BADERC- Joseph Bonventre

Joseph Bonventre, M.D.,Ph.D.

Biology of the Glomerular Mesangial Cell

Nephropathy occurs in approximately one-third of all patients with diabetes mellitus. Diabetic kidney disease is the single most frequent cause of end-stage renal disease. Diabetics account for approximately 35% of newly diagnosed patients with end-stage renal disease. Diabetic nephropathy occurs more often in diabetics of certain minority groups, in particular Native Americans, Hispanics and black Americans. The latter group have almost three times the rate of new cases as white Americans.

Mesangial expansion with mesangial cell proliferation and enhanced matrix production correlates well with the severity of renal impairment. My laboratory has had a long interest in the involvement of various signalling pathways in mesangial cells proliferation, matrix production, and synthesis of vasoactive compounds which will alter glomerular transcapillary forces.

My laboratory is also involved in the identification and characterization of phospholipases A2 (PLA2). The early increase in renal plasma flow, glomerular filtration rate, and the microangiopathy of diabetic nephropathy, may be due to abnormalities in eicosanoids, which are produced upon activation of PLA2. In addition to the effects of eicosanoids on blood flow PGE2 has been proposed to directly reduce the matrix production by mesangial cells in vitro. Thromboxane stimulates matrix production in mesangial cells whereas PGE2 has been proposed to decrease matrix production.

We identified the large molecular mass cytosolic PLA2 (cPLA2) in the mesangial cell and initially characterized its stable activation with growth factors and vasoactive peptides. This form of the enzyme is responsible for release of arachidonic acid and hence is necessary for eicosanoid synthesis. We have generated cPLA2-/- mice using techniques of homologous recombination. Mesangial cells from these mice do not produce PGE2 in response to calcium ionophores or cytokines. This model provides an excellent opportunity to study the role of eicosanoid production on glomerular disease in vivo and in vitro. cPLA2 moves from cytosol to membrane in a calcium-dependent process. Using the two-hybrid cloning system we have identified two proteins that interact with the calcium phospholipid binding region of the molecule. Targeting this region of the molecule with inhibitors will block the action of this form of PLA2 and potentially block the early hyperperfusion and filtration phase of diabetic nephropathy which may be mediated by vasodilatory eicosanoids.

Another area of research which directly relates to diabetic nephropathy is the role of ischemia in the progression of disease. Microangiopathy leads to local ischemia and tubular cell death with nephron drop-out. Chronic ischemia leads to apoptosis in other models and this is likely to be the case in diabetic nephropathy also. We have studied for many years the factors that determine ischemic cell death. Recently we have established cell models of PLA2 and/or bcl-2 overexpression to examine the role of PLA2 in cell death and the role of bcl-2 in its prevention. Two important contributors to ischemic cell death, reactive oxygen species and PLA2, induce cell death which was prevented by bcl-2 overexpression even though the PLA2 activation and arachidonic acid release was not prevented in the bcl-2 overexpressing cells These experiments and others are helping us dissect out the specific determinants critical to cell death. In addition to evaluating the processes critical for cell death we are studying the molecular mechanisms involved in the hypertrophy and hyperplasia that is characteristic of the mesangium in diabetes. We study the signaling pathways and changes in gene expression associated with endothelin-induced mesangial cell hypertrophy. As an example, in collaboration with Dr, Kyriakis at Tufts University School of Medicine we have found that transcriptional profiling identified seven genes induced with slow kinetics by endothelin. Of these, p8, which encodes a small basic helix–loop–helix protein, was most strongly and stably induced. p8 is also induced in diabetic kidney. Mesangial cell hypertrophy and p8 induction both require activation of the ERK, JNK/SAPK and PI-3-K pathways. Small interfering RNA (siRNA)-mediated RNA interference indicates that p8 is required for endothelin-induced hypertrophy. Thus, p8 is a novel marker for diabetic renal hypertrophy.

References:

1. Goruppi S, Bonventre JV, Kyriakis JM. Signaling pathways and late-onset gene induction associated with renal mesangial cell hypertrophy. EMBO J. 21:5427-36, 2002.

2. Bonventre JV. The kidney proteome: a hint of things to come. Kidney Int. 62:1470-1, 2002.

3. Hung CC, Ichimura T, Stevens JL, Bonventre JV. Protection of renal epithelial cells against oxidative injury by endoplasmic reticulum stress preconditioning is mediated by ERK1/2 activation. J Biol Chem. 278:29317-26, 2003.

4. Bonventre JV, Weinberg JM. advances in the pathophysiology of ischemic acute renal failure.J Am Soc Nephrol. 2003;14:2199-210.

5. Andreucci M, Michael A, Kramers C, Park KM, Chen A, Matthaeus T, Alessandrini A, Haq S, Force T, Bonventre JV. Renal ischemia/reperfusion and ATP depletion/repletion in LLC-PK(1) cells result in phosphorylation of FKHR and FKHRL1. Kidney Int. 2003;64:1189-98.

6. Bonventre JV. Molecular response to cytotoxic injury: role of inflammation, MAP kinases, and endoplasmic reticulum stress response. Semin Nephrol. 2003;23:439-48. 7:

7. Ichimura T, Hung CC, Yang SA, Stevens JL, Bonventre JV. Kidney injury molecule-1: a tissue and urinary biomarker for nephrotoxicant-induced renal injury. Am J Physiol Renal Physiol. 2004;286:F552-63.






			Joseph Bonventre, M.D.,Ph.D. Biology of the Glomerular Mesangial Cell Nephropathy occurs in approximately one-third of all patients with diabetes mellitus. Diabetic kidney disease is the single most frequent cause of end-stage renal disease. Diabetics account for approximately 35% of newly diagnosed patients with end-stage renal disease. Diabetic nephropathy occurs more often in diabetics of certain minority groups, in particular Native Americans, Hispanics and black Americans. The latter group have almost three times the rate of new cases as white Americans. Mesangial expansion with mesangial cell proliferation and enhanced matrix production correlates well with the severity of renal impairment. My laboratory has had a long interest in the involvement of various signalling pathways in mesangial cells proliferation, matrix production, and synthesis of vasoactive compounds which will alter glomerular transcapillary forces. My laboratory is also involved in the identification and characterization of phospholipases A2 (PLA2). The early increase in renal plasma flow, glomerular filtration rate, and the microangiopathy of diabetic nephropathy, may be due to abnormalities in eicosanoids, which are produced upon activation of PLA2. In addition to the effects of eicosanoids on blood flow PGE2 has been proposed to directly reduce the matrix production by mesangial cells in vitro. Thromboxane stimulates matrix production in mesangial cells whereas PGE2 has been proposed to decrease matrix production. We identified the large molecular mass cytosolic PLA2 (cPLA2) in the mesangial cell and initially characterized its stable activation with growth factors and vasoactive peptides. This form of the enzyme is responsible for release of arachidonic acid and hence is necessary for eicosanoid synthesis. We have generated cPLA2-/- mice using techniques of homologous recombination. Mesangial cells from these mice do not produce PGE2 in response to calcium ionophores or cytokines. This model provides an excellent opportunity to study the role of eicosanoid production on glomerular disease in vivo and in vitro. cPLA2 moves from cytosol to membrane in a calcium-dependent process. Using the two-hybrid cloning system we have identified two proteins that interact with the calcium phospholipid binding region of the molecule. Targeting this region of the molecule with inhibitors will block the action of this form of PLA2 and potentially block the early hyperperfusion and filtration phase of diabetic nephropathy which may be mediated by vasodilatory eicosanoids. Another area of research which directly relates to diabetic nephropathy is the role of ischemia in the progression of disease. Microangiopathy leads to local ischemia and tubular cell death with nephron drop-out. Chronic ischemia leads to apoptosis in other models and this is likely to be the case in diabetic nephropathy also. We have studied for many years the factors that determine ischemic cell death. Recently we have established cell models of PLA2 and/or bcl-2 overexpression to examine the role of PLA2 in cell death and the role of bcl-2 in its prevention. Two important contributors to ischemic cell death, reactive oxygen species and PLA2, induce cell death which was prevented by bcl-2 overexpression even though the PLA2 activation and arachidonic acid release was not prevented in the bcl-2 overexpressing cells These experiments and others are helping us dissect out the specific determinants critical to cell death. In addition to evaluating the processes critical for cell death we are studying the molecular mechanisms involved in the hypertrophy and hyperplasia that is characteristic of the mesangium in diabetes. We study the signaling pathways and changes in gene expression associated with endothelin-induced mesangial cell hypertrophy. As an example, in collaboration with Dr, Kyriakis at Tufts University School of Medicine we have found that transcriptional profiling identified seven genes induced with slow kinetics by endothelin. Of these, p8, which encodes a small basic helix–loop–helix protein, was most strongly and stably induced. p8 is also induced in diabetic kidney. Mesangial cell hypertrophy and p8 induction both require activation of the ERK, JNK/SAPK and PI-3-K pathways. Small interfering RNA (siRNA)-mediated RNA interference indicates that p8 is required for endothelin-induced hypertrophy. Thus, p8 is a novel marker for diabetic renal hypertrophy. References: 1. Goruppi S, Bonventre JV, Kyriakis JM. Signaling pathways and late-onset gene induction associated with renal mesangial cell hypertrophy. EMBO J. 21:5427-36, 2002. 2. Bonventre JV. The kidney proteome: a hint of things to come. Kidney Int. 62:1470-1, 2002. 3. Hung CC, Ichimura T, Stevens JL, Bonventre JV. Protection of renal epithelial cells against oxidative injury by endoplasmic reticulum stress preconditioning is mediated by ERK1/2 activation. J Biol Chem. 278:29317-26, 2003. 4. Bonventre JV, Weinberg JM. advances in the pathophysiology of ischemic acute renal failure.J Am Soc Nephrol. 2003;14:2199-210. 5. Andreucci M, Michael A, Kramers C, Park KM, Chen A, Matthaeus T, Alessandrini A, Haq S, Force T, Bonventre JV. Renal ischemia/reperfusion and ATP depletion/repletion in LLC-PK(1) cells result in phosphorylation of FKHR and FKHRL1. Kidney Int. 2003;64:1189-98. 6. Bonventre JV. Molecular response to cytotoxic injury: role of inflammation, MAP kinases, and endoplasmic reticulum stress response. Semin Nephrol. 2003;23:439-48. 7: 7. Ichimura T, Hung CC, Yang SA, Stevens JL, Bonventre JV. Kidney injury molecule-1: a tissue and urinary biomarker for nephrotoxicant-induced renal injury. Am J Physiol Renal Physiol. 2004;286:F552-63.