Int J Stem Cells.  2019 Mar;12(1):151-161. 10.15283/ijsc18067.

Effects of Human Placental Amnion Derived Mesenchymal Stem Cells on Proliferation and Apoptosis Mechanisms in Chronic Kidney Disease in the Rat

• Cetinkaya
• Unek G
• Kipmen-Korgun D
• Koksoy S
• Korgun ET

• KMID: 2447233
• DOI: http://doi.org/10.15283/ijsc18067

Abstract

BACKGROUND AND OBJECTIVES
The feature of chronic kidney failure (CKF) is loss of kidney functions due to erosion of healthy tissue and fibrosis. Recent studies showed that Mesenchymal stem cells (MSCs) differentiated into tubular epithelial cells thus renal function and structures renewed. Furthermore, MSCs protect renal function in CKF. Therefore, we aimed to investigate whether human amnion-derived mesenchymal stem cells (hAMSCs) can repair fibrosis and determine the effects on proliferation and apoptosis mechanisms in chronic kidney failure.
METHODS AND RESULTS
In this study, rat model of CKF was constituted by applying Aristolochic acid (AA). hAMSCs were isolated from term placenta amnion membrane and transplanted into tail vein of rats. At the end of 30 days and 60 days of recovery period, we examined expressions of PCNA, p57 and Parp-1 by western blotting. Immunoreactivity of PCNA, Ki67, IL-6 and Collagen type I were detected by immunohistochemistry. Besides, apoptosis was detected by TUNEL. Serum creatinine and urea were measured. Expressions of PCNA and Ki67 increased in hAMSC groups compared with AA group. Furthermore, expressions of PARP-1 apoptosis marker and p57 cell cycle inhibitory protein increased in AA group significantly according to control, hAMSC groups and sham groups. IL-6 proinflammatory cytokine increased in AA group significantly according to control, hAMSCs groups and sham groups. Expressions of Collagen type I protein reduced in hAMSCs groups compared to AA group. After hAMSC treatment, serum creatinine and urea levels significantly decreased compared to AA group. After injection of hAMSC to rats, Masson's Trichrome and Sirius Red staining showed fibrosis reduction in kidney.
CONCLUSIONS
According to our results hAMSCs can be ameliorate renal failure.

Keyword

Human amnion derived mesenchymal stem cells; Chronic kidney failure; Apoptosis; Proliferation; Rat

MeSH Terms

Amnion*
Animals
Apoptosis*
Blotting, Western
Cell Cycle
Collagen Type I
Creatinine
Epithelial Cells
Fibrosis
Humans*
Immunohistochemistry
In Situ Nick-End Labeling
Interleukin-6
Kidney
Kidney Failure, Chronic
Membranes
Mesenchymal Stromal Cells*
Models, Animal
Placenta
Proliferating Cell Nuclear Antigen
Rats*
Renal Insufficiency
Renal Insufficiency, Chronic*
Tail
Urea
Veins
Collagen Type I
Creatinine
Interleukin-6
Proliferating Cell Nuclear Antigen
Urea

Figure

• 

  Fig. 1 hAMSC characterization. Flow cytometry analysis of hAMSCs for CD90 (A, B), CD105 (C, D), CD73 (E, F), CD44 (G, H) and negative cocktail (I, J) with isotype controls.

• 

  Fig. 2 Phase contrast microscopic images of human amniotic membrane derived mesenchymal stromal cells in 4X (A) and 40X magnifications (B). hAMSCs were differentiated into adipocytes (C., as shown by Oil Red O staining), osteocytes (D., as shown by Alizarin Red S staining) and chondrocytes (E and F, as shown by Alcian Blue and Sirius Red staining).

• 

  Fig. 3 Masson’s Trichrome was performed to determine fibrosis in sham+30 (A), AA (B), AA+hAMSC +30 (C) and AA+hAMSC+60 (D) groups. Sirius Red staining was showed sham+30 (E), AA (F), AA+ hAMSC+30 (G) and AA+hAMSC +60 (H) groups respectively.

• 

  Fig. 4 At 30 day hAMSCs tracking in kidney tissue with immunolabeling of human anti-mitochondrial antibody (A, ×40, B, ×100). Positive control is human decidua tissue (C, ×40, D, ×100).

• 

  Fig. 5 Immunohistochemical staining of kidney tissue sections for the sham+30, AA, AA+hAMSC+30 and AA+hAMSC+60 groups using IL-6 (A–D), Collagen-I (E–H) I and J, H scores of IL-6 and Collagen-I. Values presented as mean ± SD. AA group compared with Sham, Control, AA+hAMSC+30, and AA+hAMSC +60 groups. Values statistically significant at: *p<0.05, Sham and Control groups compared with AA and hAMSC groups; #p<0.05.

• 

  Fig. 6 PCNA, Ki67 and TUNEL immunostaining in kidneys tissue. For PCNA, sham+30 (A), AA (B), AA+ hAMSC+30 (C) and AA+hAMSC+ 60 (D) groups, for Ki67 sham+30 (E), AA (F), AA+hAMSC+30 (G) and AA+hAMSC+60 groups (H) and for TUNEL immunostaining sham+30 (I), AA (J), AA+hAMSC +30 (K) and AA+hAMSC+60 (L) groups. M and N, H scores of PCNA and Ki67. (O) Positive cells number for TUNEL. Values statistically significant at: *p<0.05.

• 

  Fig. 7 Western blot results of PCNA, p57, total PARP and cleaved-PARP. AA administration caused increased PARP and p57 expressions but decreased PCNA compared with control, sham+30 and sham+60. After hAMSC administration PARP and p57 expressions decreased and PCNA expression increased significantly. Protein levels between AA+hAMSC+30 and AA+hAMSC +60 were not statistically significant. All results were normalized to beta actin. Graphics represent means of optical densitometry measurements. *p<0.005.

• 

  Fig. 8 Functional damage assessment. Serum creatinine (A), serum urea (B) and BUN (C) levels were determined. AA administration caused increased serum creatinine, serum urea and BUN compared to control and sham groups significantly. After hAMSC administration, serum creatinine, serum urea and BUN levels decreased significantly. Graphics represent means of optical densitometry measurements. *p< 0.005

Reference

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