Changhai Road, Shanghai, People’s Republic of …24 Parth U. Thakker, [email protected] 25...
Transcript of Changhai Road, Shanghai, People’s Republic of …24 Parth U. Thakker, [email protected] 25...
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Reno-protection of Urine-derived Stem Cells in A Chronic Kidney Disease Rat Model Induced by 1
Renal Ischemia and Nephrotoxicity 2
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Chao Zhang1,2, Sunil K. George1, Rongpei Wu1,3, Parth Udayan Thakker4, Mehran Abolbashari1, 5
Tae-Hyoung Kim1,5, In Kap Ko1, Yuanyuan Zhang1*, Yinghao Sun2, John Jackson1, Sang Jin Lee1, 6
James J. Yoo1, and Anthony Atala1 7
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1 Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Medical 10
Center Boulevard, Winston-Salem, NC, USA 11 2 Department of Urology, Changhai Hospital, the Second Military Medical University, 168 12
Changhai Road, Shanghai, People’s Republic of China 13 3 Department of Urology, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guang 14
Dong, People's Republic of China. 15 4 Department of Urology, Wake Forest Baptist Medical Center, Medical Center Boulevard, 16
Winston-Salem, NC, USA 17 5 Department of Urology, College of Medicine, Chung-Ang University, Seoul, South Korea 18
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Chao Zhang, [email protected] 21
Sunil K. George, [email protected] 22
Rongpei Wu, [email protected] 23
Parth U. Thakker, [email protected] 24
Mehran Abolbashari, [email protected] 25
Tae-Hyoung Kim, [email protected] 26
In Kap Ko, [email protected] 27
Yuanyuan Zhang, [email protected] 28
Yinghao Sun, [email protected] 29
John Jackson [email protected] 30
Sang Jin Lee, [email protected] 31
James J. Yoo, [email protected] 32
Anthony Atala, [email protected] 33
*Corresponding author: Yuanyuan Zhang, [email protected] 34
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Abstract 35
Purpose: Drug-induced nephrotoxicity can occur in patients with pre-existing renal dysfunction 36
or renal ischemia, potentially leading to chronic kidney disease (CKD) and end-stage renal 37
disease (ESRD). Prompt treatment of CKD and the related side effects is critical in preventing 38
progression to ESRD. The goal of this study was to demonstrate the therapeutic potential of 39
urine-derived stem cells (USC) to treat chronic kidney disease-induced by nephrotoxic drugs and 40
renal ischemia. 41
Materials and methods: Human USC were collected, expanded and characterized by flow 42
cytometry. A CKD model was induced by creating an ischemia-reperfusion injury and gentamicin 43
administration. Twenty-eight adult immunodeficient rats were divided into three groups: 44
PBS-treated group (n=9), USC-treated group (n=9), and sham group with age-matched control 45
animals (n=10). Cell suspension of USC (50 x 106 / 100µl / kidney) or PBS was injected bilaterally 46
into the renal parenchyma 9 weeks after CKD model creation. Renal function was evaluated by 47
collection blood and urine samples to measure serum creatinine and glomerulus filtration rate. 48
The kidneys were harvested 12 weeks after cell injection. Histologically, the extent of 49
glomerulosclerosis and tubular atrophy, the amount of collagen deposition, interstitial fibrosis, 50
inflammatory monocyte infiltration, and expression of transforming growth factor beta 1 (TGF-ß1), 51
and superoxide dismutase 1 (SOD-1) were examined. 52
Results: USC expressed renal parietal epithelial cells (CD24, CD29 and CD44). Renal function, 53
measured by GFR and serum Cr in USC-treated group were significantly improved compared to 54
PBS-treated animals (p<0.05). The degree of glomerular sclerosis and atrophic renal tubules, 55
the amount of fibrosis, and monocyte infiltration significantly decreased in USC-treated group 56
compared to the PBS group (p<0.05). The level of TGF-ß1 expression in renal tissues was also 57
significantly lower in the PBS group, while the level of SOD-1 expression was significantly 58
elevated in the USC group, compared to PBS group (p<0.05). 59
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Conclusions: The present study demonstrates the nephron-protective effect of USC on renal 60
function via anti-inflammatory, anti-oxidative stress, and anti-fibrotic activity in a dual-injury CKD 61
rat model. This provides an alternative treatment for CKD in certain clinical situations, such as 62
instances where CKD is due to drug-induced nephrotoxicity and renal ischemia. 63
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1. Introduction 64
Chronic kidney disease (CKD) is a major health problem characterized by a gradual loss of 65
kidney function over time. Over 30 million people or 15% of US adults are estimated to have 66
CKD [1]. CKD may progress to end-stage renal disease (ESRD), which is a public health 67
concern and big economic burden. ESRD costs the United States taxpayers approximately 32.8 68
billion dollars in annual Medicare expenditures [2]. Causes of CKD vary widely, however 69
drug-induced nephrotoxicity has been an increasingly recognized complication of many 70
therapeutic agents in the clinical setting. In patients with pre-existing renal dysfunction or renal 71
ischemia, the effects of drug-induced nephrotoxicity can be profound and may accelerate 72
progression to ESRD [2]. At present, hemodialysis and renal transplantation are both effective 73
treatment modalities for ESRD. However, hemodialysis is expensive with many potential 74
complications while renal transplant as a treatment modality is limited by the number organ 75
donors available [3]. 76
Cell therapy has emerged as a promising therapeutic approach for the treatment of CKD 77
with great potential [4]. Several stem cell types from a variety of sources have been used in 78
animal experiments [5-10]. Bone marrow-derived stem cells were found to significantly improve 79
renal function in mice models [5], and their safety was subsequently studied in a phase 1 clinical 80
trial [11]. In addition, stem cells derived from amniotic fluid [6, 7], endothelium [8], adipose 81
tissue [9] and primary renal cells [10] have demonstrated therapeutic effect in renal regeneration. 82
However, obtaining these stem cells requires invasive procedures, which may cause iatrogenic 83
injury to adjacent organs and increases infection risk among other complications. Thus, the 84
desire to obtain stem cells from a simple, non-invasive and inexpensive route is of utmost 85
necessity. 86
Recently, we demonstrated that stem cells exist in human urine and possess the potential for 87
clinical applications [12-20]. Urine derived stem cells (USC) are thought to originate from the 88
parietal cells in renal glomeruli [14] and have the potential for tissue regenerative effects, 89
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including robust proliferative potential, multi-potential differentiation, and the ability to exert 90
regenerative effects via paracrine factors [14, 21, 22]. USC secrete multiple paracrine factors to 91
recruit native cells to participate in tissue regeneration and induce immune-modulatory changes 92
in vivo. Studies from our group and other laboratories have demonstrated that USC or the media 93
derived from USC significantly improved renal function by reducing inflammation and fibrin matrix 94
deposition within the kidney in different rodent CKD models, including age-related kidney 95
disease [23] and streptozotocin-induced diabetic nephropathy [24, 25]. Furthermore, USC 96
enhanced renal function in an acute renal ischemia model [26]. Nephrotoxic drug induced renal 97
damage superimposed on pre-existing renal damage is one of the more common causes of 98
iatrogenic CKD [2]. However, it is challenge to revise the renal function and prevent histological 99
structure damages from the chronic renal damages in clinical settings. Compared to stem cells 100
derived from other sources such as amniotic fluid, placenta, bone marrow, adipose tissues, USC 101
have unique advantages in the treatment of CKD. USC can be obtained from simple, safe, 102
non-invasive and low-cost approaches. A large of amount of stem cells can be generated 103
through a few weeks. In addition, as the kidney tissue specific stem cells, USC might be optimal 104
for the kidney tissue repair. As such, the goal of this study was to determine therapeutic impact of 105
USC on renal parenchyma architecture and renal function in a rat CKD model, induced by dual 106
nephrotoxic drug-renal ischemia injury. 107
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2. Methods 108
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2.1 USC isolation and identification 110
Collection of human urine samples for USC isolation in this study was approved by the 111
Wake Forest University Health Sciences Institutional Review Board. USC culture and 112
characterization have been described previously [12-20]. In brief, USC were collected from 113
healthy adult males (n=6) and expanded in culture media comprised of keratinocyte serum-free 114
medium (Gibco, Gaithersburg, MD) and Dulbecco's Modified Eagle's medium (DMEM, high 115
glucose, Gibco, Gaithersburg, MD) with 5% fetal bovine serum (FBS) (Gibco, Gaithersburg, MD) 116
containing supplements [12], see Table 1. Expanded USC were trypsinized and incubated with 117
anti-human antibody labeled with cell surface markers of renal parietal epithelial cells (CD24, 118
CD29 and CD44) (BD, Franklin Lakes, NJ) [27], mesenchymal stem cells (MSC) (CD73, CD90, 119
CD105, and CD146) (BD, Franklin Lakes, NJ), embryonic stem cells (SSEA4), hematopoietic 120
stem cell (CD31, CD34, CD45) (BD, Franklin Lakes, NJ) and STRO-1 (BioLegend, San Diego, 121
CA). Fluorescein isothiocyanate (FITC) or phycoerythrin (PE) conjugated isotype antibodies 122
were used to determine background fluorescence. All cells were analyzed using BD FACS 123
Caliber analytical fluorescence activated cell sorter (BD, Franklin Lakes, NJ). 124
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2.2 Osteogenic and adipogenic differentiation of USC 126
Adipogenic induced differentiation 127
USC were seeded at a density of 21,000 cells/cm2 and cultured in serum containing DMEM 128
low-glucose medium with 1 µM dexamethasone, 500 µM 3-isobutyl-1-methylxanthine, 10 µg/ml 129
insulin, and 100µM indomethacin for 28 days with medium changes every third day. 130
Differentiated cells were then fixed with 4% paraformaldehyde for 30 min at room temperature 131
and stained with fresh 0.3% Oil Red O solution for 50 min. An inverted microscope was used to 132
identify red stained areas, indicating fat droplets. Adipose derived stem cells were used as 133
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positive controls. 134
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Osteogenic induced differentiation 136
USC were seeded at a density of 4,000 cells/cm2 and cultured in serum containing DMEM 137
low-glucose medium with 100 nM dexamethasone, 10 mM β-glycerophosphate and 50 µM 138
ascorbic acid-2-phosphate (Wako Chemicals, Richmond, VA) for 28 days with medium changes 139
every third day. Differentiated osteocytes were then fixed by ice-cold 95% ethanol for 5 minutes 140
at 4 ℃ and stained for calcium deposits with 2% Alizarin Red Solution (pH 4.0). An inverted 141
microscope was used to identify orange-red stained areas, indicating calcium deposits. 142
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2.3 An athymic rat model of dual injury-induced CKD 144
Animal experiments were approved by Wake Forest Institutional Animal Care and Use 145
Committee (Protocol no A11-085). Animals were housed in a temperature-controlled 146
environment with free access to food and water. A 12-hour light and 12-hour dark cycle was 147
provided. 148
To avoid the potential risk of immunoreaction caused by the implanted xenogenic cells, 149
athymic rats (RNU316) were used for this study. These athymic nude rats were T-cell deficient 150
and demonstrated depleted cell populations in thymus-dependent areas of peripheral lymphoid 151
organs. In total, 58 male athymic rats (age 9 weeks, Charles River, NC) were used in this study. 152
To optimize the doses of gentamicin and renal ischemia time frame, 30 animals were used for 153
creation model of dual injury-induced CKD before USC or PBS injection. Among these animals, 154
22 animals died and 8 animals recovered renal function (failure of model creation) during the 155
model creation processes. 156
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Once the model was successfully established, 28 animals were divided into three groups: a 158
PBS-treated group (n=9), an USC-treated group (n=9), and an age-matched control group 159
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(n=10). A CKD model was induced by creating an ischemia-reperfusion injury and gentamicin 160
injection as demonstrated in Figure 1 [28]. Briefly, the rats were anesthetized and laid in the 161
supine position with continuous 4% isoflurane via nose cone. A midline incision was made and 162
intestines were retracted to expose both kidneys. Using vascular clamps, the renal pedicles were 163
clamped bilaterally, for 60 minutes and then released to induce ischemia-reperfusion injury. The 164
clamps were then removed and the incision was closed. Two weeks after surgically-created 165
renal ischemia injury, gentamicin (100 mg/kg/day, Phoenix Pharmaceutical Saint Joseph, MO) 166
was injected subcutaneously for 5 consecutive days. Serum creatinine levels were used to 167
evaluate renal function. Blood and urine samples were collected every 2 weeks and analyzed 168
with blood chemistry machine (Beckman Coulter Inc., Brea, CA). Glomerulus filtration rate (GFR) 169
was calculated as follows: GFR=urine creatinine x urine volume/serum creatinine/time. 170
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2.4 Injections of USC into rat renal parenchyma 172
Eight weeks after the dual injury model was created, a USC cell suspension was injected 173
into the kidneys for the USC-treated group. The abdominal contents were exposed once again 174
and both kidneys were identified. A cell suspension of USC was administered into the 175
parenchyma of both kidneys. Each kidney received a total of 5x106/ml in 100 µl of PBS, 8 weeks 176
after CKD creation. To distribute injected cells evenly, 50% of the aforementioned cell 177
suspension was injected into the upper pole and lower pole of each kidney. PBS (100 µl) only 178
was injected in the same fashion as above for the PBS-control group. The model was not created 179
in aged-matched control animals and no injections were performed. Blood and urine samples 180
were collected to test renal function parameters i.e. serum creatinine and GFR, at 2 week 181
intervals. Kidneys were harvested from each animal for histological evaluation, 12 weeks after 182
injection. 183
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2.5 Histological and Immuno-histochemical analysis 185
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Harvested kidney tissues were fixed in 10% paraformaldehyde solution and processed for 186
paraffin embedding. Five µm sections were obtained. Slides were stained with hematoxylin and 187
eosin (H&E), and numbers of glomeruli were counted under high power fields (200X) microscope 188
(Leica, Germany). Normal glomeruli are uniform in size, visceral (inner) structures and Bowman's 189
space. Amounts of collagen deposited were assayed by computerized ImageJ (ImageJ software 190
package, NIH) based on Masson’s Trichrome staining. Immunohistochemistry was performed 191
with anti-human leukocyte antigen (HLA) and anti CD68 antibodies as a marker of macrophage 192
(BD Biosciences, CA). All slides were blocked in serum-free protein blocker (DAKO, Carpinteria, 193
CA) for 20 minutes. 194
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Transforming growth factor beta 1 (TGF-ß1, DAKO) was tested as dysregulation of TGF-β 196
activation and signaling may result in apoptosis of resident functional cells but proliferation of 197
fibroblast. In addition, superoxide dismutase 1 (SOD-1, DAKO) as an antioxidant enzyme 198
protecting the cell from reactive oxygen species toxicity were tested in USC-treated group, 199
compared to PBS-treated control. Anti-superoxide dismutase 1 antibody (EPI722Y) was used at 200
a 1:400 dilution. After incubation with the primary antibodies, tissue sections were incubated with 201
biotin-conjugated secondary antibodies (1:300 dilution) (Vector Laboratories, Burlingame, CA, 202
USA) at room temperature for 60 min, followed by incubation with HRP-conjugated streptavidin 203
(Vector Laboratories) for 30 min at room temperature and further developed with a DAB 204
substrate kit (Vector Laboratories). 205
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Positive cells were counted under lower and high power fields (100X, 400X). Total number 207
of glomeruli with abnormal structures, increased glomerular size, pathological changes of renal 208
tube, intestinal fibrosis, collagen deposits and inflammatory were quantified. The degree of 209
histological abnormalities on nephrons, such as dilation or collapse, epithelium and obliterated 210
lumen necrosis in renal tubule and glomerular was evaluated. Quantitative comparison of 211
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histology and immunohistochemical staining was measured by digital image analysis. 212
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2.6 Statistical analysis 214
All data were analyzed using software Statistical Package for the Social Sciences (SPSS) 215
version 16.0 for windows (SPSS, Inc., Chicago, IL). Normality of the analyzed data was 216
corroborated with Shapiro-Wilk test. When parametric analysis was possible, data were 217
expressed as mean ± SD and one-way analysis of variance (ANOVA) was used for comparison 218
among the three groups. Least Significant Difference test was applied to further compare 219
differences among three groups. P<0.05 was considered statistically significant. 220
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Results 221
Individual USC appeared consistently as rice grain-like at primary culture (Supplementary 222
Figure 1). USC at passage 3-5 were strong positive for glomerular parietal epithelial cells (CD24, 223
CD29 and CD44), and also classical cell surface markers for MSC (CD73, CD90, CD105, CD146 224
and SSEA-4), but lack of the expression of hematopoietic markers (CD 31, CD34 and CD45). 225
These results were consistent with our previous reports (Supplementary Figure 1) [12-20]. In 226
addition, USC differentiated into osteogenic and adipogenic cell lineages, respectively (Figure 227
2). 228
For evaluation of renal function, serum creatinine (Cr) was evaluated and GFR was 229
calculated every 2 weeks. Creatinine rise and GFR depression were used as indicators of 230
effective CKD model creation. Creatinine levels increased significantly in all animals assigned to 231
USC and PBS groups after one week, post-model creation and remained elevated compared to 232
AMC (p<0.01). Likewise, GFR declined significantly for animals assigned to USC and PBS 233
groups after one week, post-model creation (p<0.01). The changes in creatinine and GFR were 234
similar in both USC and PBS groups until injection was performed at 13 weeks post-model 235
creation. In the USC-treated group, serum creatinine decreased and GFR increased significantly 236
improved compared to PBS-treated animals two weeks after cell implantation (p=0.027 and 237
p=0.037, respectively). Th difference in GFR and Cr remained significantly different between 238
these groups until the study endpoint 12 weeks after cell implantation. GFR in the USC-treated 239
group improved to 50% of that in AMC whereas PBS-treated animals only exhibited a 25% 240
recovery of GFR compared to AMC (Figure 3). 241
242
Grossly, the surface of the kidneys in PBS-treated group was coarse and pale, while 243
kidneys in USC-treated group appeared similar to AMC (Figure 4). In addition, the kidney weight 244
in PBS-treated group (1.48±0.31g) was significantly lower compared to those of the AMC 245
(1.98±0.19 g) and USC-treated groups (1.69±0.22 g) 12 weeks after injection therapy. The 246
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kidney weight in USC-treated group significantly increased compared to that in PBS-treated 247
group (p<0.01) despite not returning to kidney weights exhibited in the AMC group. 248
249
Immunocytochemical analysis using HLA staining revealed that the implanted human USC 250
were present around Bowman’s capsule or scattered within renal tubules for the entire study 251
period, however numbers of the grafted cell decreased significantly, 12 weeks after injection 252
compared to immediately post-injection (Figure 5). To determine the reno-protective effects of 253
USC, we evaluated the changes in glomerular, renal tubular, and tubulo-interstitial structure 254
among the three groups. In the PBS-treated group, about 38% of glomeruli displayed normal 255
architecture (4.0±2.0 normal glomeruli/high power field [HPF]) within the renal cortex while 62% 256
(6.6±2.0) demonstrated glomerulosclerosis evidenced by wrinkling and collapse of the basement 257
membrane and constricted glomerular capillaries, compared to AMC. This pathological changes 258
in nephrons were significantly lower than the USC-treated group (6.3±2.1) and the AMC group 259
(10.6±2.2) (p<0.01). Conversely, the USC-treated group demonstrated 60% normal glomeruli 260
with 40% displaying glomerular sclerosis and dilated, atrophic tubules (Figure 6) compared to 261
the AMC group (p<0.01). 262
263
Collagen deposition in the renal parenchyma was investigated using Masson’s Trichrome 264
staining (Figure 7). Collagen deposition was elevated in PBS-treated animals (28.4±6.3). This 265
was significantly higher than in USC-treated group (8.3±3.0) and the AMC group (1.3±0.3) 266
(p<0.01), which represented over a 20-fold increase. There was also significantly higher collagen 267
deposition in the USC-treated group compared to the AMC group which was only a 6-fold 268
increase. To assess monocyte infiltration (Figure 8), numbers of cells expressing CD68+ was 269
significantly higher in the PBS group (8.7±2.54) compared to both USC-treated group (4.7±2.12) 270
and AMC group (2.0±1.05) (p<0.01). The USC-treated group exhibited an elevated numbers of 271
cells expressing CD68+compared to the AMC group as well, however albeit not as drastic. 272
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To evaluate tissue fibrosis within the renal tissues, TGF-ß1 as an inflammatory marker 273
(Figure 9) was used. TGF-ß1 expression was significantly high in the PBS-treated groups in 274
both the cortex and medulla (1.2±0.1 and 1.3±0.08) which represented a 58% and 69% increase 275
compared to AMC (0.8±0.05 and 0.7±0.06). Expression of TGF- ß1 in the cortex and medulla of 276
the USC-treated group (0.95±0.09 and 0.99±0.06), respectively, represented as 23% and 29% 277
increase compared to AMC. The relative increase in TGF-ß1 for PBS-treated animals was 278
significantly higher than for the USC-treated group (p<0.01). 279
280
Superoxide dismutase (SOD-1) as important mediator in the oxidative stress response was 281
assessed (Figure 9). SOD-1 expression in the PBS-treated group in the cortex and medulla was 282
1.49±0.17 and 1.53±0.14, which represented 84% and 89% of that in the AMC group (1.8±0.16 283
and 1.7±0.11). Expression of SOD-1 in the USC-treated group was 2.35±0.1 and 2.43±0.11 in 284
the cortex and medulla, respectively. This represented a 33% and 42% increase in SOD-1 285
expression compared to AMC. The relative changes in SOD-1 expression were significantly 286
different among the three study groups (p<0.01). 287
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Taken together, there is a tendency between the creatinine clearance and both the 289
inflammatory and antioxidant parameter amount three groups, i.e. renal function improved with 290
less inflammatory and increase level of antioxidate in USC treated group than those in 291
PBS-treated group, competed to AMC (Figure 10) . 292
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Discussion 293
Chronic kidney disease is caused by progressive injury to nephrons including glomeruli and 294
renal tubules, interstitium and vasculature. Etiologies include genetic predisposition, metabolic 295
derangements, renal ischemia, environmental and infectious causes, autoimmune conditions, 296
and drug-related nephrotoxicity. Repeated or superimposed insults may ultimately lead to 297
chronic kidney disease, which ultimately may progress to end-stage renal disease without 298
appropriate therapy. Of particular interest to us was drug-induced nephrotoxicity. 299
300
Drug-induced nephrotoxicity occurs more frequently in patients with pre-existing renal 301
dysfunction or ischemia due to acute kidney injury, chronic kidney disorder, diabetic nephropathy, 302
congestive heart failure, among other causes. Gentamicin is notorious for its nephrotoxic 303
side-effects, which is characterized by proximal convoluted tubular necrosis and glomerular 304
congestion, resulting in decreased GFR and renal dysfunction [29]. Similarly, renal ischemia 305
often causes acute tubular necrosis [30]. Dual insults with renal ischemia and drug nephrotoxicity 306
may lead to severe CKD and ESRD without prompt treatment and thus it is necessary to develop 307
an effective approach to prevent progression of CKD to ESRD. 308
309
Several animal models of CKD have been reported in the literature [31], however, due to the 310
robust regenerative capacity of rodents, it is challenging to study long-term efficacy of cellular 311
therapy. The subtotal nephrectomy model [32-34] requires a complex procedure and does not 312
reflect the mechanism of renal failure most often observed in humans. The streptozotocin model 313
mimics tissue injury observed in diabetic nephropathy. However, as CKD caused by 314
drug-induced nephrotoxicity increases, the need for another animal model is required. Herein, 315
we have established a model using ischemia-reperfusion injury combined with 316
gentamicin-induced nephrotoxicity to provide an easy and reliable animal model to study CKD 317
[28]. 318
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Given that definitive treatment i.e. renal transplant is not commonplace; it is of utmost 319
importance to prevent progression of CKD to ESRD. The nephron is the functional unit of the 320
kidney and thus we quantified the number of normal nephrons in each treatment group. In the 321
USC treated group, 70% of glomeruli displayed normal architecture while this number decreased 322
to 40% in the PBS-treated group. In contrast, numbers of abnormal nephrons significantly lower 323
in USC-treated group than those in PBS group. Likewise there was a significant decrease in 324
serum Cr and increase in GFR in the USC group compared to PBS treated animals. It is likely 325
that the preservation of glomerular architecture, evidenced by a lower level of glomerulosclerosis 326
and dilatated, atrophic tubules, in the USC treated group compared to the PBS group is 327
responsible for the chemical (serum Cr) and functional (GFR) findings. 328
329
Tissue fibrosis and collagen deposition are important contributors to progressive renal 330
failure. This is a complex process involving fibroblasts, immune reactions and free radical 331
damage to the renal parenchyma. We evaluated these processes through gross examination of 332
the kidneys and through the chemical investigation of mediators of fibrosis and free radical 333
damage. Monocyte infiltration as cells expressing CD68+ was elevated in the PBS group to a 334
greater extent than in USC treated group, compared to AMC. TGF- ß1 as a surrogate for tissue 335
fibrosis followed a similar trend. Superoxide dismutase is an important mediator in inhibiting free 336
radical induced tissue damage and its expression was lowest in the PBS treated group and 337
highest in the USC treated group. Given the lower levels of TGF- ß1 and CD68+ cells in the USC 338
group compared to the PBS group, it is unsurprising the see a lower level of collagen deposition 339
and improved renal function in the latter (Figure 9). Furthermore, collagen and fibrosis lead to 340
glomerular and tubular atrophy, explaining the gross appearance and the weights of the kidneys 341
in the PBS group compared to the USC and AMC groups. 342
343
Our study is not without limitations which may need to be addressed prior to beginning 344
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safety trials. First, we only administered a single dose of a given concentration of USC. It is 345
feasible that multiple injections may further improve the renoprotection imbued by USC. These 346
injections may need to be distributed over several weeks to months for maximal effect. Second, it 347
is possible that increasing the number of injected cells will improve kidney function even further. 348
Third, very few studies have been conducted investigating routes of administration for urologic 349
stem cell therapy and thus it may be that intravenous therapy may provide improved benefit over 350
intrarenal injection. Finally, molecular analysis of pro-regenerative, anti-apoptotic or anti-fibrotic 351
pathways needs further investigation. 352
353
Herein, we have demonstrated that local implantation of human USC significantly improved 354
renal function, imbued a protective effect on nephrons, reduced renal scaring, and mollified the 355
inflammatory response in a renal ischemia-reperfusion, gentamicin, dual injury-induced CKD 356
rodent model (Figure 10). Through our chemical and functional analysis, it is clear that USC 357
have some impact on the aforementioned parameters insofar as USC treatment ameliorates 358
renal degradation seen in the group that received no treatment. 359
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360
361
Table 1. Antibodies, mediums, and serums used in this study.
Items
Antibody
Type
Source
Catalog No.
A list of
Antibodies
CD24 FITC-conjugate BD 560992
CD29 PE-conjugated BD 555443
CD31 FITC-conjugated BD 560984
CD34 APC-conjugated BD 560940
CD44 PE-conjugated BD 550989
CD45 APC-conjugated BD 340943
CD73 PE-conjugated BD 550257
CD90 APC-conjugated BD 561971
CD105 PE-conjugated BD 560839
CD146 PE-conjugated BD 550315
SSEA4 PE-conjugated BD 560128
STRO-1 FITC-conjugated Biolegend 340105
HLA Rabbit monoclonal Abcam 52922
CD68 Mouse monoclonal AbD Serotec MCA341R
TGF-ßI Rabbit monoclonal Abcam 170874
SOD-1 Rabbit monoclonal Abcam EP1727Y
Medium and Serum
Source
Catalog No.
Culture
Mediums
KSFM Gibco 17005042
DMEM Gibco 41966-052
Serums
Fetal bovine serum
Gibco
26140079
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362
363
Table 2. Pathohistological changes in kidney of USC-treated rats, compared to those in controls 12
weeks after cell therapy.
Pathological
changes
PBS-treated
Group*
USC-treated
Group*
Age-matched
Control*
No. of rats n=9 n=9 n=10
Ratio of normal nephrons
(%) (Fig. 6)
38% (4.0±2.0)
60% (6.33±2.1)
100% (10.6±2.2)
Ratio of abnormal
nephrons (%) (Fig. 6)
62% (6.6±2.0)
40% (4.27±2.1)
0% (0.0±0.0)
Ratio of collagen
deposition (%) ( Fig. 7)
322% (16.2±4.7)
152% (7.6±3.3)
100% (5.0±2.0)
Ratio of inflammatory
(%) (Fig. 8)
435% (8.7±2.5)
235% (4.7±2.1)
100% (2.0±1.1)
Increased interstitial
fibrosis (cortex vs.
medulla) (%) (Fig. 9)
158% (1.22±0.1)
vs.
182% (1.32±0.08)
123% (0.95±0.09)
vs.
128%(0.99±0.06)
100% (0.77±0.05)
vs.
100%(0.72±0.06)
Abbreviations:
PBS-Phosphate buffered saline; USC-Urine-derived stem cells.
Notes: Glomerular sclerosis, dilated or atrophic renal tubule structure; Inflammatory response -
measured by monocyte infiltration. One-way ANOVA statistical analysis was formed among three
groups, based on the average relative intensity of ten fields per sample, each taken at a 200x
magnification for immunostaining. Percentages were relative to AMC. All values are reported as mean
± SEM.
*All differences listed in this table were statistically significant between the PBS, USC-treated groups,
and AMC, p<0.05 or p<0.01.
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Figures and legends: 364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
Figure 1. A brief flow chart of the study design. The CKD model was created using dual-injury 384
of renal ischemia and nephrotoxicity using 5 consecutive days of gentamycin injection, 2 weeks 385
after renal ischemia-reperfusion injury. Eight weeks after confirmation of model creation using 386
serial serum Cr and GFR, animals received either USC or PBS via direct injection into renal 387
parenchyma. Renal function was performed at different time points. Finally, histological and 388
immunocytochemical analysis were performed 12 weeks after injection. Age matched animals 389
were applied as controls (AMC). 390
391
Gentamycin
injection daily
for 5 days
USC or PBS
Groups
AMC
Group
Renal ischemia
/reperfusion
USC or PBS
injection
Histology
analysis
2 wks 8 wks
12 wks
3rd
wk 11th
wk 23th
wk 0
20
392
A 393
100 µm
21
394
395 396
B 397
398
399
Supplement Figure 1. (A) Human USC all displayed ''rice grain''-like morphology in the early 400
passage (p2). (B) USC expressed markers of glomerular parietal epithelial cells (CD24, CD29 401
and CD44), and also classical cell surface markers of MSC (CD73, CD90, CD105, CD146 and 402
SSEA-4) and negative expression of hematopoietic markers (CD 31, CD34 and CD45). 403
22
404
405
406
407
408
409
410
411
412
413
414
415
Figure 2. In vitro differentiation potential of USC. (A) Adiopogenic differentiation of USC was 416
demonstrated with oil red O (lipid droplet). (B) Osteogenic differentiation of USC was 417
demonstrated with Alizarin Red. (C) Non-induced USC acted as a control group. 418
23
419
420
421
422
423
Figure 3. Dynamic changes of renal function at different time points. A CKD model was 424
successfully established in athymic rats. (A) Serum creatinine levels were significantly lower in 425
USC-treated rats, compared to PBS-treated group on weeks 4 and 6 after cell therapy, 426
respectively. (B) Glomerular filtration rate was significantly higher in USC-treated group at each 427
time period after cell therapy, compared to the PBS-treated group (* p<0.01). 428
A
B
S
eru
m C
reatin
ine (
mg
/dl)
G
FR
(m
l/m
in)
Final evaluation 12 weeks after cell therapy
USC Injection
Gentamycin
Clamp bilateral renal vessels
Final evaluation 12 weeks after cell therapy
USC Injection
Gentamycin
Clamp bilateral renal vessels
A
* *
* * * *
24
429
430
431
Figure 4. The gross appearance and weight changes of the kidneys. (A) The gross 432
appearance of kidneys in the three treatment groups was noted. (B) The weight of the kidneys in 433
USC-treated rats was significantly elevated compared to the PBS-treated animals but also 434
significantly lighter than that in AMC group, * Significance at the p<0.05 level. 435
436
B
We
igh
t o
f k
idn
ey *
*
25
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
Figure 5. Tracking implanted USC within renal tissues by immuncytochemistry analysis 452
with anti-HLA A antibody. More grafted USC (brown) were found in the tubule interstitial 453
regions one week (A) and two weeks (B) after implantation compared to 12 weeks. Number of 454
the implanted USC decrease with time during the 12 week follow-up. A few cells were still 455
identified in the renal tubules and the medulla (C) as well as around Bowman’s capsule in the 456
renal cortex (D). 457
50 µm
50 µm
50 µm
A B
C D
26
458
459
460
461
462
463
464
465
466
467
468
469
470
471
Figure 6. of Potho-histological changes in nephrons within renal cortex and medulla after 472
USC implantation. (A) About 60% of glomeruli increased in size with collapse of some 473
glomerular tufts (short arrow) within the renal cortex and 62% of renal tubules were dilated (long 474
arrow) within the medulla in PBC-treated rats. In contrast, a majority of glomeruli and renal 475
tubules displayed normal structure and only 40% of nephrons displayed abnormal configuration 476
in USC-treated group. (B) Expressed as the average numbers of relative abnormal nephrons in 477
six fields per sample at 200x magnification. Numbers of normal nephron within renal tissue under 478
high power field with H&E staining. * Significance at the p<0.05 level. 479
PBS USC AMC A
Me
du
lla
C
ort
ex
B B
PBS USC AMC
No
. o
f n
orm
al
ne
ph
ron
s/
Lo
w p
ow
er
fie
ld
B
27
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
Figure 7. Histological appearance of collagen deposition and fibrotic changes of the 497
kidneys. (A) Among of collagen deposition in Masson Trichrome staining was significantly 498
elevated in PBS-treated rats, and minimal changes in kidney of rats treated with USC-treated 499
group compared to that in AMC. (B) Percentage of collagen deposition within the renal 500
parenchyma. * Significance at the p<0.05 level 501
502
503
A
Me
du
lla
C
ort
ex
B
PBS USC AMC
PBS USC AMC
Pe
rce
nta
ge
of
co
lla
ge
n
de
po
sit
ion
28
504
505 506
Figure 8. CD68 expression as a surrogate for monocyte infiltration within the kidney. (A) 507
Number of infiltrated monocyte expressing positive for CD68 significantly increased in 508
PBS-treated group, compared to AMC. (B) Number of cells expressing CD68 significantly 509
decreased in USC group compared (C) compared to PBS group. (D) Analysis of CD68 510
expression in the three groups with immunocytochemical staining. * Significance at the p<0.05 511
level 512
513
D
Pe
rce
nta
ge
of
mo
no
cyte
infi
ltra
tio
n
29
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
Me
du
lla
PBS USC AMC
TGF-1 expression in renal tissues
50 m
50 m 50 m
PBS
50 m
USC
50 m
50 m
A
AMC
*
*
Co
rte
x
Rela
tive inte
nsity o
f T
GF
-ß1 e
xpre
ssio
n
30
553
554
555
PBS USC AMC
Co
rte
x
Me
du
lla
50 m 50 m
50 m 50 m 50 m
50 m
A
SOD-1 expression in renal tissues
PBS USC AMC
* *
Rela
tive inte
nsity o
f S
OD
1 e
xpre
ssio
n
31
Figure 9. Effect of USC implantation on anti-fibroblast and redox properties in CKD 556
rodent model. (A) Amount of TGF-β1 expression significantly increased in renal cortex and 557
medullar tissue of PBS treated animals compared to that of AMC group. However, USC 558
implantation significantly decreased levels of TGF-β1 expression, compared PBS treatment. (B) 559
Amount of SOD-1 expression levels significantly increased in renal tissue of USC-treated groups 560
compared to that of PBS group. Semi-quantification is expressed as the average relative 561
intensity of six fields around defect site per sample, each taken at a 200x magnification. 562
Representative images are shown at 200x magnification. *p < 0.01 compared to AMC as a unit 563
set. 564
32
565
566
Figure 10. Relationship between serum creatinine and both the oxidative stress parameter 567
and the inflammatory parameters with regression line analysis. Antioxidant and 568
antiinflammatory activity of USC is behind the renoprotective activity, compared to those of 569
PBS-treated and AMC groups. 570
SOD1
Serum Creatinine
TGF-ß1
No
rma
lize
d v
alu
es
33
Abbreviations 571
AMC, age-matched control 572
CKD, chronic kidney disease 573
ESRD, end-stage renal disease 574
H&E, hematoxylin and eosin 575
HLA, human leukocyte antigen 576
HPF, high power field 577
FITC, Fluorescein isothiocyanate 578
GFR, Glomerulus filtration rate 579
iPSC, induced pluripotent stem cells 580
PBS, phosphate buffered saline 581
PE, phycoerythrin 582
SSEA-4, stage-specific embryonic antigen-4 583
SPSS, Statistical Package for the Social Sciences 584
USC, Urine-derived stem cells 585
34
Declarations 586
Acknowledgement 587
The authors would like to acknowledge all parties that participated in this study. Authors also 588
would like to thank Dr. Marshall Z. Schwartz in Department of Urology at Wake Forest School of 589
Medicine for his editing and helpful discussions 590
35
Funding 591
Not applicable 592
36
Availability of data and materials 593
All data generated and/or analyzed during this study are included in this published article and its 594
supplementary files. 595
37
Competing interests 596
The authors declare that there are no competing interests associated with this manuscript. 597
38
Author contributions 598
CZ, SG, MA, TK, and PT carried out the cell culture, molecular studies, the animal experiments 599
and drafted the manuscript. CZ, SG, and PT carried out immunoassays and performed statistical 600
analyses. AA and YZ participated in the design of the study. YZ, IK, and PT conceived the study, 601
participated in its design, and completed the manuscript. JY, YS, JJ and AA participated in 602
administration and coordination. All authors read and approved the final manuscript. 603
39
Consent for publication for data from individual person 604
Not applicable 605
40
Ethics approval and consent to participate 606
Collection of human urine samples in this study was approved by the Wake Forest University 607
Health Sciences Institutional Review Board and was performed in accordance with the 608
Declaration of Helsinki (2004). Signed, informed consent was collected from patients. 609
41
Ethics approval for animal study 610
Rodent experiments were approved by Wake Forest Institutional Animal Care and Use 611
Committee (IACUC). 612
42
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