) or EOC-conditioned medium (Therapy group, n?=?15). After an initial 2-week set of thrice-weekly injections of EOC-conditioned
medium, Therapy group animals were then randomly assigned to receive either a repeat
2-week course of thrice-weekly conditioned or unconditioned medium injections. Repeat
administration (of either conditioned medium or unconditioned medium) to all Therapy
group animals was mandated by the study protocol if mean urinary protein excretion
rose fourfold relative to the level following completion of the initial treatment
course. This cutoff for repeat therapy was chosen (1) to establish when the effect
of initial EOC-conditioned medium treatment wanes and (2) to define a threshold for
repeat treatment that was clinically relevant. Given the large variability typically
seen in urinary protein excretion rates in the subtotal nephrectomy model 8], we reasoned that using a lower threshold might trigger repeat treatment at a time
when the effects of initial EOC medium treatment might not have clearly waned.

No Therapy group animals received the initial course of thrice-weekly UCM injections
beginning 4 weeks post-SNX and another 2 weeks of unconditioned medium injections
when the Therapy groups received their second course of CM. Animals were then monitored
for four additional weeks. Accordingly, three groups of animals were studied: a No
Therapy group (UCM?+?UCM, n?=?14), an Initial Therapy Only group (EOC CM?+?UCM, n?=?8), and a Repeat Therapy group (EOC CM?+?EOC CM, n?=?7) (Fig. 1).

Fig. 1. Study design. UCM unconditioned medium, EOC CM early outgrowth cell-conditioned medium, SNX subtotal nephrectomy, wks weeks

Outcome measurements

Systolic blood pressure was measured using a tail-cuff plethysmograph (ADInstruments,
Colorado Springs, CO) 8], 11], 12]. Urinary protein excretion was determined as protein:creatinine ratios measured on
spot urine collected before surgery and 4, 6, 8, 10, 12, and 14 weeks after. Glomerular
filtration rate (GFR) was measured just prior to termination using a modified FITC-inulin
plasma clearance method 8].

To assess structure, the kidneys, excised at termination, were immersion fixed in
10 % neutral buffered formalin. Glomerular endothelial cell density, glomerulosclerosis,
and tubulointerstitial fibrosis were assessed on JG-12 or type IV collagen immunostained
formalin-fixed sections, respectively, as previously described 8]. Analyses of kidney structure were performed in a masked fashion using computer-assisted
image analysis, as described previously 12].

Statistical analysis

All data are shown as mean?±?SEM unless otherwise stated. Differences between groups
were analyzed by ANOVA with a post hoc Tukey’s test. A change was considered statistically
significant if p??0.05.