Safety and tolerability of autologous bone marrow mesenchymal stromal cells in ADPKD patients

Autosomal dominant polycystic kidney disease (ADPKD) is a genetic ciliopathy disease characterized by progressive formation and enlargement of cysts in multiple organs. The kidneys are particularly affected and patients may eventually develop end-stage renal disease (ESRD). We hypothesize that bone marrow mesenchymal stromal cells (BMMSCs) are renotropic and may improve kidney function via anti-apoptotic, anti-fibrotic, and anti-inflammatory effects. In this study, we aim to assess the safety and tolerability of a BMMSC infusion in ADPKD patients. We performed a single-arm phase I clinical trial with a 12-month follow-up. This study enrolled six eligible ADPKD patients with an estimated glomerular filtration rate (eGFR) of 25–60 ml/min/1.73 m2. Patients received autologous cultured BMMSCs (2 × 106 cells/kg) through the cubital vein according to our infusion protocol. We investigated safety issues and kidney function during the follow-up visits, and compared the findings to baseline and 1 year prior to the intervention. There were no patients lost to follow-up. We observed no cell-related adverse events (AE) and serious adverse events (SAE) after 12 months of follow-up. The mean eGFR value of 33.8 ± 5.3 ml/min/1.73 m2 1 year before cell infusion declined to 26.7 ± 3.1 ml/min/1.73 m2 at baseline (P = 0.03) and 25.8 ± 6.2 ml/min/1.73 m2 at the 12-month follow-up visit (P = 0.62). The mean serum creatinine (SCr) level of 2 ± 0.3 mg/dl 1 year before the infusion increased to 2.5 ± 0.4 mg/dl at baseline (P = 0.04) and 2.5 ± 0.6 mg/dl at the 12-month follow-up (P = 0.96). This indicated significant changes between the differences of these two periods (12 months before infusion to baseline, and 12 months after infusion to baseline) in SCr (P = 0.05), but not eGFR (P = 0.09). This trial demonstrated the safety and tolerability of an intravenous transplantation of autologous BMMSCs. BMMSC efficacy in ADPKD patients should be investigated in a randomized placebo-controlled trial with a larger population, which we intend to perform. ClinicalTrials.gov, NCT02166489. Registered on June 14, 2014.


Background
Autosomal dominant polycystic kidney disease (ADPKD) is a genetic ciliopathy disease characterized by progressive formation and enlargement of cysts in multiple organs. The kidneys are particularly affected and patients may eventually progress to end-stage renal disease (ESRD). ADPKD is the most common genetic renal disease [1] that affects approximately 4 to 7 million individuals [2]. Mutations in polycystins 1 and 2 impair homeostasis and intracellular calcium signaling, which subsequently cause cyst formation, inflammation, and fibrosis in the kidneys [3,4].
Despite recent advances in the identification of ADPKD pathogenesis, current treatments have been unable to completely cure or control disease progression. Tolvaptan, a vasopressin II receptor antagonist (V2RA), is the only drug that has passed a phase III clinical trial. Though it has the ability to reduce progression of height-adjusted total kidney volume (htTKV), it is associated with numerous adverse effects [5].
Transplantation of mesenchymal stromal cells (MSCs) is a recent approach to treat renal disease [6,7]. MSCs are plastic-adherent cells that must at least express CD73, CD90, and CD105 and lack expression of CD45, CD34, CD14 and human leukocyte antigen (HLA)-DR. They have the potential for in vitro differentiation to osteoblasts, adipocytes, and chondroblasts [8]. Several studies report their ability to treat renal diseases by different mechanisms [9]. Anti-apoptotic [10], anti-fibrotic [11], and anti-inflammatory [12] capabilities of MSCs [7] provide the basis for their administration to control the progression of polycystic kidney disease (PKD). Franchi et al. have reported that transplanted MSCs restored kidney function in a PKD model and improved damaged vasculature of these kidneys [13]. Previously we showed the promising efficacy of transplanted MSCs in experimental models of chronic kidney disease (CKD) and acute kidney injury (AKI) [14][15][16]. We had indicated that MSC-derived conditioned medium induced renal tubular cell regeneration after nephrotoxicity induction [14], then we showed the bone marrow MSC (BMMSC) lowered serum creatinine (SCr) and urea levels and accelerated regulatory T cells (Tregs) in a monkey AKI model [15]. In a recent study, our results indicated that MSCs resulted in decreased CKD progression in a monkey model [16]. Carvalhosa et al. reported that cells lining cysts in ADPKD consisted of polycystic CD133+ progenitor [17]. Our unpublished data showed that in vitro co-culture of MSCs and tubular CD133+ progenitor cells from ADPKD patients resulted in reduced cyst formation and proliferation potential.
We hypothesize that MSCs are renotropic. Therefore, this study aims to assess the safety and tolerability of MSC infusion in ADPKD patients.

Study design
We performed a single-arm phase I clinical trial at one trial center with a 12-month follow-up period. We investigated safety issues and kidney function during the follow-up visits, and compared the findings to baseline and 1 year prior to intervention. Table 1 shows the study assessment schedule. We evaluated 27 ADPKD patients for potential participation in this trial. We had diagnosed all these patients according to unified imaging diagnostic criteria [18]. Patients were under our observation for at least 1 year prior to study enrollment. Their blood pressures (BP) were well controlled. From these potential participants, we recruited six eligible ADPKD patients who provided written informed consent for study participation. This study enrolled patients from June 2014 to January 2015. All patients have been on a special diet that included decreased salt, protein, and caffeine consumption, and high water intake, recommended by our nutritionist. We strongly recommended that patients keep track and not change their medications, supplements, or make any lifestyle changes. This trial was registered with clinical trial number NCT02166489.
We evaluated both safety and tolerability of the cell infusion according to physical examination, adverse event (AE) assessments, and laboratory changes. Potential efficacy included changes in kidney length (KL) measured by ultrasound imaging, estimated glomerular filtration rate (eGFR) by the modification of diet in renal disease (MDRD) study formula, and glomerular filtration rate (GFR) by diethylenetriaminepentaacetic acid (DTPA) scan.
We abstracted data that pertained to the "1 year prior to infusion" point from patients' medical files in the clinic. We scheduled patient follow-up visits for clinical and laboratory assessments at specified intervals of 1, 3, 6, 9, and 12 months after the cell infusion.

Enrollment criteria
Inclusion criteria were: (1) both genders; (2) ADPKD confirmed by sonography imaging or genetic testing; (3) age 18 to 60 years; (4) eGFR 25-60 mL/min/1.73 m 2 ; (5) ability to understand and willingness to sign a consent form. Exclusion criteria consisted of: (1) pregnancy or breastfeeding; (2) positive history of associated cardiovascular disease; (3) diabetes that required medical intervention; (4) other systemic diseases that involved the kidneys such as cancers, autoimmune diseases, blood diseases, and liver diseases; (5) hospitalization due to illness 2 months prior to study entry; (6) life expectancy less than 2 years; and (7) allergies to the cell culture ingredients.

Primary and secondary endpoints
Primary endpoints consisted of the numbers, type, and severity of AEs related to the cell infusion. We recorded AEs and serious adverse events (SAE) during follow-up visits and whenever patients reported any symptoms. We recorded the type and grade of AEs according to the Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. We sent these reports to the Data Safety Monitoring Board (DSMB) of the study. Secondary endpoints consisted of changes in eGFR (as an existing surrogate endpoint) [19] from baseline to 12 months after the cell infusion.

Kidney ultrasound imaging and kidney DTPA scan
Additional file 1 lists this information.
Bone marrow aspiration (BMA) procedure, cell preparation, and culture Once the patients' laboratory viral tests were normal (Additional file 1: Table S1), an oncologist performed a bone marrow aspiration (BMA) under local anesthesia (2% lidocaine solution) and an intravenous sedation that consisted of 0.1 mg/kg midazolam and 25-50 mg intravenous fentanyl. The BMA was obtained from the posterior superior iliac crest with the patient placed in Table 1 Assessment schedule   SV  BV1  V1  BV2  V2  V3  V4  V5  V6  V7   Day  -56  -49  -42  0  1  30  90  180  270  360   Time window  NA  ±3d  ±7d  ±1d  ±1d  ±7d  ±7d  ±7d  ±7d  ±7d Informed consent x Physical examination Medical history x Laboratory assessments  Figure S1 show MSC characterization of the enrolled patients. The cells were tested for possible microbial contamination prior to infusion. We used quality control tests (QCT) in accordance with the recommendations for cell and tissue therapy promotion and validation tests of the Iranian Health Ministry Pharmacopoeia Commission and the Department of Health and Human Services, Food and Drug Organization (FDO). Tests included the sample microbial test, limulus amebocyte lysate (LAL) gel clot assay, mycoplasma detection, and karyotype analysis. Passages 1 to 3 of the cultured BMMSCs were washed with phosphate-buffered saline and trypsinized with trypsin/EDTA (0.05%; 25300-062, Gibco). The cells were diluted in 10 ml of normal saline and loaded into 10 ml sterile syringes. For each patient, we prepared approximately 1-2 × 10 6 cells/kg, which were delivered to the institute's operating room in a cold box at a temperature of approximately 4°C.

Statistical analysis
Data are written as mean ± standard deviation (SD). Following normal distribution of parametric data, we used the paired t test to compare variables at two time points (baseline and 12 months after cell infusion). We compared variables at three time points (12 months before infusion, baseline, and 12 months after infusion) by the repeated measures ANOVA test. A twosided P value of <0.05 was considered statistically significant. We used SPSS for statistical analyses (Version 16, SPSS Inc., Chicago, IL, USA).

Patient characteristics
All six patients completed the trial. No patients were lost to follow-up. Table 2 lists the patients' characteristics and demographic data at the time of enrollment. Additional file 1: Table S1 shows the cell parameters. All patients' BP was under tight control prior to enrollment. Patients' antihypertensive medications did not change during the trial. There were five patients with children. None of these patients underwent any procedures to prevent the transmission of the disease gene to their children. One patient has not attempted to have any children. None experienced external renal complications due to PKD prior to enrollment. We did not observe any external renal complications from PKD during the 12 months of follow-up. Although one patient had autosomal dominant polycystic liver disease (ADPLD), we did not observe any changes in the size of her liver by ultrasonography at the 12-month follow-up.

Safety and tolerability
We discharged all patients 2 h after the cell infusion and observed no AEs during this time. The bone marrow puncture site healed within 72 h and the intravenous infusion site healed within 24 h without any related complications. In total, we recorded 1 SAE and 37 AEs during the 12-month follow-up in all patients (Additional file 1: Table S2).
We sent the related AE reports to the DSMB of the trial as scheduled. The DSMB raised the issue that vomiting and dizziness AEs occurred shortly after the DTPA kidney scan in the majority of patients at the baseline and follow-up visits. They recommended that we cancel the DTPA kidney scans for the rest of the trial.
According to DSMB comments, all other AEs were unrelated to the intervention and probably attributed to patients' underlying disease progression or other medical conditions. All AEs resolved spontaneously or by the use of over-the-counter medications.
One patient experienced an SAE. Patient number 3 who had a positive history for low back pain took a single dose of a celecoxib tablet (200 mg) and one acetaminophen tablet (500 mg) due to severe back pain 3 weeks after his cell infusion. His blood urea nitrogen (BUN) and SCr levels elevated 24 h after celecoxib and acetaminophen. We advised him against using these types of medications. Instead, we prescribed physiotherapy for pain relief. After 1 week, his BUN and SCr values returned to previous levels. We referred him to the orthopedic surgeon who performed a lumbosacral magnetic resonance imaging (MRI) scan without contrast. According to the MRI report, the patient had L4/L5 disc demyelination and a central disc protrusion with significant canal and left foraminal stenosis. He underwent surgery 1 week later. After surgery, his pain reduced and cleared completely within 10 days.

Primary endpoint
We did not detect any SAEs or AEs related to the cell infusion in any of the patients during the 12-month follow-up. The results showed no significant differences in laboratory parameters, which we evaluated for safety issues after 1, 3, 6, 9 (Additional file 1: Table S3) and 12 months compared to baseline (Table 3).

Secondary endpoint
The single-dose autologous MSC infusion did not induce any significant changes in eGFR, nor reductions in SCr at 12 months compared to baseline in all patients (Table 4).

eGFR, sCr, and blood pressure (BP)
Patients had a mean eGFR value of 33.8 ± 5.3 ml/min/ 1.73 m 2 1 year before the cell infusion, which declined to 26.7 ± 3.1 ml/min/1.73 m 2 at baseline (P = 0.03) and 25.8 ± 6.2 ml/min/1.73 m 2 at the 12-month visit (P = 0.62). Figure 1 shows the patients' eGFR changes. The mean SCr level of 2 ± 0.30 mg/dl within the same period increased to 2.5 ± 0.4 mg/dl at baseline (P = 0.04) and 2.5 ± 0.6 mg/dl at the 12-month follow-up (P = 0.96). There was a significant change between the differences of these two periods (-12, baseline and +12, baseline) in SCr (P = 0.05) but not in eGFR (P = 0.09; Table 4). Systolic (P = 0.55) and diastolic (P = 0.43) BP did not significantly change 12 months after the cell infusion (Table 4). We observed no significant changes in renal function parameters and BP during the year after intervention (Additional file 1: Table S4).

Kidney length (KL)
There was a slight increase in the median left KL at baseline (18.3 ± 2.4 cm) to 12 months after the cell infusion (18.7 ± 2.6 cm; P = 0.06). The median right KL at baseline was 19.1 ± 2 cm which increased slightly to 19.5 ± 2 cm at 12 months (P = 0.13; Table 4).

Discussion
To the best of our knowledge, this is the first clinical trial that has shown the safety and tolerability of a single BMMSC intravenous infusion in ADPKD patients. However, the mean eGFR level noticeably decreased from 1 year before intervention to baseline (P = 0.03). We observed a slight, nonsignificant decrease for eGFR at 12-month follow-up that could not be considered exactly due to the intervention.
We observed enlargement of both kidneys in all patients at 12 months after the cell infusion that was probably due to usual cyst growth. We used KL to evaluate disease progression and, to some extent, represent the size of the kidney [20,21]. However, KL is not as appropriate as HtTKV as proposed by the Consortium for Radiologic Imaging Studies of Polycystic Kidney Disease (CRISP) [22].
The HALT study showed a crucial role for hypertension (HTN) control in management of ADPKD [23]. Accordingly, we tightly controlled HTN in our patients prior to their enrollment. This did not change during the follow-up period.
Franchi et al. performed the only preclinical study that used allogeneic MSCs in a PKD rat model. They showed the beneficial effect of a single intravenous infusion for improvements in systolic HTN and fibrosis. Although the infusion did not affect cyst size and number, it had a significant improvement on cortical and parenchymal vasculature density, which resulted in better tubular function and improvements in creatinine clearance seen in the intervention group in the autosomal recessive BMI body mass index, BP blood pressure, eGFR estimated glomerular filtration rate, DTPA diethylenetriaminepentaacetic acid, ADPKD autosomal dominant polycystic kidney disease, DM diabetes mellitus, HTN hypertension, ADPLD autosomal dominant polycystic liver disease, CKD chronic kidney disease, NA not applicable, N no, Y yes a Neg: 0 mg/dl; Trace: 15-30 mg/dl; +: 30-100 mg/dl; ++: 100-300 mg/d; +++: 300-1000 mg/dl; ++++: >1000 mg/dl b Under controlled diet is defined as: no: on normal diet; yes: patients have been under the recommendation of nutritionist by taking low protein, low salt diet, with low caffeine and high intake of water polycystic kidney disease (ARPKD) model [13]. They also reported the safety of the MSC infusion with no mortality related to their protocol, which supported our safety results.
There is a growing body of clinical research which indicates that a systematic MSC infusion is safe under different conditions [24], including renal diseases [7,9]. No AEs such as acute infusion toxicity, organ complications, infections, death or malignancies associated with systematic infusion of these cells have been reported in human studies [24]. In concordance with their results, our study showed the safety of an intravenous MSC infusion following comprehensive assessment of AEs, SAEs, and clinical and paraclinical parameters. There are also reports of potential efficacy and safety of MSC infusions in patients with different etiologies of CKD [9,[25][26][27]. However, there is a lack of data demonstrates the impact of MSCs on PKD.
Recent experimental studies reported that MSCs predominantly improved kidney tubular regeneration by secretion of growth factors, cytokines, and chemokines which decrease apoptosis, inflammation, and fibrosis [7], and subsequently ameliorate kidney function (creatinine clearance, eGFR, and HTN) and structure in CKD models [7,9]. The potential capabilities of MSCs [35] are not limited to antifibrotic and anti-inflammatory effects on CKD models [11,12] and its beneficial impact on apoptosis in an AKI model [10,36] which could be useful for PKD.
Rapamycin decreases the proliferation of CD133+ and CD24+ cells and cyst formation in vitro through inhibition of the mechanistic target of rapamycin (mTOR) in PKD [17]. In our in vitro study, we observed that MSCs could reduce the proliferative potential and cyst formation of CD133+ progenitor cells from ADPKD patients (unpublished data). The mitogen-activated protein kinase (MAPK) family is involved in proliferation, apoptosis, and fibrosis in PKD. MSC-derived conditioned medium has been shown to inhibit p38 mitogen-activated protein kinases (p38) and extracellular signal-regulated kinase (ERK) activity (MAPK family) as well as induce antiapoptotic effects in an AKI rat model [37].
Various studies illustrated that downregulation of proinflammatory cytokines reduced cyst formation and inflammation in ADPKD [38][39][40]. MSCs have the potential to decrease both the activity of the nuclear factor kappa B (NF-ĸB) pathway [11] and pro-inflammatory cytokines, which eventually lessen inflammation in a CKD model [12].
Following ADPKD progression, renovascular architecture disrupts where there is the vessel network around the cyst [13,41]. Vascular endothelial growth factor (VEGF) is an antiapoptotic molecule [42] that can normalize the blood vessel network surrounding the cysts, BUN, SCr, and the cyst disorder as well as reduce macrophage pericystic infiltration in an ARPKD model [43]. VEGF C has been shown to decrease in an ARPKD mice model [43]. Human MSCs are a source of VEGF and may be useful for appropriate angiogenesis [44].  RAS inhibition improves BP and TKV, and prevents the spread of cyst formation in experimental animal models and humans [23,45]. MSCs reduce renin, angiotensin-converting enzyme (ACE), and angiotensin II type 1 (AT1) receptor expression, and lead to reductions in BP, inflammation, and fibrosis [46,47]. The MSC inhibitory effect on RAS is more stable than ACE inhibitors [47].
Taken together, following the safety and tolerability results of our study, the next step is to test the efficacy of MSC in ADPKD patients.

Limitations
The current trial limitations included our inability to demonstrate the efficacy due to the study design that consisted of a small sample size, lack of a control group, short follow-up period, the single cell infusion, and not using htTKV as a surrogate endpoint.

Conclusions
This trial demonstrated that an intravenous infusion of autologous MSCs was safe and well tolerated in ADPKD patients. We could not assess the efficacy due to the trial design. Our findings should be investigated in a randomized placebo-controlled trial with a larger population, which we intend to perform.

Additional files
Additional file 1: Table S1. Method of kidney ultrasound imaging. DTPA kidney scan method. Cell parameters for the study patients. Table S2. Safety assessment of the mesenchymal stromal cell (MSC) infusion in 1 year following baseline visit of study patients. Table S3. Laboratory parameters at baseline, 1, 3, 6, 9, and 12 months of follow-up. Table S4. Changes in renal function parameters and blood pressure (BP) 1 year before, and at 1, 3, 6, 9, and 12 months after mesenchymal stromal cell (MSC) infusion. (DOCX 70 kb) Additional file 2: Figure S1.