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) [14–16]. 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 Our unpublished data showed that in vitro co-culture of MSCs and tubular CD133+ progenitor cells from ADPKD patients resulted in reduced cyst formation and proliferation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.

Methods

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.

Table 1 Assessment schedule
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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 m2; (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 the lateral decubitus position. Samples were placed inside a cold box and sent to the clean room. The oncologist discharged all patients 4 h after the procedure. We telephoned patients 2 days later to ask about any complications related to the BMA procedure. None of the patients reported any complications. Bone marrow (BM) mononuclear cells were plated at 1 × 106 cells/cm2 in a 150-cm2 culture flask with alpha modified Eagle medium (α-MEM; 22571-020, Gibco BRL, Karlsruhe, Germany) supplemented with 100 IU penicillin and 100 IU streptomycin (15070-063, Gibco), 10% Hyclone defined fetal bovine serum (FBS; SH30070.03, Thermo Fisher Scientific, Waltham, MA, USA), and 1% L-glutamine (25030-024, Gibco). Phase-contrast microscopy of the BMMSC cultures demonstrated a low heterogeneous population that predominately consisted of long, spindle-shaped cells. For each patient, we characterized the cells for immunophenotypic properties that included surface marker expressions for MSCs - phycoerythrin (PE)-conjugated CD105, CD44, CD73 (Becton Dickinson, Franklin Lakes, NJ, USA) and fluorescein isothiocyanate (FITC)-conjugated CD90 (Dako, Glostrup, Denmark) along with those that were negative for hematopoietic stem cell (HSC) surface markers PE-conjugated CD34 and FITC-conjugated CD45. Negative isotype controls included nonspecific mouse IgG1-FITC/IgG1-PE and IgG2a-FITC that were substituted for the primary antibody. All samples were analyzed by flow cytometry (BD FACS Caliber, BD Biosciences, San Jose, CA, USA) and win-MDI2.9 software. Additional file 2: 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 × 106 cells/kg, which were delivered to the institute’s operating room in a cold box at a temperature of approximately 4 °C.

Cell infusion

We infused autologous cultured MSCs (1–2 × 106/kg) through the cubital vein according to our infusion protocol.

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 two-sided P value of <0.05 was considered statistically significant. We used SPSS for statistical analyses (Version 16, SPSS Inc., Chicago, IL, USA).

Results

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.

Table 2 Patient characteristics and demographic data at the time of enrollment
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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).

Table 3 Laboratory parameters at baseline and 12-month follow-up
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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).

Table 4 Changes in renal function parameters and blood pressure (BP) from 1 year before mesenchymal stromal cell (MSC) infusion, baseline and the 12-month follow up
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eGFR, sCr, and blood pressure (BP)

Patients had a mean eGFR value of 33.8 ± 5.3 ml/min/1.73 m2 1 year before the cell infusion, which 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 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).

Fig. 1
figure 1

Estimated glomerular filtration rate (eGFR) changes in autosomal dominant polycystic kidney disease (ADPKD) patients from 1 year prior to mesenchymal stromal cell (MSC) infusion up to 12 months after the infusion

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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 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, 25–27]. However, there is a lack of data demonstrates the impact of MSCs on PKD.

ADPKD progression is associated with several pathways such as apoptosis [28], fibrosis [2, 29, 30], inflammation [2, 31, 32], cyst proliferation, renin–angiotensin system (RAS) activation [33, 34], and kidney vasculature impairment [13]. MSCs may affect these pathways through different mechanisms, as briefly discussed in the following.

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 pro-inflammatory cytokines reduced cyst formation and inflammation in ADPKD [38–40]. 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.