a new type of kidney stone: rocks in aki anthony valeri, md
TRANSCRIPT
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A NEW TYPE OF KIDNEY STONE: ROCKs IN AKI
Anthony Valeri, MD
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Rho
• Belongs to a family of small GTP-binding proteins
• Several subfamilies – Ras, Rho, Rab, Ran
• Controls multiple signaling pathways
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Rho GTPases
• Modulate the activation of Rho by regulation of the interconversion of Rho-GDP to/from Rho-GTP (a molecular switch)
• Associated with changes in the actin cytoskeleton (stress fibers)
• Mediates a variety of cell processes:– Cell adhesion– Cell contraction– Cell motility and migration– Cell proliferation
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Rho and Rho-GTPases
• RhoA regulates the assembly of actin stress fibers
• Cycles between the inactive GDP-bound and active GTP-bound forms
• Cycling controlled by regulatory proteins:– GDI (guanine nucleotide dissociation inhibitor) – binds
to Rho-GDP and prevents its translocation from the cytosol to the cell membrane
– GEF (guanine nucleotide exchange factor) – activates RhoA (by phosphorylation of Rho-GDP to Rho-GTP)
– GAP (GTPase activating protein) – inactivates RhoA (by dephosphorylation of Rho-GTP to Rho-GDP)
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ROCK (ROK) • Rho-associated coiled-coil kinase• A SER-THR kinase – phosphorylates multiple targets• Downstream target of Rho• Structure:
– Amino-terminal kinase domain– Rho-binding domain (RBD) – binding of Rho-GTP activates ROCK– Carboxy-terminal cysteine-rich domain (CRD)
• Regulates cell shape, polarity and migration via cellular contraction, protrusions and focal adhesions
• Regulates cell growth (via regulation of cytokines after nuclear division and G1-to-S cycle progression) and apoptosis
• Regulates monocyte/macrophage phagocytic activity and endothelial cell permeability by affecting tight and adherens junctions through actin cytoskeletal contractions
• Effects mediated via control of actin cytoskeleton (stress fibers) assembly (45-50% homology to other actin cytoskeleton kinases)
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ROCK
• ROCK, in turn, regulates cell contraction via SER-THR phosphorylation of multiple targets (abducin, ezrin-radixin-moesin (ERM) protein, LIM kinase, MLC phosphatase, Na/H-exchanger (NHE)-1)
• Phosphorylates MLC phosphatase which inhibits its activity, thereby, preventing myosin II de-phosphorylation
• Increased MLC phosphorylation enhances actin-myosin association and prevents actin depolymerization (disassembly)
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Copyright ©2006 American Heart Association
Loirand, G. et al. Circ Res 2006;98:322-334
Contraction signaling in vascular smooth muscle cells
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Copyright ©2006 American Heart Association
Loirand, G. et al. Circ Res 2006;98:322-334
Known mechanisms of ROCK activation and inactivation
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Targets of ROCKTargets Effect Function
Cytoskeleton regulating proteins
AdducinIncreased aducin/F-actin
interaction Assembly of spectrin/F-actin network; Increase of cell motility
ERM
Decrease of intra-or intermolecular head-to-tail association of ERM Actin filament/membrane interaction; microvilli formation
MARCKS ? Cytoskeletal rearrangement
NHE1Stimulation of its Na+-H+
exchanger activity Actin stress fiber formation
EF-1 alpha Inhibition of its binding to F-actin Increase of actin stress fiber formation?
LIM-kinases 1 and 2 Stimulation of kinase activity
Actin polymerization (through phosphorylation and inactivation of cofilin); Coordination of microtubule destabilization and actin formation
RhoE Increase RhoE stabilityPotentialization of the inhibitory effect of RhoE on actin stress fiber
formation and Ras-induced transformation
Intermediate filaments
GFAP Inhibition of its filament formation Regulation of cytokinesis
NF-L Inhibition of its filament formation ?
Desmin Inhibition of its filament formation ?
Vimentin Inhibition of its filament formation Regulation of cytokinesis
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Contraction regulating proteins
MYPT-1 Inhibition of MLCP activityCa2+ sensitization of smooth muscle contraction/ stress
fiber formation
CPI-17 Inhibition of MLCP activity Ca2+ sensitization of smooth muscle contraction
MLCStimulation of actomyosin ATPase
activityCa2+ sensitization of smooth muscle contraction/ stress
fiber formation
Calponin Inhibition of calponin binding to actin Ca2+ sensitization of smooth muscle contraction
TroponinInhibition of actomyosin ATPase
activity Inhibition of tension generation of cardiac myocytes
Microtubule regulating proteins
Tau Reduction of its activity Regulation of microtubule dynamics
MAP 2 ? ?
Neuronal proteins
CRMP-2 ? Growth cone collapse
Signaling proteins
PTEN Stimulation of phosphatase activityDecrease of intracellular PtdIns(3,4,5)P3 level; tumor
suppression
ERM indicates ezrin, radixin, moesin; MARCKS, myristoylated alanine-rich C-kinase substrate; NHE1, Na+-H– exchanger; EF-1{alpha}, elongation factor 1-{alpha}; GFAP, glial fibrillary acidic protein; NF-L, neurofilaments; MAP 2, microtubule-associated protein 2; CRMP-2, collapsin response mediator protein 2.
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Copyright ©2006 American Heart Association
Loirand, G. et al. Circ Res 2006;98:322-334
Major cell functions regulated by ROCKs after stimulation by hormones or neuromediators, growth factors, interaction with the extracellular matrix
(ECM), or mechanical stretch
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ROCK isoforms
• ROCK 1• aka ROKß or p160-
ROCK• Chromosome 18• 1,354 aa protein• Found predominantly in
lung, liver, spleen, kidney and testes (ubiquitous)
• Centrosome localization• Cleaved by caspase-3
during apoptosis
• ROCK 2• aka ROKα or Rho-
kinase• Chromosome 12• 1,388 aa protein• Found predominantly in
heart and brain• Activated by RhoA• Cytoplasmic localization –
translocates to cell membrane when activated
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Copyright ©2006 American Heart Association
Loirand, G. et al. Circ Res 2006;98:322-334
The molecular structure of ROCKs
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• Systemic HTN (via mechanical stress) and AII:– Activates T-type Ca+2 channels in VSMC– Activates ROCK in VSMC – increase in p27kip1 (CDK inhibitor)
– stimulates cell proliferation – contributing to neointimal hyperplasia
– May mediate some of the vasculo- and reno- toxic effect of AII
• Contributes to the agonist-induced Ca+2 sensitization of VSMC – increased vasoconstriction
• Rho/ROCK pathway – downregulates endothelial NOS (eNOS) expression in response to chronic hypoxic stimuli or cell proliferation
Vascular Effects of ROCK
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CV Effects of ROCK• Involved in angiogenesis, atherosclerosis,
cerebral and coronary vasospasm, cerebral ischemia, ED, HTN, glomerulosclerosis, myocardial hypertrophy and I/R injury, neointimal hyperplasia, pulmonary HTN, vascular remodeling
• Many effects of statins may be mediated by ROCK inhibition
• ROCK inhibitors:– Prevent cerebral vasospasm after SAH– Inhibits atherosclerosis and arterial remodeling after
vascular (endothelial) injury
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Copyright ©2005 American Heart Association
Shimokawa, H. et al. Arterioscler Thromb Vasc Biol 2005;25:1767-1775
Role of Rho/Rho-kinase pathway in the pathogenesis of cardiovascular diseases
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Copyright ©2005 American Heart Association
Shimokawa, H. et al. Arterioscler Thromb Vasc Biol 2005;25:1767-1775
Involvement of Rho-kinase pathway in the pathogenesis of arteriosclerosis
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Metabolic Effects of ROCK
• Inhibits insulin signaling via direct phosphorylation of insulin receptor substrate-1 (IRS-1)– Uncouples the insulin-R from PI3K and inhibits PI3K/Akt– GLUT4 activation
• ROCK activity reduced by PPARγ agonists (glitazones) – with reduced systemic BP (?use in the metabolic syndrome)
• Enhances IGF-1 induced cAMP element binding protein (CREB) phosphorylation (linked to cardiac hypertrophy)
• p190B Rho-GAP (inactivates RhoA – inhibits ROCK)– Causes adipogenesis– Causes myocyte differentiation
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Other Effects of ROCK
• Evidence to suggest involvement in Alzheimer’s, bronchial asthma, cancer demyelinating disease, glaucoma and osteoporosis
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Study Level
Studies with ROCK
Molecular level
Gene expression
Promotor region analysis
Single nucleotide polymorphism
Role in signal transduction
Cellular level
VSMC contraction
VSMC proliferation/migration
Cell adhesion and motility
Cytokinesis
Animal studies
Coronary vasospasm
Cerebral vasospasm
Arteriosclerosis/restenosis
Ischemia/reperfusion injury
Hypertension
Pulmonary hypertension
Stroke
Heart failure
Renal disease
Glaucoma
Erectile dysfunction
Clinical studies
Angina
Hypertension
Pulmonary hypertension
Stroke
Heart failure
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ROCK and the Kidney
• Rho/ROCK pathway has effects on:– Renal circulation– Mesangial cells– Podocytes– Renal tubular cells
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ROCK and the Kidney (Renal Circulation)
• Constitutively active in the renal circulation
• Partially mediates AII-constriction of efferent arteriole
• Effect on PGC unknown
• ROCK inhibitors may help in slowing progression of CKD – dilates both the afferent and efferent arteriole (greater effect on afferent arteriole)
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ROCK and the Kidney(Mesangial Cells)
• Mesangial cells are smooth muscle-like cells that produce ECM and collagen (the mesangial matrix) – partly mediated by TGFß
• Mechanical stress caused by increased PGC leads to increased MAPK activity, stress fiber formation and cell proliferation by mesangial cells – RhoA is a modulator of MAPK
• ROCK mediates the actin cytoskeletal rearrangement leading to EMT (transdifferentiation of mesangial cells to myofibroblasts) – increased αSMA expression – leading to glomerulosclerosis
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ROCK and the Kidney(Podocytes)
• F-actin controls cellular morphology
• Y-27632 (ROCK inhibitor) has been shown to inhibit the reorganization of the podocyte cytoskeleton induced by mechanical stress in culture
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ROCK and the Kidney(Renal Tubular Cells)
• Effects on cell proliferation, migration and apoptosis
• Regulates formation of stress fibers, focal adhesions and peripheral bundles by reorganization of the actin cytoskeleton – based on in-vitro studies in the MDCK cell line
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ROCK and the Kidney(Inflammation)
• Infiltrating monocytes/macrophages stimulate EMT leading to interstitial fibrosis
• May be mediated, in part, by ROCK
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ROCK and the Kidney(Renal Intersititium)
• ROCK inhibition prevents the TGFß-induced increase in CTGF in fibroblasts leading to reduced fibroblast cell proliferation and ECM production/deposition (fibrogenesis)
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ROCK and the Kidney
• Thus, by effects on:– Glomerular hemodynamics– Mesangial cell proliferation, EMT, ECM deposition– ROCK inhibitors may be of benefit in
glomerulosclerosis
• And, by effects on:– Renal tubular cell EMT, inflammatory cell
(monocyte/macrophage) infiltration, fibroblast proliferation and ECM deposition
– ROCK inhibitors may be of benefit in tubulointerstitial fibrosis
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ROCK inhibitors
• Fasudil and Y-27632
• Selective ROCK inhibitors
• Targets the Rho-GTP-dependent binding domain of BOTH ROCK 1 and ROCK 2
• Though at high concentrations, also inhibits PKA and PKC
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Copyright ©2005 American Heart Association
Shimokawa, H. et al. Arterioscler Thromb Vasc Biol 2005;25:1767-1775
Broad pharmacological properties of Rho-kinase inhibitors
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Copyright ©2005 American Heart Association
Shimokawa, H. et al. Arterioscler Thromb Vasc Biol 2005;25:1767-1775
Therapeutic targets of Rho-kinase inhibitors
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ROCK Inhibition in Renal Models
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UUO
• Fasudil and Y-27632 (ROCK inhibitors):– Reduce tubulointerstitial fibrosis
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SHR with 5/6 Nx
• Model of HTN glomerulosclerosis• SHR – increased ROCK activity in the aorta• Increased PGC – mechanical stress – activates
Rho/ROCK pathway• Fasudil (ROCK inhibitor):
– Reduces UVprotein– Reduces glomerulosclerosis and tubulointerstitial fibrosis– Reduces infiltration of ED-1 (+) cells (monocyte/macrophage
marker)– Reduces PNCA (+) cells (marker of proliferating cells)– All these changes occur despite no change in systemic BP– Reduces p27kip1 (CDK inhibitor) – leading to inhibition of cell
proliferation and monocyte/macrophage recruitment
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DOCA-SHR rats• Model of malignant HTN• Fasudil (ROCK inhibitor):
– No effect on systemic BP– Reduces UVprotein and improves GFR– Reduces periarteriolar fibrosis and glomerulosclerosis– Reduces inflammatory cell infiltrate (by ED-1 staining for
monocytes/macrophages)– Reduces oxidative stress (as measured by urinary excretion of 8-
isoprostane – PGF-2-like compound produced by the free radical peroxidation of aa in cell membranes and circulating LDL))
• DOCA-SHR – upregulation of NADPH oxidase subunits – p40, p47, p67phox
• AII – upregulates NAPDH oxidase via the Rho/ROCK pathway– Reduces TGFß, COL I, and COL III expression (reduced
fibrogenesis)– Improves eNOS expression
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SHR-SP
• Model of HTN CV disease
• On high salt diet – develop LVH and CVA
• Fasudil (ROCK inhibitor):– No change in systemic BP– Prolonged survival compared to untreated
animals
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Dahl Salt Sensitive Rats
• Model of HTN nephrosclerosis• Fasudil (ROCK inhibitor):
– reduces UVprotein and improves GFR– reduces histologic damage without change in
systemic BP• Less afferent arteriolar and glomerulo- sclerosis• Less TA/IF
– reduces ROCKß (ROCK1) mRNA expression– reduces TGFß, COL I, COL III mRNA
expression
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eNOS
• eNOS expression reduced in animal models and human CKD
• Rho/ROCK pathway lead to downregulation of eNOS expression
• Fasudil (ROCK inhibitor) – improves eNOS expression
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Beneficial effects of Rho/ROCK inhibition
• Reduces cell migration (influx of inflammatory cells)
• Reduces oxidative stress
• Increases eNOS expression
• Reduces TGFß – collagen cascade (ECM deposition and fibrogenesis)
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I/R Renal Injury
• PCT cells lose polarity and detach from the TBM as a result of cytoskeletal reorganization – a process known to be regulated by the Rho GTPases
• Rho GTPases:– Control actin cytoskeleton organization– Alter cell proliferation and migration – affecting
infiltration of inflammatory cells– Role in EMT of renal tubular cells into myofibroblasts
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I/R Renal Injury
• Rho GTPases – activate RhoA – which activate ROCK
• ROCK involved in:– Vasomotor tone – via contraction of VSMC– Migration and activation of immune cells
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Clinical Use of ROCK inhibitors(Limitations)
• Systemic administration of ROCK inhibitors may have adverse effects on:– Vasomotor tone/BP– Alteration of systemic immune function
(leukopenia)
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Clinical Use of ROCK inhibitors(in Renal Disease)
• Renal specific targeting of ROCK inhibitors has the advantage of:– Avoiding interaction with nontarget cells– Increase drug delivery to the target organ
allowing potentially increased therapeutic efficacy at a lower drug dose
– Done by coupling the drug to a low MW carrier – lysozyme (LZM) – which filters through the glomerulus and is taken up by PCT cells via the megalin receptor
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Inhibition of Renal Rho Kinase Attenuates
Ischemia/Reperfusion-Induced Injury
Prakash, de Borst, Lacombe et al
JASN 19: 2086-2097, 2008
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Methods
• I/R renal injury model – X-clamping of (L) RA/RV x 45 min (unilateral and bilateral models)
• ULS (Universal Linkage System) – to link the ROCK inhibitor (Y-27632) to LZM (1:1)
• Y-27632-LZM conjugate or free Y-27632 given 2 hours before surgery and then once daily on post-op days 1, 2 and 3
• Rats sacrificed on day 4 for histologic analysis
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Copyright ©2008 American Society of Nephrology
Prakash, J. et al. J Am Soc Nephrol 2008;19:2086-2097
Figure 1. (A) Y27632 was chemically modified and conjugated to LZM
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Copyright ©2008 American Society of Nephrology
Prakash, J. et al. J Am Soc Nephrol 2008;19:2086-2097
Figure 2. (A and B) Serum (A) and renal levels (B) of Y27632-LZM after administration of Y27632-LZM intravenously at a dosage of 20 mg/kg (equivalent to 555 {micro}g/kg Y27632) to rats
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Copyright ©2008 American Society of Nephrology
Prakash, J. et al. J Am Soc Nephrol 2008;19:2086-2097
Figure 3. Renal gene expression of MCP-1, TGF-{beta}1, {alpha}-SMA, procollagen I{alpha}1, TIMP-1, and Kim-1 in normal rats and vehicle-treated, Y27632-LZM-treated, and Y27632-treated rats after unilateral I/R injury
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Table 1. Effect of various treatments on kidney weight/body weight ratio, serum creatinine levels, and blood cell counts after 4 days
Parameter Normal I/R + Vehicle I/R + Y27632-LZM I/R + Y27632
Ischemic kidney wt/body wt (%) 0.43 ± 0.01 0.68 ± 0.03b 0.59 ± 0.04c 0.78 ± 0.04
Contralateral kidney wt/body wt (%) 0.43 ± 0.02 0.49 ± 0.06 0.46 ± 0.02 0.54 ± 0.02
Serum creatinine levels (µmol/L) 17.50 ± 0.29 24.40 ± 0.80b 22.30 ± 0.61d 27.40 ± 0.87e
RBC counts (x1012/L) N.D. 6.35 ± 0.19 6.33 ± 0.19 6.33 ± 0.06
WBC counts (x109/L) N.D. 12.09 ± 1.04 12.08 ± 1.99 9.13 ± 0.68e
a Data are means ± SEM. For red blood cell (RBC) and white blood cell (WBC) count groups, I/R + vehicle (n = 7) and I/R + Y27632-LZM (n = 4). ND, not determined.
b P < 0.001 versus normal. c P < 0.01. d P < 0.001 versus I/R + Y26732. e P < 0.05 versus I/R + vehicle
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Copyright ©2008 American Society of Nephrology
Prakash, J. et al. J Am Soc Nephrol 2008;19:2086-2097
Figure 4. (A through D) Representative photomicrographs of Kim-1 immunostaining (periodic acid-Schiff [PAS] counterstained) in normal (A), vehicle-treated (B), Y27632-LZM-treated (C), and Y27632-treated (D) rats
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Copyright ©2008 American Society of Nephrology
Prakash, J. et al. J Am Soc Nephrol 2008;19:2086-2097
Figure 5. (A through D) Representative photomicrographs of vimentin staining in normal (A), vehicle-treated (B), Y27632-LZM-treated (C), and Y27632-treated (D) rats
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Copyright ©2008 American Society of Nephrology
Prakash, J. et al. J Am Soc Nephrol 2008;19:2086-2097
Figure 6. (A through D) Representative photomicrographs of E-cadherin staining in normal (A), vehicle-treated (B), Y27632-LZM-treated (C), and Y27632-treated (D) rats
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Copyright ©2008 American Society of Nephrology
Prakash, J. et al. J Am Soc Nephrol 2008;19:2086-2097
Figure 7. Representative photomicrographs for the double immunostainings of vimentin/megalin (left column) and E-cadherin/megalin (right column) in normal, vehicle-treated, Y27632-LZM-treated, and Y27632-treated I/R
rats
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Copyright ©2008 American Society of Nephrology
Prakash, J. et al. J Am Soc Nephrol 2008;19:2086-2097
Figure 8. (A) Representative photomicrographs of ED-1 and {alpha}-SMA immunostainings in normal, vehicle-treated, Y27632-LZM-treated, and Y27632-treated I/R rats
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Copyright ©2008 American Society of Nephrology
Prakash, J. et al. J Am Soc Nephrol 2008;19:2086-2097
Figure 9. (A) Representative photomicrographs of collagen I, collagen III, and fibronectin staining in normal (n = 4), vehicle-treated (n = 8), Y27632-LZM-treated (n = 7), and Y27632-treated (n = 6) rats
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Copyright ©2008 American Society of Nephrology
Prakash, J. et al. J Am Soc Nephrol 2008;19:2086-2097
Figure 10. (A through D) Representative photomicrographs of double staining for p-MLC2 and megalin in normal (A), vehicle-treated (B), Y27632-LZM-treated (C), and Y27632-treated (D) rats
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Summary• In a U/L or B/L model of renal I/R injury, the Y-27632-LZM
conjugate leads to:– Reduced dedifferentiation of renal tubular cells (reduced KIM-1
and vimentin expression and retained E-cadherin expression)– Reduced ROCK activity in renal tubular cells and interstitium– Reduced inflammation and fibrogenesis (reduced mRNA
expression of MCP-1, TGFß, Procollagen-Iα1, TIMP-1, αSMA; reduced monocyte/macrophage infiltration; reduce protein expression of αSMA, COL I, COL III and fibronectin
– BUT, did NOT lead to improved renal function (in U/L model, and actual worsening of renal function in B/L model)
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Conclusion
• ROCK inhibition via a renal targeted delivery system (Y-27632-LZM) inhibits:– Tubular damage– Inflammation– Fibrogenesis– In an I/R renal injury model
• Actual clinical use/benefit remains an open question