**1. Introduction**

92 Novel Insights on Chronic Kidney Disease, Acute Kidney Injury and Polycystic Kidney Disease

Magenheimer BS, St John PL, Isom KS, Abrahamson DR, De Lisle RC, Wallace DP, Maser

Mangos S, Lam PY, Zhao A, Liu Y, Mudumana S, Vasilyev A, Liu A, Drummond IA. (2010)

Norman J. (2011) Fibrosis and progression of autosomal dominant polycystic kidney disease

Patel V, Li L, Cobo-Stark P, Shao X, Somlo S, Lin F, Igarashi P. (2008) Acute kidney injury

Ramasubbu K, Gretz N, Bachmann S. (1998) Increased epithelial cell proliferation and

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Shannon BM, Patton BL, Harvey, SJ and Miner JH. (2006) A Hypomorphic Mutation in the

Torres VE and Harris PC. (2009) Autosomal dominant polycystic kidney disease: the last 3

Vijayakumar S, Parkhi K, and Stolar J. Abnormal expression and assembly of laminin-332 and laminin-511 in ARPKD. *JASN* 22: Kidney week 2011 abstracts, 576A-577A, (2011) Wang S, Zhang J, Nauli SM, Li X, Starremans PG, Luo Y, Roberts KA, Zhou J. (2007)

dependent cystic dilation. J Am Soc Nephrol. 17:3424-37.

(ADPKD). Biochim Biophys Acta. 1812:1327-1336.

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RL, Grantham JJ, Calvet JP. (2006) Early embryonic renal tubules of wild-type and polycystic kidney disease kidneys respond to cAMP stimulation with cystic fibrosis transmembrane conductance regulator/Na(+),K(+),2Cl(-) Co-transporter-

The ADPKD genes pkd1a/b and pkd2 regulate extracellular matrix formation. Dis

and aberrant planar cell polarity induce cyst formation in mice lacking renal cilia.

abnormal extracellular matrix in rat polycystic kidney disease. J Am Soc Nephrol.

Epidermal growth factor receptor activity mediates renal cyst formation in

Mouse Laminin 5 Gene Causes Polycystic Kidney Disease. J Am Soc Nephrol 17:

Fibrocystin/polyductin, found in the same protein complex with polycystin-2, regulates calcium responses in kidney epithelia. Mol Cell Biol. 27(8):3241-52. Wilson PD, Schrier RW, Breckon RD, Gabow PA. (1986) A new method for studying human polycystic kidney disease epithelia in culture. Kidney Int. 30(3):371-8. Wilson PD, Hreniuk D, Gabow PA. (1992) Abnormal extracellular matrix and excessive growth of human adult polycystic kidney disease epithelia. J Cell Physiol. 150:360-9. Yamaguchi T, Nagao S, Kasahara M, Takahashi H, Grantham JJ. (1997) Renal accumulation

and excretion of cyclic adenosine monophosphate in a murine model of slowly

polycystin-1, polycystin-2, polaris, and cystin, are co-localized in renal cilia. J Am

progressive polycystic kidney disease. Am J Kidney Dis. 30(5):703-709 Yoder BK, Hou X, Guay-Woodford LM. (2002) The polycystic kidney disease proteins, Occurring with an incidence between 1/400 – 1/1000 live births autosomal dominant polycystic kidney disease (ADPKD) is the most common potentially lethal genetic disorder affecting the kidney (Ecder et al., 2007). The disease results from mutation in either of two genes *PKD1,* located on chromosome 16p13.3 or *PKD2,* located on chromosome 4q21 and is inherited in an autosomal dominant manner (European Polycystic Kidney Disease Consortium, 1994; Mochizuki et al., 1996). The resulting disrupted expression of the respective encoded proteins polycystin 1(PC1) and polycystin 2(PC2) leads to development of multiple fluid filled cysts in the kidney. As the cysts continure to grow throughout life the normal kidney parenchyma is gradually lost and ensuing decrease of renal function occurs. ADPKD accounts for 4-10% of end-stage renal disease (ESRD) worldwide (Freedman et al., 2000; Konoshita et al., 1998). In 50% of cases loss of renal function, necessitating renal replacement therapy occurs by age 60 (Gabow et al., 1992). Renal cysts are often evident on ultrasound or magnetic resonance imaging (MRI) in children, who typically do not become symptomatic until reaching young adulthood (Chapman et al., 2003; Fick-Brosnahan et al., 2001; Seeman et al., 2003). While renal cysts are an invariable characteristic of ADPKD, cysts may also occur in other organs with differing degrees of severity. Hepatic cysts are found in 75% of patients with ADPKD by age 60, while pancreatic, arachnoid, seminal vesicle, and prostate cysts occur with a lower frequency (Ecder et al., 2007). ADPKD is a systemic disorder with abnormalities occuring in several organs and a significantly increased risk for cardiovascular complications among affected patients. The reader is referred to several comprehensive reviews on the clinical and and genetic determinants of ADPKD for more detailed description of disease attributes (Chapin & Caplan, 2010; Ecder et al., 2007; Pei, 2011).

The process of cystogenesis involves proliferation of the epithelial cells that line the kidney tubules. This process initially results in localized dilation of the tubule. Continued epithelial cell proliferation and fluid secretion into the cyst results in cyst growth, until the cyst pinches off from the tubule. While the development and growth of renal cysts is the key feature of this disorder, the exact mechanism and identity of the factors influencing this process remain to be determined. However, it is apparent that vascular changes including expansion and remodeling of the existing vascular network must occur in order to support the structural changes occurring in the ADPKD kidney. Accordingly, it is not surprising that

Angiogenesis and the Pathogenesis of Autosomal Dominant Polycystic Kidney Disease 95

In this section we will describe some of the most important angiogenic growth factors and their respective receptors with emphasis on the role of VEGF, Ang-1, and Ang-2 in the

VEGF is a central mediator of angiogenesis inducing endothelial cell proliferation, sprouting and promoting vascular leakiness (Otrock et al., 2007). The VEGF family includes VEGF A, VEGF B, VEGF C, VEGF D and placenta growth factor (PlGF) each coded by a separate gene

VEGFR-2/Flk (with lower

VEGF B VEGFR-1/Flt-1 Embryonic angiogenesis VEGF C VEGFR-3/Flt-4 Mitosis, Migration,

VEGF D VEGFR-3/Flt-4 Lymphatic vasculature

The gene encoding VEGF A comprises of eight exons which by differential splicing encodes seven transcript variants that give rise to isoforms of differing amino acid length, VEGF-A206, VEGF-A189, VEGF-A183, VEGF-A165, VEGF-A148, VEGF-A145 and VEGF-A121 respectively (Bevan et al., 2008; Hoeben et al., 2004). A further variant VEGF-A110 is derived by proteolytic cleavage. The major circulating isoform VEGF-A165, is also abundant in the extracellular matrix. The VEGF polypeptides are homodimers although heteodimeric forms of VEGF-A and PlGF have also been described (DiSalvo et al., 1995). The biological functions of VEGF are mediated by binding to the tyrosine kinase receptors, VEGF receptor-1/fmslike tyrosine kinase-1 (VEGFR-1/Flt1), VEGF receptor-2/fetal liver kinase-1 (VEGFR-2/Flk-1) and VEGF receptor-3/ fms-like tyrosine kinase-4 (VEGFR-3/Flt4) (Ortega et al., 1999). The various members of the VEGF family bind to different VEGF receptors as shown in Table 1. VEGF-A (also referred to as VEGF) is expressed by mural cells including vascular smooth muscle cells and pericytes. In addition, in the kidney VEGF is expressed by both glomerular epithelial cells (podocytes) and by tubular epithelial cells (Robert et al., 2000).

PlGF VEGFR-1 Vasculogenesis

Table 1. Receptor affinity and actions of VEGF family members.

Angiogenesis

Mitosis Permeability Chemotactic for macrophages and granulocytes

Endothelial cell migration

Differentiation, Survival of lymphatic endothelial cells

around broniole in lung

angiogenesis

Angiogenesis in ischaemia, Inflammation, Wound healing, Cancer related

**Family Member Receptor Action** 

affinity)

**3. Angiogenic growth factors** 

**3.1 Vascular Endothelial Growth Factor (VEGF)** 

VEGF A VEGFR-1/Flt-1 and

kidney in health and disease.

(Table 1).

cyst growth in ADPKD has been likened to growth of a benign tumor (Grantham & Calvet, 2001). Indeed, there are many similarities between tumor growth and cyst growth, both processes being marked by increased cell proliferation, changes in apoptosis, and angiogenesis. In this chapter we will focus on the process of angiogenesis, defined as the growth of new blood vessels by invasion and sprouting of the existing vessels, as distinct from embryonic vasculogenesis or de novo growth of blood vessels.

### **2. Angiogenesis**

In order to understand the various signals and processes that define angiogenesis it is necessary to consider the main function of blood vessels, namely the supply of oxygen and nutrients to all the cells in the body. Much of our current knowledge of angiogenesis stems from studies of tumor biology. The fact that the diffusion limit of oxygen is approximately 100m indicates that all blood vessels must be located within 100-200 m of mammalian cells to ensure viability (Torres Filho et al., 1994). Subsequent studies by Judah Folkman et al. determined that tumor growth beyond 1-2-mm was angiogenesis dependent (Folkman, 2006). In health the endothelial cells that line the blood vessel lumen and the pericytes that surround the outer surface of the endothelial cells are in a "quiescent " state. This state is maintained by a balance of "pro" and anti-angiogenic growth factors that include vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), and various other chemokines and growth factors. Angiogenesis in the adult is defined by sprout formation or by splitting of a pre-existing blood vessel (Persson & Buschmann, 2011). The process of angiogenesis proceeds in several distinct stages and is initiated by a decrease in partial pressure of oxygen, which is detected by oxygen sensors on the endothelial cell. In the ADPKD kidney the growing cysts compress the renal vasculature resulting in decreased oxygenation. Hypoxia results in stabilization of the hypoxia-inducible factor (HIF-1). The HIF family, which in addition to HIF-1, also includes HIF-2 and HIF-3 are transcription factors. Structurally the HIF's comprise of a heterodimer of a regulatory subunit and a constitutively expressed subunit (Wang & Semenza, 1995). Angiogenesis is initiated by binding of HIF-1 to a hypoxia response element in the promoter of an angiogenic growth factor such as VEGF as reviewed by Hoeben et al. (Hoeben et al., 2004). In the case of new vascular sprout formation, when an angiogenic signal is detected by a quiescent blood vessel, the pericytes detach from the blood vessel wall and from the basement membrane. This is mediated by metalloproteinase (MMP) induced proteolytic degradation (Persson & Buschmann, 2011). Endothelial cells undergo several changes, loosening their cell junctions and allowing dilation of the vessel. VEGF increases endothelial cell permeability allowing escape of plasma proteins and formation of a provisional extracellular matrix (ECM). Endothelial cells next migrate onto the ECM surface mediated by integrin. Degradation of the ECM by proteases releases additional angiogenic growth factors from the ECM providing an angiogenic gradient that mediates migration and proliferation of the endothelial cells. One endothelial cell called a "tip cell" is instrumental in leading the migration, ECM degradation and consequent direction of growth of the vascular sprout. Maturation of the vessel requires return of the endothelial cells to a quiescent state, pericytes to attach and cover the vessel and down regulation of proteases by expression of tissue inhibitors of metalloproteinases (TIMP's). These changes are mediated by downregulated expression of VEGF and increased levels of Ang-1, transforming growth factor (TGF-), and platelet derived growth factor (PDGF) (Chung et al., 2010).
