THE MOLECULAR MECHANISM OF LEPTIN SECRETION AND EXPRESSION INDUCES BY ARISTOLOCHIC ACID IN KIDNEY FIBROBLAST

Chung-shi yang; et al.

BY: ALEJANDRA ALVAREZ

SILVANA ZAPATA Estudiantes medicina UPB

III sermestre

OBJETIVE

The aim of this study is to explorethe effect of AA on leptinproduction and to dissect the AA-induced downstream signaling invitro.

INTRODUCTION

Leptin is a 16 kDa peptidehormone of cytokine familymainly secreted byadipocyte into blood streamand functions as a centralmediator that negativelyregulates satiety inhypothalamus1.

1: Zhang Y.; et al. 1994.

INTRODUCTION

Aristolochic acid (AA) is a famous botanical toxin which has been characterized to associate with the development of aristolochic acid nephropathy (AAN) 2

2: Vanherweghem JL, et al. (1993)

INTRODUCTION

A fibroblast is a type of cell that synthesizes the

extracellular matrix and collagen, the structural framework (stroma) for

animal tissues, and plays a critical role in wound

healing. Fibroblasts are the most common cells of

connective tissue in animals3, 4.3: Lan HY.;(2003) .

4: Roberts IS.; (1997).

INTRODUCTION

Leptin has been considered to playan important role in progressive renalfibrosis. Renal fibroblast, in particularunder the progression of fibrosisinduced by AA.

Findings imply the fibroblast-producedleptin maybe one of the factor whichpromotes the progression of renal fibrosisinduced by AA5.

5: Merabet E, et al. (1997)

MATERIALES Y METODOS

1. Cultivo celular :

Se obtuvieron fibroblastosNRK-49f a través de unacolección de cultivo tipoamericano .

2. La viabilidad celular :

Fue evaluada mediante la reducción dependiente de mitocondrias a purpura formazan y la incorporación de BrdU respectivamente .

3. Medición de la secreción de Leptina:

Fue determinada mediante el método de ELISA .

4. Western blot:

Fue utilizado para medir la expresión de la leptina inducida por AA.

• 5. RT-PCR y PCR en tiempo real:

La RT-PCR de RNAm a cDNA

PCR en tiempo real cuantificar concentración de RNAm.

6. Transfección:

Introducción de siRNA para bloquear la expresión del gen de la leptina.

7. Inmuno precipitación:

•Anticuerpo anti-Akt

RESULTADO 2.B

(B) Leptin expression was examined by Western Blot. NRK-49f kidney fibroblasts were treated without or with 12 mM AA for 24 and 48 h.

RESULTADO 2C

(C) Expression of leptin mRNA. Fibroblasts were treated with 0, 6 and 12 mM AA for 12and 24 h, the level of leptin mRNA was determined by RT-PCR

RESULTADO 3A

(A) Enhancement of C/EBP a DNA binding activity by AA. Fibroblasts were treated with 0, 6 and 12 mM AA for 3 h, nuclear extracts were isolated and C/EBP a DNA binding activity was analyzed by DAPA.

RESULTADO 3B

(B) Knockdown of C/EBP a expression by siRNA. NRK-49f cells were transfected with 0, 90 and 180 nM of C/EBP a siRNA or control scramble siRNA for 8 h, and then cells were treated with 12 mM AA for another 48 h, the expression level of C/EBP a was examined by immunoblotting (upper panel) and RTPCR (lower panel).

RESULTADO 4A

• (A) Examination of Akt activation. NRK-49f cells were incubated with 0 and 12 mM AA for 1, 3 and 6 h. The phosphorylated activation form of Akt was detected by immunoblotting

RESULTADO 4B

• (B) The levels of phosphorylated PDK1 and PI3K-p85 were evaluated by Western Blot at indicated periods of AA treatment.

RESULTADO 4C

• (C) The interaction of phosph-PDK1 with Akt. After 1 h of 0 and 12 mM AA treatments, cells were immunoprecipitated with anti-Akt antibody. The immunoprecipitated pellets were further immnoblotted with anti-phospho PDK1 or anti-Akt antibody. The input and mouse IgG were served as positive and negative control, respectively.

RESULTADO 5A

• (A) Knockdown of Akt by siRNA. Cells were transfected with 0, 60 and 120 nM of Akt siRNA or control scramble siRNA for 8 h, and then cells were treated with AA for another 48 h, the expression level of Akt was examined by immunoblotting (upper panel) and RT-PCR (lower panel).

RESULTADO 5C

• (C) Analysis of Akt activation. Cells were pretreated with 10 mMLY294002 (PI3K inhibitor) or 50 nM rapamycin (mTOR inhibitor) 1 h prior 12 mM AA addition. After 3 h incubation, level of Aktphosphorylation was determined by immunoblotting.

RESULTADO 5E

• (E) immunoblotting, respectively.

RESULTADO 5F

• (F) Expression of leptin mRNA. After the 1 h pretreatment of indicated inhibitors, cells were then treated with AA for another 24 h. RNA was isolated, and the level of leptin mRNA was assessed by RT-PCR.

RESULTADO 6A

• (A) DAPA

RESULTADO 6B

• (B) ChIP.

DISCUSION

Mapa silvana zapata

Mapa Alejandra Alvarez

CONCLUSION• Leptin is an important hormone in obesity, kidney

disease and has implications in different organs ofthe body.

• The Molecular Biology is very important in theinvestigation and diagnosis of different diseases.

• Leptin is part of the regulation of AA and this isinvolved in renal fibrosis.

• Genetic research to deliver various changes to findthe solution to many diseases.