Losartan through Hsp70 Avoids Angiotensin II Induced Mesenchymal Epithelial Transition in Proximal Tubule Cells from Spontaneously Hypertensive Rats

Cell Physiol Biochem. 2019;53(4):713-730. doi: 10.33594/000000167.

Abstract

Background/aims: Renal injury related to hypertension is characterized by glomerular and tubulointerstitial damage. The overactivation of the renin-angiotensin system mainly by angiotensin II (AII) seems to be a main contributor to progressive renal fibrosis. Epithelial to mesenchymal transition (EMT) is a mechanism that promotes renal fibrosis. Owing to heat shock protein 70 (Hsp70) cytoprotective properties, the chaperone exhibits an important potential as a therapeutic target. We investigate the role of Hsp70 on Angiotensin II induced epithelial mesenchymal transition within the Losartan effect in proximal tubule cells (PTCs) from a genetic model of hypertension in rats (SHR).

Methods: Primary cell culture of PTCs from SHR and Wistar Kyoto (WKY) rats were stimulated with AII, treated with Losartan (L), (L+AII) or untreated (Cc). The functional Hsp70 role in Losartan effect, after silencing its expression by cell transfection, was determined by Immunofluorescence; Western blotting; Gelatin Zymography assays; Scratch wound assays; flow cytometry; and Live Cell Time-lapse microscopy.

Results: (L) and (L+AII) treatments induced highly organized actin filaments and increased cortical actin in SHR PTCs. However, SHR PTCs (Cc) and (AII) treated cells showed disorganized actin. After Hsp72 knockdown in SHR PTCs, (L) was unable to stabilize the actin cytoskeleton. We demonstrated that (L) and (L+AII) increased E-cadherin levels and decreased vinculin, α-SMA, vimentin, pERK, p38 and Smad2-3 activation compared to (AII) and (Cc) SHR PTCs. Moreover, (L) inhibited MMP-2 and MMP-9 secretion, reduced migration and cellular displacement, stabilizing intercellular junctions. Notably, (L) treatment in shHsp72 knockdown SHR PTCs showed results similar to SHR PTCs (Cc).

Conclusion: Our results demonstrate that Losartan through Hsp70 inhibits the EMT induced by AII in proximal tubule cells derived from SHR.

Keywords: Angiotensin II; Hsp70; Losartan; Mesenchymal epithelial transition; Proximal tubule cells.

MeSH terms

  • Actin Cytoskeleton / drug effects
  • Angiotensin II / pharmacology*
  • Animals
  • Cadherins / metabolism
  • Cell Movement / drug effects
  • Cells, Cultured
  • Epithelial-Mesenchymal Transition / drug effects*
  • Focal Adhesions / drug effects
  • Gene Expression Regulation / drug effects
  • HSP70 Heat-Shock Proteins / antagonists & inhibitors
  • HSP70 Heat-Shock Proteins / genetics
  • HSP70 Heat-Shock Proteins / metabolism*
  • Kidney Tubules, Proximal / cytology
  • Kidney Tubules, Proximal / metabolism
  • Losartan / pharmacology*
  • Male
  • Matrix Metalloproteinase 2 / metabolism
  • RNA Interference
  • RNA, Small Interfering / metabolism
  • Rats
  • Rats, Inbred SHR
  • Rats, Inbred WKY
  • Vinculin / metabolism

Substances

  • Cadherins
  • HSP70 Heat-Shock Proteins
  • RNA, Small Interfering
  • Angiotensin II
  • Vinculin
  • Matrix Metalloproteinase 2
  • Losartan