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  • 1. Abbiamo farmaci che sappiano riparare il danno ai podociti? Ariela Benigni Dipartimento di Medicina Molecolare Istituto di Ricerche Farmaclogiche Mario Negri, Laboratori Negri Bergamo Torino, 23 gennaio 2009 1
  • 2. Glomerular hypertension Podocytes GBM Endothelial cells Disease progression 2
  • 3. Glomerular hypertension Mechanical strain * 2.5 1.2 (pg per µg of cell lysate) (adjusted for tubulin) 1.0 2.0 0.8 AT1R level Ang II 1.5 Podocyte number 0.6 1.0 0.4 0.5 0.2 0 0 Ctr MS Ctr MS Durvasula et al, Kidney Int, 2004 Pore dimension Proteinuria Disease progression 3
  • 4. AT1R PLC DAG IP3 TRPC6 Ca2+ Cytoskeleton TRPC6: Transient Receptor Potential cation channel Winn et al, Science, 2005 Nitschke et al., Kidney Int, 2000 4
  • 5. actin ZO-1 merge 40 Albumin flux (µg/hour) 30 µ Control 20 10 0 40 Albumin flux (µg/hour) * 30 µ Ang II 20 10 0 Macconi et al., Am J Pathol, 2006 5
  • 6. 1 - apoptosis 2 - phenotype changes 6
  • 7. UNINEPHRECTOMIZED MWF/ZTM RATS - 1 * 700 Urinary Protein Excretion 600 500 (mg/24 hrs) 400 300 200 100 ** 0 Control UNx UNx + Lis * 100 Percentage of glomeruli affected by sclerosis 80 60 40 20 ** 0 Control UNx UNx + Lis * p < 0.05, **p < 0.01 vs control 7 Remuzzi A. et al., Kidney Int, 1995
  • 8. UNINEPHRECTOMIZED MWF/ZTM RATS - 2 Control 100 UNx + Lis 80 Survival (%) 60 40 20 UNx 0 0 3 6 9 12 15 Time (months after UNx) 8 Remuzzi A. et al., Kidney Int, 1995
  • 9. Ramipril Ramipril 45 ∆ GFR = -0.44 ± 0.54 40 (ml/min/month) ∆ GFR = - 0.10 ± 0.50 GFR 35 ∆ GFR = -0.81 ± 1.12 30 ∆ GFR = -0.14 ± 0.87 25 Conventional Ramipril FOLLOW-UP CORE Ruggenenti et al., Lancet, 1998 9
  • 10. REGRESSION 10 patients with increasing GFR ∆ Proteinuria P = 0.01 65 (pre vs post break point) ∆GFR -0.21 + 0.09 +0.49 + 0.19 60 (ml/min/month) GFR (ml/min/month) 0 55 50 45 -20 40 % 35 -40 30 25 Break point 20 -60 -30 -20 -10 0 10 20 30 months Ruggenenti et al., J Am Soc Nephrol, 1999 10
  • 11. MWF 50W MWF 60W MWF+LIS 50-60 w Remuzzi A. et al., Kidney Int, 2006 11
  • 12. 100 % of Capillary Tuft Volume Affected by Sclerosis 75 MWF 50W 50 25 0 0 10 20 30 40 50 60 70 80 90 100 Reconstructed Glomeruli 12
  • 13. 100 % of Capillary Tuft Volume Affected by Sclerosis 75 MWF 50W MWF 60W 50 25 0 0 10 20 30 40 50 60 70 80 90 100 Reconstructed Glomeruli 13
  • 14. 100 % of Capillary Tuft Volume Affected by Sclerosis 75 MWF 50W MWF 60W 50 25 MWF + LIS 50-60W 0 0 10 20 30 40 50 60 70 80 90 100 Reconstructed Glomeruli 14
  • 15. Sclerosis was effectively reabsorbed and a consistent amount of glomerular tissue regained normal structure This suggests neoformation of glomerular capillary segments 15
  • 16. INSIGHT INTO ACE-INDUCED RENAL REPAIR/ANGIOGENESIS Renal cells Adult differentiated Resident progenitor/stem Extra renal cells Endothelial progenitor and/or bone marrow- derived stem 16
  • 17. 30 VV endothelial cells (%) 20 10 * 0 40 W 60 W LIS 40-60 W WGA = cell membranes RECA-1= endothelial cells Macconi et al., 2008 17
  • 18. WT1+ cells/glom 160 120 ** 80 40 0 40W 60W LIS 40-60W WT1 = podocyte marker (nuclei) Macconi et al., Am J Pathol, 2009 18
  • 19. Migration of parietal cells to capillary tuft through the vascular pole 19
  • 20. Migration of parietal cells from the Bowman’s capsule to capillary tuft 20
  • 21. PARIETAL CELLS WITH PODOCYTE PHENOTYPE 12 * Parietal podocytes/PEC (%) 10 8 6 4 2 0 MWF MWF MWF + Lis 40 W 60 W 60 W These cells were identified by staining for PGP 9.5 (parietal epithelial cell marker) and WT1 (podocyte marker) Macconi et al., Am J Pathol, 2009 21
  • 22. ISOLATION AND CHARACTERIZATION OF MULTIPOTENT PROGENITOR CELLS CD133+CD24+ FROM THE BOWMAN’S CAPSULE OF ADULT HUMAN KIDNEYS Sagrinati et al., J Am Soc Nephrol, 2006 22
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  • 24. Brenner’s seminal paper NEJM Retarding renal Promoting kidney disease progression repair 1983 2003 24
  • 25. The presence of VEGF is crucial for normal renal development Differentiating glomerular epithelia produce VEGF and may attract endothelial cells into the glomeruli Administration of anti-VEGF antibody during early kidney development in mice leads to formation of abnormal glomerular structures and diminished nephrogenesis Kretzler et al., Kidney Int, 1998 25
  • 26. PODOCYTE ARE THE MAJOR SOURCE OF VEGF IN THE GLOMERULUS Kretzler et al., Kidney Int, 1998 26
  • 27. PODOCYTE VEGF BINDS TO COGNATE RECEPTORS EXPRESSED ON GLOMERULAR ENDOTHELIAL CELLS How podocytes can signal “up stream” in the glomerular endothelium A concentration gradient favors diffusion of VEGF from the podocyte to glomerular endothelial cells Eremina et al., N Engl J Med, 2008 27
  • 28. 28
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  • 30. Glomerular endothelial cells differ from most other endothelial cells in that they are extremely flattened and densely perforated by transendothelial pores, the fenestrae, necessary for the unique permeability characteristics of the glomerular filtration barrier Ballerman et al., Nephrol Physiol, 2007 Mature fenenestrated endothelium is located adjacent to podocytes expressing VEGF at high levels Breier et al., Development, 1992 30
  • 31. VEGF induces endothelial fenestrations by activating the fusion of intracellular organelles thought to represent the precursors of fenestrae 31
  • 32. The New York Times - May 3, 1998 32
  • 33. THE CASE OF THE HUMANIZED ANTI-VEGF ANTIBODY BEVACIZUMAB Bevacizumab is effective in the treatment of patients with many cancers, such as metastatic colorectal cancer, non-small-cell lung cancer, and breast cancer It is also promising for renal cell carcinoma and prostate cancer Zhu et al., Am J Kidney Dis, 2007 33
  • 34. PROTEINURIA IS A COMMON SIDE-EFFECT OF HIGH DOSE BEVACIZUMAB A randomized, double-blind, phase 2 trial in patients with metastatic renal-cell carcinoma Bevacizumab: 10 mg/kg every two weeks Adverse events Bevacizumab Placebo (n=39) (n=40) Patients (n) 15 25 Proteinuria * 1 14 Hypertension 6 13 Malaise 0 5 Hematuria Proteinuria: > 1+ or > 150 mg/24 hours * Yang et al., N Engl J Med, 2003 34
  • 35. RISK OF PROTEINURIA IN PATIENTS WITH CANCER GIVEN BEVACIZUMAB IS DOSE-DEPENDENT A meta-analysis of 5 trials in 1,850 patients (1966-2006 year) Relative risk Hurwitz et al., 2004 Johnson et al., 2003 Kabbinavar et al., 2003 Kabbinavar et al., 2005 Yang et al., 2003 Combined P < 0.001 P = 0.003 Bevacizumab Control Bevacizumab Control high dose low dose 100 1 100 1 Zhu et al., Am J Kidney Dis, 2007 35
  • 36. PROTEINURIA AFTER BEVACIZUMAB THERAPY Urinary albumin/creatinine 6 4 ratio 2 0 Post Pre Bevacizumab (9 months) Eremina et al., N Engl J Med, 2008 36
  • 37. VEGF Inhibition and Renal Thrombotic Microangiopathy The glomerular microvasculature is particularly susceptible to injury to thrombotic microangiopathy, but the mechanisms by which this occurs are unclear We report the cases of six patients who were treated with bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor, in whom proteinuria and glomerular disease characteristic of thrombotic microangiopathy developed Eremina et al., N Engl J Med, 2008 37
  • 38. Disease Clinical and biochemical Kidney biopsy parameters - Normal renal function at Thrombotic Patient 1 Hepatocellular baseline microangiopathy (59 years) carcinoma - Urinary P/C from 0.5 to 3.4 - New onset hypertension - Low platelet count - Normal renal function at Thrombotic Patient 2 Recurrent baseline microangiopathy (74 years) hepatocellular - Urinary P/C from 0.4 to 2.7 carcinoma Patient 3 Bronchoalveolar - Normal renal function at Thrombotic baseline microangiopathy (56 years) carcinoma - Minimal proteinuria (0.6 g/24h) - Hypertension worsened - Anemia Eremina et al., N Engl J Med, 2008 38
  • 39. Disease Clinical and biochemical Kidney biopsy parameters - Diabetic nephropathy at Thrombotic Patient 4 Small-cell lung baseline microangiopathy (62 years) carcinoma - Acute renal failure (s. creat from 1,4 to 5,7 mg/dl) - Proteinuria 3+ - Normal renal function at Thrombotic Patient 5 Metastatic baseline microangiopathy (61 years) pancretic cancer - Proteinuria up to 4.6 g/24h - Low platelet count Thrombotic - Proteinuria from 0.2 to 0.8 Patient 6 Metastatic microangiopathy mg/24 h (59 years) ovarian cancer - Normal platelet count Eremina et al., N Engl J Med, 2008 39
  • 40. To show that local reduction of VEGF within the kidney is sufficient to trigger the pathogenesis of thrombotic microangiopathy, we used conditional targeting to delete VEGF from renal podocytes in adult mice This resulted in pronounced proteinuria and thrombotic glomerular injury These observations provide evidence that glomerular injury in patients who are treated with bevacizumab is probably due to direct targeting of VEGF by antiangiogenic therapy Eremina et al., N Engl J Med, 2008 40
  • 41. VEGF inhibition/blockade 120 ET-1 (pg/106 cells) Ab-VEGF 60 Loss of glomerular Endothelial C endothelial fenestrae ET-1 0 Collino et al., Am J Physiol Renal Physiol, 2008 Nephrin Microvascular Protein expression injury traffic on podocyte Protein overload Podocyte on podocytes ET-1 Altered Ct) 2 glomerular ET-1 gene (2- permselectivity Albumin 1 Thrombotic C 0 microangiopathy Proteinuria Morigi et al., Am J Pathol, 2005 41
  • 42. 10 YEARS OF MARKET APPROVALS OF CANCER DRUGS BY EMEA 1995-2004 - 14 cancer drugs - 27 indications Survival benefit 0-3.7 months Apolone et al., Br J Cancer, 2005 42
  • 43. February 15, 2006 A Cancer Drug Show Promise, at a Price That Many Can’t Pay By ALEX BERENSON 43
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  • 45. These slides are belonging to Ariela Benigni, Ph.D. Mario Negri Institute for Pharmacological Research, Bergamo, Italy. Using these slides is only authorized by mentioning the source 45