Secreted factors from bone marrow stromal cells upregulate IL-10 and reverse acute kidney injury

Abstract

Acute kidney injury is a devastating syndrome that afflicts over 2,000,000 people in the US per year, with an associated mortality of greater than 70% in severe cases. Unfortunately, standard-of-care treatments are not sufficient for modifying the course of disease. Many groups have explored the use of bone marrow stromal cells (BMSCs) for the treatment of AKI because BMSCs have been shown to possess unique anti-inflammatory, cytoprotective, and regenerative properties in vitro and in vivo. It is yet unresolved whether the primary mechanisms controlling BMSC therapy in AKI depend on direct cell infusion, or whether BMSC-secreted factors alone are sufficient for mitigating the injury. Here we show that BMSC-secreted factors are capable of providing a survival benefit to rats subjected to cisplatin-induced AKI. We observed that when BMSC-conditioned medium (BMSC-CM) is administered intravenously, it prevents tubular apoptosis and necrosis and ameliorates AKI. In addition, we observed that BMSC-CM causes IL-10 upregulation in treated animals, which is important to animal survival and protection of the kidney. In all, these results demonstrate that BMSC-secreted factors are capable of providing support without cell transplantation, and the IL-10 increase seen in BMSC-CM-treated animals correlates with attenuation of severe AKI.

Figure 1

BMSC-CM confers a significant survival benefit to rats undergoing lethal cisplatin-induced AKI. At hour 0, cisplatin dissolved in physiological saline (0.75 mg/mL) was administered i.p. to male, SAS-SD rats at a lethal dose (7.5 mg/kg). At hours 3, 9, 24, 48, and 72, 1 mL of either physiological saline (n = 12), fibroblast conditioned medium (n = 10) or BMSC-conditioned medium (n = 14) were administered i.v. to the cisplatin-treated AKI rats. They were monitored for survival for 30 days. *P < 0.05 for BMSC-CM compared to both FB-CM and saline by Logrank test.

Figure 2

BMSC-CM prevents onset of severe AKI. Blood was collected from rats subjected to lethal cisplatin (7.5 mg/kg) i.p. via tail snip 1, 3, and 5 days following cisplatin administration. The rats were treated at 3, 9, 24, 48, and 72 hours following cisplatin administration, with 1 mL of either physiological saline (n = 4), or BMSC-conditioned medium (n = 3) i.v. (a) BUN and (b) creatinine were measured to monitor the extent of renal injury as a function of time after cisplatin. *P < 0.01 by Student's t-test.

Figure 3

BMSC-CM protects the native architecture of the kidney during AKI. Hematoxylin and eosin staining of sections of kidneys from rats administered a lethal dose of cisplatin (7.5 mg/kg) and sacrificed at 5 days following cisplatin administration. The rats were treated at 3, 9, 24, 48, and 72 hours following cisplatin administration, with 1 mL of either physiological saline (n = 4), or BMSC-conditioned medium (n = 3) i.v. Scale bar = 200 μm.

Figure 4

BMSC-CM prevents apoptosis in the outer medulla. (a) TUNEL staining was performed on sections of formalin fixed and paraffin-embedded kidneys from rats administered a lethal dose of cisplatin (7.5 mg/kg) and sacrificed at 5 days following cisplatin administration. The arrows indicate TUNEL positive cells. The rats were treated at 3, 9, 24, 48 and 72 hours following cisplatin administration, with 1 mL of either physiological saline (n = 4), or BMSC conditioned medium (n = 3) i.v. (b) Quantification of the TUNEL positive cells per field of view. Scale bar = 200 μm. *P < 0.01 by Student's T-test.

Figure 5

Vehicle treated have a greater number of PCNA+ cells 5 days after cisplatin administration. (a) PCNA staining was performed on sections of formalin fixed and paraffin-embedded kidneys from rats administered a lethal dose of cisplatin (7.5 mg/kg) and sacrificed at 5 days following cisplatin administration. The rats were treated at 3, 9, 24, 48 and 72 hours following cisplatin administration, with 1 mL of either physiological saline (n = 4), or BMSC conditioned medium (n = 3) i.v. (b) Quantification of the PCNA positive cells per field of view. Scale bar = 200 μm.

Figure 6

BMSC-CM significantly upregulates IL-10 in AKI rats, and BMSC-CM is not an exogenous source of IL-10. (a) Rat serum IL-10 was measured via ELISA from rats administered a lethal dose of cisplatin (7.5 mg/kg) and then treated with either vehicle or BMSC-CM at 3, 9, 24, 48, and 72 hours after cisplatin administration. Blood was collected via tail snip, and the serum was analyzed on day 3. *P < 0.05 by Student's t-test. (b) IL-10 was measured via ELISA in whole BMSC-CM.

Figure 7

IL-10 antibody abates the survival benefit of BMSC-CM-administered to rats undergoing lethal cisplatin AKI. At hour 0, cisplatin dissolved in physiological saline (0.75 mg/mL) was administered i.p. to male, SAS-SD rats at a lethal dose (7.5 mg/kg). At hours 3, 9, 24, 48, and 72, 1 mL of either physiological saline (n = 12), BMSC-conditioned medium (n = 14) or BMSC-conditioned medium containing 4 μg/mL neutralizing anti-rat IL-10 antibody (n = 10) were administered i.v. to the cisplatin AKI rats. They were monitored for survival for 30 days. *P < 0.05 for BMSC-CM compared to saline by Logrank test.

Figure 8

IL-10 antibody reduces the tissue sparing capacity of BMSC-CM. (a) H&E histological analysis of kidneys from cisplatin-treated rats administered either BMSC-CM plus a neutralizing IL-10 antibody (n = 3), BMSC-CM (n = 3), or saline (n = 3). Animals were sacrificed at 5 days after cisplatin for tissue harvesting. (b) Quantification of tissue injury in the outer cortex (OC), inner cortex (IC), and outer stripe of the outer medulla (OSOM) for rats treated with either BMSC-CM plus a neutralizing IL-10 antibody, BMSC-CM, or saline. Scale bar = 200 μm. *P < 0.05 by Student's t-test.

Figure 9

Protection of kidney function by BMSC-CM during AKI is abated with IL-10 antibodies. (a) Serum BUN and (b) serum creatinine values for rats subjected to cisplatin AKI and subsequently treated with either saline, BMSC-CM, or BMSC-CM with IL-10 neutralizing antibodies. Animals were sacrificed at 5 days after induction of AKI for blood collection. *P < 0.05 by Student's t-test.

Figure 10

Kidney injury molecule-1 (KIM-1) positive staining is more extensive when IL-10 antibodies are coadministered with BMSC-CM compared to BMSC-CM alone. Immunohistochemical staining for KIM-1 of kidneys from AKI rats treated with either BMSC-CM plus a neutralizing IL-10 antibody (n = 3), BMSC-CM (n = 3), or saline (n = 3). Animals were sacrificed at 5 days after induction of AKI for tissue harvesting. Scale bar = 200 μm.