A NEW TYPE OF KIDNEY STONE: ROCKs IN AKI Anthony Valeri, MD

A NEW TYPE OF KIDNEY STONE: ROCKs IN AKI

Anthony Valeri, MD

Rho

• Belongs to a family of small GTP-binding proteins

• Several subfamilies – Ras, Rho, Rab, Ran

• Controls multiple signaling pathways

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

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)

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)

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)

Copyright ©2006 American Heart Association

Loirand, G. et al. Circ Res 2006;98:322-334

Contraction signaling in vascular smooth muscle cells



Known mechanisms of ROCK activation and inactivation

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

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.



Major cell functions regulated by ROCKs after stimulation by hormones or neuromediators, growth factors, interaction with the extracellular matrix

(ECM), or mechanical stretch

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



The molecular structure of ROCKs

• 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

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


Shimokawa, H. et al. Arterioscler Thromb Vasc Biol 2005;25:1767-1775

Role of Rho/Rho-kinase pathway in the pathogenesis of cardiovascular diseases



Involvement of Rho-kinase pathway in the pathogenesis of arteriosclerosis

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

Other Effects of ROCK

• Evidence to suggest involvement in Alzheimer’s, bronchial asthma, cancer demyelinating disease, glaucoma and osteoporosis

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

ROCK and the Kidney

• Rho/ROCK pathway has effects on:– Renal circulation– Mesangial cells– Podocytes– Renal tubular cells

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)

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

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

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

ROCK and the Kidney(Inflammation)

• Infiltrating monocytes/macrophages stimulate EMT leading to interstitial fibrosis

• May be mediated, in part, by ROCK

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)

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

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



Broad pharmacological properties of Rho-kinase inhibitors



Therapeutic targets of Rho-kinase inhibitors

ROCK Inhibition in Renal Models

UUO

• Fasudil and Y-27632 (ROCK inhibitors):– Reduce tubulointerstitial fibrosis

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

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

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

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 glomerulosclerosis• Less TA/IF

– reduces ROCKß (ROCK1) mRNA expression– reduces TGFß, COL I, COL III mRNA

expression

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

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)

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

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

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)

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

Inhibition of Renal Rho Kinase Attenuates

Ischemia/Reperfusion-Induced Injury

Prakash, de Borst, Lacombe et al

JASN 19: 2086-2097, 2008

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

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



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



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

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



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



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



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



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



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



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



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

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)

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

Documents

A NEW TYPE OF KIDNEY STONE: ROCKs IN AKI Anthony Valeri, MD