Over-Activation of Hypoxia-Inducible Transcription Factor 1 alpha (HIF)-1α by Chronic Hypoxia Mediates Chronic Ischemic Renal Injury Romesh Dhaduk Department of Pharmocology and Toxicology Virginia Commonwealth University Short-term Educator Program for Underrepresented Persons (STEP-UP) Mentors: Dr. Pin-Lan Li and Dr. Ningjun Li
Background Once the renal damage reaches a certain threshold, the progression of chronic renal disease is consistent and irreversible. Ultimately leads to fibrosis. Mechanisms are not yet known. According to the United States Renal Data System: Total Medicare spending in 2006 - nearly $355 billion End Stage Renal Disease (ESRD) cost $23 billion. Need efficient therapeutic strategies to reverse or prevent the progress of chronic renal injury.
Background Hypoxia-inducible transcription factor 1 alpha (HIF-1α). Transcription factor. Extremely prevalent in the kidney. Hypoxia detected in all kinds of chronic renal diseases. HIF-1α is up-regulated in different chronic renal diseases. Activation of HIF-1α stimulates the fibrotic factors.
Collagen I/III level (normalized intensity) ANG II + Ang II + Ang II + A Scrambled Scrambled Scrambled Scrambled HIF1alpha- sirna ANG II + + Naïve Control sirna sirna sirna AngII HIF-1a sirna AngII sirna 1 2 3 4 5 6 HIF1- -actin B Ang II + Ang II + Scrambled Scrambled HIF1alpha- Naïve sirna sirna sirna 1 2 3 4 5 6 7 8 Collegan I/II I TIMP -1 C 2.5 2.0 * - actin 1.5 1.0 0.5 * 0.0 Naïve Scrambled sirna Ang II + scrambled sirna Ang II + HIF1alphasiRNA
Background Chronic hypoxia is possibly responsible for the overactivation of HIF-1α in chronic kidney diseases. HIF-1α may be a pathogenic factor that mediates chronic renal injury. At present, no direct evidence showing the contributing role of HIF-1α in this process. Therefore, in the present study we use 2 kidneys 1-clip rat as a chronic renal ischemia model to test our hypothesis, which is whether HIF-1α is increased in clipped kidneys and whether HIF-1α shrna blocks renal injury.
Hypothesis Hypoxia HIF-1α Fibrogenic factors Extra Cellular Matrix
Animal Model Making a clip on the left renal artery Plasmid transfection by intra renal artery injection
mrna level Bioluminescent Signal After Transfection of Luciferase Plasmids as Reporter genes. A 12000 11000 10000 9000 8000 7000 Color Bar Min = 6541.8 Max = 12482 B Click # CAR20090902162232 Wed, Sep 02, 2009 16:22:54 Em filter=open Bin:HS (16), FOV19.9, f1, 1m Camera: IS0648N4048, Spectral Instruments TE 2.0 Luciferin + O 2, ATP Oxyluciferin + Light 1.6 (Substrate) Luciferase 1.2 (Reporter Gene) Series: NJ-luciferase Experiment: 3/27/2009 Label: blue-2 Comment: body photo Analysis Comment: Weeker-back-5 bkg sub flat-fielded cosmic 0 5 10 14 (days)
Mean Artery Blood Pressure (mmhg) Blood Pressure Changes 200 180 160 140 120 100 80 60 ACEI Ctrl LE HE 40 1 3 5 8 10 12 14 16 18 20 Day after Surgery
HIF1a Level in Cortex HIF-1α Expression in Each Group A HIF-1 B 1 2 3 4 5 6 7 8 Ctrl L LE HE 5.0 4.0 3.0 -Actin Ctrl=Normal Animal+ Luciferase, L=Clipped Animal+Luciferase, LE=Clip+ACEI+Luciferase, HE=Clip+ACEI+HIF-1 shrna 2.0 1.0 0.0 Ctrl L LE HE
Glomerular Damage Index (GDI) Glomerular Damage in Each Group A Ctlr L B 4 3 LE HE 2 1 0 Ctrl (N=4) L(n=3) LE(n=7) HE(n=7)
Collagen Staining Area Ratio (%) Effect of Silencing HIF-1α on Collagen Distribution A 25 B LE 20 15 10 HE 5 0 Ctrl(n=4) L(n=3) LE (n=7) HE (n=7)
Effect of Silencing HIF-1α on CD5, B Cell Marker 100X 200X Clip+ACEI+Luciferase Clip+ACEI+HIF-1α shrna Clip+ACEI+Luciferase Clip+ACEI+HIF-1α shrna Cortex Medulla
Conculsion Clip over-activates hypoxia-inducible transtription factor 1 alpha (HIF-1α) expression by chronic hypoxia. Over-activation of HIF-1α contributes to chronic ischemic renal injury. Inflammation is involved in this renal injury. Silencing HIF-1α can protect chronic ischemic renal injury.
Future Directions Use disease models such as diabetic nephropathy, hypertensive nephropathy and see whether silencing HIF- 1α can protect against chronic ischemic renal injury. HIF prolyl hydroxylase (PHD): Oxygen sensor that regulate HIF-1a levels in response to changes of oxygen concentrations In normoxia, targets HIF-1α for destruction. To determine whether PHD will also be involved in CKD via regulation of HIF-1alpha.
Acknowledgements Dr. Pin-Lan Li and Dr. Ningjun Li; Dr. Zhengchao Wang and Dr. Qing Zhu; Lori P. Payne; All other staff in the lab; This work is supported by NIH and STEP-UP.