Autosomal dominant polycystic kidney disease: Disrupted pathways and potential therapeutic interventions

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is a monogenic inherited renal cystic disease that occurs in different races worldwide. It is characterized by the development of a multitude of renal cysts, which leads to massive enlargement of the kidney and often to renal failure in adulthood. ADPKD is caused by a mutation in PKD1 or PKD2 genes encoding the proteins polycystin‐1 and polycystin‐2, respectively. Recent studies showed that cyst formation and growth result from deregulation of multiple cellular pathways like proliferation, apoptosis, metabolic processes, cell polarity, and immune defense. In ADPKD, intracellular cyclic adenosine monophosphate (cAMP) promotes cyst enlargement by stimulating cell proliferation and transepithelial fluid secretion. Several interventions affecting many of these defective signaling pathways have been effective in animal models and some are currently being tested in clinical trials. Moreover, the stem cell therapy can improve nephropathies and according to studies were done in this field, can be considered as a hopeful therapeutic approach in future for PKD. This study provides an in‐depth review of the relevant molecular pathways associated with the pathogenesis of ADPKD and their implications in development of potential therapeutic strategies.

Autosomal dominant polycystic kidney disease (ADPKD) is a monogenic inherited renal cystic disease that is characterized by the development of a multitude of renal cysts, which leads to renal failure in adulthood. Recent studies showed that cyst formation and growth result from deregulation of multiple cellular pathways like proliferation, apoptosis, metabolic processes, cell polarity, and immune defense. This study provides an in‐depth review of the relevant molecular pathways associated with the pathogenesis of ADPKD and their implications in development of potential therapeutic strategies.

Abbreviations

ACE

Angiotensin converting enzyme

ARB

Angiotensin receptor blockers

AC‐VI

Adenylate cyclase 6

ADPKD

Autosomal dominant polycystic kidney disease

AKI

Acute kidney injury

ARPKD

Autosomal recessive polycystic kidney disease

AMP

Adenosine monophosphate

AMPK

Adenosine monophosphate‐activated protein kinase

ATP

Adenosine tri‐phosphate

AVP

Arginine vasopressin

BM‐MSCs

Bone marrow mesenchymal stem cells

BP

Blood pressure

BUN

Blood urea nitrogen

cAMP

Cyclic adenosine monophosphate

CDK

Cyclin‐dependent kinase

CFTR

Cystic fibrosis transmembrane conductance regulator

COX

Cyclooxygenase

EGF

Epidermal growth factor

EGFR

Epidermal growth factor receptor

ER

Endoplasmic reticulum

ErbB

Avian erythroblastosis oncogene B

ERK

Extracellular‐signal regulated kinase

ESRD

End‐stage renal diseases

GFR

Glomerular filtration rate

GPCR

G‐protein coupled receptor

GSK

Glycogen synthase kinase

HDAC

Histone deacetylase

HGF

Hepatocyte growth factor

Id2

Inhibitor DNA binding 2

IGF‐1

Insulin growth factor‐1

JAK

Just another kinase

JNK

Jun N‐terminal kinase

KDR

Kinase insert domain receptor

MAPK

Mitogen‐activated protein kinase

MCP‐1

Monocyte chemoattractant protein‐1

MDCK

Madin‐Darby canine kidney cells

Met

Mesenchymal to epithelial transition

mTOR

Mechanistic target of rapamycin

NFκB

Nuclear factor κB

PC1

Polycystin1

PC2

Polycystin 2

PDE

Phosphodiesterase

PKA

Protein kinase A

PKD

Polycystic kidney disease

RAAS

Renin–angiotensin–aldosterone system

Rb

Retinoblastoma

SCr

Serum creatinine concentration

STAT

Signal transducer and activator of transcription

TKV

Total kidney volume

TLV

Total liver volume

TNF

Tumor necrosis factor

TRP

Transient receptor potential

TSC

Tuberous sclerosis complex

VEGF

Vascular endothelial growth factor

VEGFR

Vascular endothelial growth factor receptor

V2R

Vasopressin V2 receptor

Wnt

Wingless‐related integration site

1. INTRODUCTION

Autosomal dominant polycystic kidney disease (ADPKD) is a monogenic inherited renal cystic disease that occurs in different races worldwide. ADPKD is the most common hereditary kidney disease with an estimated prevalence of 1 in 400 (when considering observed and estimated autopsy cases) to 1 in 1,000 (when only considering clinically diagnosed cases) and around 3–4 cases per 10,000 in the general population (Chebib & Torres, 2016). It is characterized by the development of a multitude of renal cysts, which leads to massive enlargement of the kidney. Approximately 50% of patients develop end‐stage renal disease (ESRD) between the fifth and sixth decade of life and ADPKD is the fourth leading cause necessitating renal replacement therapy which includes dialysis or kidney transplantation. Cyst formation begins in utero and the cysts continuously enlarge over the patient's lifetime via an autonomous function in each cyst (Akoh, 2015). The symptoms of ADPKD are often correlated with renal enlargement and during the patient's life; blood may accumulate in the renal cysts following trauma or pyogenic infection. The majority of patients also experience other symptoms such as kidney pain and gross hematuria. Comorbidities associated with ADPKD are hypertension, urinary tract infection, and proteinuria. Hypertension occurs in 50% of patients with early stage disease (i.e., those aged 20–35 years), and in 100% of patients with end‐stage renal diseases (ESRD). Cyst enlargement also occurs in the liver, with a prevalence of 95% (by the age of 35 years; Gevers, 2013).

1.1. ADPKD genetics

In almost all patients, the pathogenesis of ADPKD stems from a mutation in PKD1 or PKD2 genes encoding polycystin‐1 (PC1) and polycystin‐2 (PC2) proteins, respectively. PKD1 is located on chromosome 16 and its mutant form is found in 85% of ADPKD cases. PKD2 is located on chromosome 4 and its mutant form is found in around 15% of cases. These mutations lead to the formation of distinctive fluid‐filled renal cysts. However, mutations are not detected in around 1% of patients (Rossetti & Harris, 2013). Recently, a third ADPKD gene, GANAB, was reported in patients with ADPKD that are negative for PKD1 and PKD2 mutations. GANAB accounts for ~0.3% of total ADPKD and it is associated with a milder manifestation (Iliuta et al., 2017). It encodes the glucosidase II (alpha subunit) that forms a functional holoenzyme in the endoplasmic reticulum (ER). Its dysfunction associated with defects of PC1 maturation and localization. A more systematic and detailed study of the phenotypic features of patients with ADPKD and GANAB mutations is needed (Cordido, Besada‐Cerecedo, & García‐González, 2017; Porath et al., 2016). DNAJB11 is also another player gene in ADPKD which encodes a co‐chaperone protein involved in protein processing of ER. It has a role in the appropriate trafficking of PC1 (Cornec‐Le Gall et al., 2018). These new findings demonstrate that ADPKD is more complex than previously thought, with a number of rare genes that should be more investigated.

1.2. Polycystin proteins

The proteins encoded by PKD1 and PKD2 genes are PC1 and PC2, respectively. These two proteins are extensively expressed in epithelial cells and could be also found in endothelial cells, smooth muscle, and cardiomyocytes. PC1 and PC2 are protein complexes that work together and are also involved in a network of other proteins in the cell. Certain amount of each of these proteins is required for appropriate function and higher or lower levels lead to disruption of their functionality (Lantinga‐van Leeuwen et al., 2004; Nigro, Castelli, & Boletta, 2015).

PC1 is a large transmembrane protein (500 kD) with 11 transmembrane domains and it has a structure similar to that of G‐protein coupled receptor (GPCR) family. PC1 is located in the plasma membrane of primary cilia and in cell junctions, such as desmosomal junctions; therefore, PC1 is a multifunctional protein with important roles in cell–cell/matrix adhesion and ciliary functions (Aksentijevich, Pras, & Gruberg, 1994; Fedeles, Gallagher, & Somlo, 2014). PC2 with a molecular weight of 110 kD and six transmembrane domains is considered a member of the transient receptor potential (TRP) channels family. It is a calcium‐selective channel and functions in multiple subcellular locations including the plasma membrane, ER, and primary cilia (Busch, Köttgen, & Hofherr, 2017). PC2 plays important roles in the structure of centrosome and mitotic spindle and its malfunction leads to a decrease in intracellular calcium concentration (Grieben et al., 2017; Mochizuki et al., 1996; Yang & Ehrlich, 2016).

This study provides an overview of molecular pathways associated with the development and growth of cysts. Also, therapeutic approaches that have been proposed to control each signaling pathways associated with ADPKD progression, are presented.

2. MECHANISMS ASSOCIATED WITH ADPKD PATHOGENESIS

2.1. The role of polycystins in the pathogenesis of ADPKD

2.1.1. Polycystins in primary cilia

Primary cilia are hairy structures that are found on the apical surface of cell membrane (Malicki & Johnson, 2017). The primary cilium (or nonmotorized) senses stimuli (such as accumulation of growth factors or fluid flow) and transmit their message to the cell (Marshall & Nonaka, 2006). In kidney cells, cilium acts as a mechanosensor on the renal epithelial tubular cells, which bends when stimulated by the urine flow (Singla & Reiter, 2006; Yoder, Hou, & Guay‐Woodford, 2002). PC1 and PC2 on the membrane of primary cilia, form a complex which is capable of sensing urine flow rate and convey this mechanical signal to the intracellular calcium signaling (Retailleau & Duprat, 2014). In this model, the large extracellular PC1 domain is considered the mechanosensor of urine flow and its subsequent structural change stimulates PC2. Consequently, intracellular calcium is discharged from IP3‐dependent reservoirs into the cytoplasm and the signal is transmitted as a wave through cellular connections (Lemos & Ehrlich, 2018). Changes in intracellular calcium concentration influence gene expression, cell cycle, cell differentiation, and cell death, which are abnormally regulated in ADPKD. On the other hand, Ma et al. (Ma, Tian, Igarashi, Pazour, & Somlo, 2013) defined a novel and unexpected relationship between integrity of cilia structure and polycystin function. This pathway specifically effects cyst growth dependent on the presence of intact cilia devoid of either PC1 or PC2. It is notable that this cilia‐dependent cyst growth was not explained by activation of the MAPK/ERK, mTOR, or cAMP pathways. Studies suggested the hypothesis that intact cilia elaborate a positive signal for rapid cyst growth that is normally inhibited by polycystin action. The activity of this cyst promoting signal requires structurally preserved cilia and is maximally active when cilia are devoid of polycystins, the same happening in ADPKD. So, it can be concluded that intact cilia are required to promote rapid cyst growth following loss of PC1 or PC2. It means loss of cilia suppresses cyst growth (following inactivation of polycystins; Ma et al., 2013).

In addition, polycystins expressed on the surface of the primary cilia, are essential for maintaining epithelia lining tubule cells in differentiated and polarized state. In ADPKD, well‐differentiated, polarized epithelia with low rates of cell division and apoptosis, become cystic epithelia containing partially dedifferentiated cells with improper polarization and high rates of division and apoptosis. These features are fully discussed below (Nauli et al., 2003; Praetorius & Spring, 2001; Saigusa & Bell, 2015).

As stated above, in ADPKD, the amount of intracellular calcium decreases due to loss of function of the polycystin complex. Thus, approaches that restore intracellular calcium levels may halt ADPKD progression. Triptolide is an active diterpene in a traditional Chinese medicine and induces calcium release from the ER through PC2. its effective role in pkd models (Pkd1^−/− cells, Pkd1 knockout mice) and clinical trials has been proven. However, triptolide has side effects such as infertility and immunosuppression (Leuenroth, Bencivenga, Igarashi, Somlo, & Crews, 2008). TRPV4 channel activators also increase intracellular calcium (Gradilone et al., 2011). Moreover, calcimimetics (like R‐568) are allosteric activators of the calcium sensing receptor through coupling to Gq proteins. Calcium sensing receptors activate phospholipase C‐protein kinase signaling and mobilize calcium from intracellular stores (Gattone et al., 2009). These molecules have therapeutic role in ADPKD. Table 1 displays name, function, and effect of drugs that exert their effects via this mechanism.

Table 1.

Treatment related to intracellular calcium regulation

Name	Mechanism	Effect on PKD model	Reference
Triptolide (Tripterygium wilfordii)	Induces PC2 mediated calcium release from the ER	1. In Pkd1−/− cells inhibit cyst growth 2. In Pkd1 knockout mice reduced cystogenesis 3. Clinical trial (NCT00801268)	Leuenroth et al. (2008); Leuenroth et al. (2007); Torres (2010)
			NCT00801268
TRPV4 channel activators	Increase intracellular calcium	1. In PCK rats attenuate the development of renal cysts and fibrosis	Gradilone et al. (2011)
Calcimimetics: R‐568	Allosteric activators of the calcium sensing receptor	1. In Han:SPRD Cy/+ rats and pcy mice Inhibits cyst growth and fibrosis 2. In PCK rats and Pkd2−/WS25 mice no effect on cyst growth	Gattone et al. (2009); Chen et al. (2011); Wang, Harris, Somlo, Batlle, and Torres (2009); Torres (2009)

Note. ER: endoplastic reticulum; PC2: polycystin 2; PCK: polycystic kidney; PKD: polycystic kidney disease; TRPV: transient receptor potential cation channel subfamily.

2.1.2. Polycystins in the cell cycle

The structure of the primary cilium and centrosome regulate cell division and formation of mitotic spindle; hence, they play an important role in the cell cycle (Phua et al., 2017). Deployment of polycystins in the primary cilium connect the polycystin complex to the cell cycle (Bukanov, 2008). PC1 in connection with PC2, is able to activate Jak2 that in turn activates STAT1; this process eventually leads to induction of P21 expression and regulation of cell cycle through apoptosis induction as well as cell cycle arrest (Bhunia et al., 2002; Park et al., 2007). Moreover, the carboxyl tail resulting from PC1 proteolytic cleavage, activates STAT6 (with a direct connection to p100), which then activates STAT6‐dependent gene expression (Low et al., 2006). Generally, STAT6 is translocated between primary cilia and the nucleus, depending on apical fluid flow. Based on our knowledge about regulation of PC1 tail cleavage by fluid flow, we can conclude that PC1‐mediated regulation of STAT6 activity plays a role in sensing changes in luminal fluid flow and causing corresponding changes in gene expression. In normal kidney tubules, the urine flow and normal expression of PC1, keep STAT6 in the cilia. In the absence of urine flow or in case of mutant PC1, STAT6 is translocated to the nucleus and it induces gene transcription. STAT6 is aberrantly activated in cyst‐lining epithelial cells; so, increased level of STAT6 can be regarded as a pathogenic factor in ADPKD (Chauvet et al., 2004).

On the other hand, Id2 (a component of DNA binding inhibitors) attaches to the phosphorylated PC2. This attachment causes keeping of Id2 in the cytoplasm and prevents its transmission to the nucleus. In the absence of PC2 or in case of malfunctioning PC2, Id2 is released to the cytoplasm and transferred to the nucleus where it inhibits Epro. Epro affects growth‐suppressive genes. In the ADPKD, these genes are not expressed; so, the amount of cyclin‐dependent kinase2 (Cdk2) increases and the cell enters the proliferation phase (Li et al., 2005). Studies have shown that inhibiting Id2 by RNAi reduces cell proliferation in pkd1 mutant cells. As mentioned above, PC2 can hold Id2 in a phosphorylated state. PC2 phosphorylation process is controlled by factors such as PC1, glycogen synthase kinase 3, and casein kinase 2. For example, increased expression of PC1 leads to increased PC2 phosphorylation. Therefore, treatments that affect PC2 phosphorylation could be effective in patients with PC1 mutations. The PC2‐Id2 pathway is related to various proteins which are important in ADPKD pathogenesis such as catenin, E‐cadherin, C‐myc, and Rb which can be the cause of increased cell proliferation. Thus, PC1 and PC2 indirectly affect cell cycle regulation (Lee, Battini, & Gusella, 2011).

Roscovitine (Seliciclib, CYC202) is a blockade of the G1/S phase in the cell cycle. It increases the levels of p21 which is downregulated in PKD. Studies showed that Roscovitine reduces cystic indexes in the mice model of PKD (Bukanov, Smith, Klinger, Ledbetter, & Ibraghimov‐Beskrovnaya, 2006; Park et al., 2007). Cdc25A is a phosphatase that plays an essential role in cell cycle progression by activating Cdks. Menadione as a drug lowers the levels of Cdc25A and inhibits renal and hepatic cyst growth in PKD models (Masyuk et al., 2012). Table 2 shows these therapeutic approaches that intervene with cell cycle and their impact on the PKD models.

Table 2.

Treatment related to cell cycle regulation

Name	Mechanism	Effect on PKD model	Reference
Roscovitine	CDK inhibitor	In cpk mice, jck mice, Pkd1 cKO mice reduce renal and hepatic cystic indexes	Bukanov, Smith, Klinger, Ledbetter, and Ibraghimov‐Beskrovnaya (2006); Park et al. (2007)
Menadione	Lowers the level of Cdc25A	In PCK rats and Pkd2ws25/− mice inhibits renal and hepatic cyst growth	Masyuk et al. (2012)

Note. CDK: cyclin‐dependent kinase; PKD: polycystic kidney disease.

2.1.3. Polycystins in cell junctions

PC1 plays an important role in cell–cell interconnections through its placement in desmosomal junctions (DJs) and adherens junctions (AJs). DJs provide a link between the intermediate filaments of adjacent cells and AJs whose cytoplasmic faces is linked to actin cytoskeleton (Streets et al., 2003). In ADPKD, the AJs and DJs are impaired but tight junctions remain intact. Dysfunction of these cellular interactions can be associated to the dysregulated epithelial growth and altered tubular architecture. This happening is common to almost all forms of renal cystic disease. In ADPKD, in epithelial cells, PC1 is ineffective and at a reduced concentration of calcium, desmosomal proteins which are removed from cell–cell connections could be found in intracellular vesicles (Huan & van Adelsberg, 1999). Studies showed that in ADPKD cells, E‐cadherin is removed from the connections and in turn, N‐cadherin which is expressed at higher levels, forms a complex with beta‐catenin (Prozialeck, Lamar, & Appelt, 2004; Roitbak et al., 2004). However, the replacement of N‐cadherin with E‐cadherin weakens cellular connections and was not sufficient to maintain epithelial cell–cell adhesion (Russo et al., 2005). PC1 also reacts with focal adhesion proteins that are responsible for attachment of the cell to basement membrane. In ADPKD, due to the removal of the FAK protein, PC1 complex cannot connect to them, and consequently cells connection with the matrix is weakened (Wilson, 2004).

2.2. cAMP in ADPKD pathogenesis

Cyclic adenosine monophosphate (cAMP) is one of the most important secondary messengers that plays an important role in regulating key cellular processes such as proliferation, differentiation, translation, and transfer of fluids. Considerable evidence suggest an increased level of cAMP in ADPKD kidney cells making it a stimulating factor for cyst growth and disease progression (Bukanov, 2008; Yamaguchi et al., 2000). Ca²⁺ reduction, due to mutations in the PKD genes, increases the activity of the Ca²⁺‐inhibitable AC6 and decrease the activity of Ca²⁺/ calmodulin‐dependent phosphodiesterases (PDEs) so results in increased level of cAMP in ADPKD (Wallace, 2011). That's why reduction in intracellular Ca²⁺ causes increased accumulation of intracellular cAMP. The hydrolytic capacity of cAMP PDE exceeds the maximum rate of synthesis by adenylate cyclase (AC). As a therapeutic approach, acylamino‐3‐thiophenecarboxylates, a nonselective PDE activator, was shown to reduce cAMP and inhibit the growth of Madin‐Darby canine kidney (MDCK) cysts (Table 4). Moreover, somatostatin is a peptide hormone that acts on five Gi protein‐coupled receptors (GiPCR; SSTR1–5), present on renal tubular epithelial cells and inhibiting the AC6 activity, so prevents cAMP production. It has a very short half‐life (3 min). Octreotide, lanreotide, and Pasireotide are more stable synthetic peptides developed for clinical use. Also, they have tolerable side effects such as diarrhea and increased fasting plasma glucose concentration. For example, Octreotide is a long‐acting somatostatin analog capable of reducing cAMP production via Gi activation and halts the expansion of hepatic cysts in PCK rat models. It was also examined in clinical trials for ADPKD (NCT01377246; Table 4).

Table 4.

The cAMP signaling pathway targeted therapy

Name	Mechanism	Effect on PKD model	Reference
OPC‐41061 (Tolvaptan)	Vasopressin V2 receptor antagonist	1. Inhibited AVP‐induced chloride secretion cyst growth 2. Administration for one week reduced TKV by 3.1% 3. TEMPO trial: TKV and GFR progression slowed Phase III (NCT00428948)	Reif et al. (2011); Irazabal et al. (2011); Torres (2010); Wang (2005)
			NCT00428948
OPC‐31260	Vasopressin V2 receptor antagonist	1. In the PCK rat and pcy and PKD2WS25 mouse: Reduced renal cAMP and inhibited kidney cyst progression	Wang (2005); Gattone, Wang, Harris, and Torres (2003)
Mozavaptan	Vasopressin V2 receptor antagonist	In rodent models: Attenuated PKD progression	Meijer et al. (2011); Gattone et al. (2003)
Satavaptan	Vasopressin V2 receptor antagonist	Blocked tubular expression of sFRP4 (which is overexpressed in polycystic kidneys and promotes cystogenesis of zebrafish pronephros)	Romaker et al. (2009)
Forced water intake	Suppression of AVP	In PCK rat: Attenuated PKD progression (3.5‐fold increase in urine output)	Nagao (2006)
Octreotide	Somatostatin analogs	1. Inhibits kidney cyst growth (TLV: decreased, TKV: decreased, eGFR: no change) 2. Octreotide clinical trial (NCT01377246)	Masyuk et al. (2007); Caroli et al. (2010); Ruggenenti et al. (2005); van Keimpema et al. (2009); Hogan et al. (2010); Chrispijn et al. (2012)
Lanreotide			NCT01377246
Pasireotide
2‐(Acylamino)‐3‐thiophenecarboxylates small molecule	PDE activator	Inhibit the growth of MDCK cysts (lack of selectivity and potential toxicity in vivo)	Tradtrantip, Yangthara, Padmawar, Morrison, and Verkman (2009)
Tetrazolo‐CFTRinh‐172 Ph‐GlyH‐101	CFTR inhibitor	In 3D cultures of MDCK cells, metanephric organ cultures, conditional Pkd1 knockout mice: Slowed cyst growth.	Yang, Sonawane, Zhao, Somlo, and Verkman (2008)
VX‐809	CFTR corrector	In Pkd1‐knockout mice and In vitro: Significant reduction in the AC3, cAMP levels, intracellular Ca2+, cell proliferation, apoptosis, and ER stress	Yanda, Liu, and Cebotaru (2018)
TRAM‐34	KCa3.1 channel inhibitor	In MDCK and ADPKD cells inhibit cyst formation and enlargement in collagen gels and chloride secretion	Albaqumi et al. (2008)
Senicapoc	KCa3.1 channel inhibitor	Used successfully in a Phase 2 trial and has shown little or no toxicity in a Phase 3 trial for sickle cell disease.	Ataga et al. (2012)
Ouabain	Na+, K+‐ATPase inhibitor	Blocks cAMP‐dependentnet fluid secretion	Nguyen, Wallace, and Blanco (2007)
Furosemide (Lasix)	NKCC1 inhibitor	Blocks cAMP‐dependentnet fluid secretion	Sullivan, Wallace, and Grantham (1998)

Note. AC3: adenylyl cyclase 3; CFTR: cystic fibrosis transmembrane conductance regulator; cAMP: cyclic adenosine monophosphate; ER: endoplasmic reticulum; GFR: glomerular filtration rate; MDCK: Madin‐Darby canine kidney cells; PDE: phosphodiesterase; TKV: total kidney volume; TLV: total liver volume.

2.2.1. Effects of increased cAMP on cell proliferation

cAMP inhibits cell proliferation in normal kidney tubules but stimulates cell proliferation in the epithelial cells of human ADPKD kidney. Generally, the effect of cAMP on the cell proliferation may vary based on the cell type and conditions (Houslay & Milligan, 1997; Pinto et al., 2016).

In cystic epithelial cells, cell proliferation is induced through activation of the MEK/ERK pathway due to the differences in calcium concentration between cystic and normal kidney cells (Hanaoka & Guggino, 2000; Yamaguchi et al., 2000). An initial step in the MEK/ERK pathway is activation of Ras, a small GTPase protein that recruits Raf to the plasma membrane and activates Raf. Activated Raf activates MEK, which, in turn, stimulates ERK (also known as MAPK). Translocation of activated ERK to the nucleus leads to phosphorylation of transcription factors affecting gene expression and ultimately cell proliferation. The main intermediates in this pathway are Rafs, which are divided into three groups: A‐Raf, B‐Raf, and Raf‐1 (c‐Raf; Davies et al., 2002). Raf kinases are responsible for regulating Rafs (by inhibitory or activator phosphates; Dumaz & Marais, 2005). In fact, cAMP‐induced ERK regulation and cell proliferation is mediated by Rafs. Though B‐Raf and Raf‐1 are homologous in the amino acid sequence, the effect of cAMP on them is different. Raf‐1 has three inhibitory phosphorylation sites for protein kinase A (PKA; cAMP‐dependent protein kinase) and interaction of PKA with any of these sites prevents the Raf‐1 activation. Since B‐Raf lacks these sites, cAMP and PKA have no inhibitory effect on B‐Raf and PKA‐induced phosphorylation activates B‐Raf (Wellbrock, Karasarides, & Marais, 2004). B‐Raf has a higher tendency to have connection with MEK and can extend the ERK pathway (generally lower levels of phosphorylation are required to activate B‐Raf compared with Raf‐1). On the other hand, in normal cells, proper concentrations of calcium in the cytoplasm activate IP3 kinase and AKT (protein kinase B), thereby, inhibiting B‐Raf through its inhibitory phosphorylation (Chong, Lee, & Guan, 2001; Dumaz & Marais, 2003; Mason, 1999). Thus, the ERK pathway is suppressed, whereas in ADPKD due to the effect of PC1 and PC2 on calcium concentration, intracellular calcium reduction induces cAMP‐dependent cell proliferation. In case calcium levels are reduced, cAMP can activate B‐Raf because AKT is not active and has no inhibitory effect on B_Raf so the ERK/MEK can activate cell proliferation. This cascade actually re‐blocks in the normal concentration of calcium (Yamaguchi, 2005; Yamaguchi et al., 2004).

From a therapeutic point of view, B‐Raf, which appears to be uniquely activated in ADPKD cyst cells and centrally located for ERK activation, can be ideal option to reduce the mitogenic effects of cAMP. Sorafenib (BAY 43–9006) is a multikinase inhibitor that was developed as a Raf inhibitor. This compound also has activity against receptor tyrosine kinases (like vascular endothelial growth factor receptor [VEGFR] and PDGFR). Sorafenib (also called Nexavar) has been used for the treatment of advanced renal cell and hepatocellular carcinomas. In the case of PKD, it can completely block in vitro cyst growth of human ADPKD cystic cells cultured within a three‐dimensional collagen gel. Studies demonstrated that relatively low concentrations of Sorafenib inhibited cAMP‐dependent activation of B‐Raf and caused a concentration‐dependent inhibition of cell proliferation induced by cAMP (Yamaguchi, Reif, Calvet, & Wallace, 2010; Table 3). PLX5568 is a selective small molecule inhibitor of Raf kinases. PLX5568 attenuated cyst enlargement in vitro and in a rat model of ADPKD without improving kidney function, presumably due to increased renal fibrosis (Buchholz et al., 2011). In this regard, p38 MAPK, a mitogen‐activated protein kinases family of serine/threonine kinase, which has been reported to be activated in human and mouse cystic kidneys, stimulates secretion of several fibrogenic cytokines from renal tubular cells and stimulates collagen production in the cystic kidney of inv mutant mice (Sugiyama, Kohno, & Yokoyama, 2012). Studies showed that administration of selective p38 MAPK inhibitor, FR167653, in a NPHP2 mouse model reduced renal fibrosis but not cyst expansion, cell proliferation, and apoptosis. Hence, effective therapies for the treatment of ADPKD will need to target fibrosis as well as the growth of cysts (Table 3).

Table 3.

Pharmacological inhibitors of MAPK pathways in the PKD therapy

Name	Mechanism	Effect on PKD model	Reference
PLX5568	Raf kinase inhibitor	In vitro and in Han: SPRD rats: Attenuated cyst enlargement, failed to prevent renal enlargement, promoted hepatic, and renal fibrosis and did not improve renal function	Buchholz et al. (2011)
Sorafenib or Bay 43–9006	Raf kinase inhibitor activity against tyrosine kinases receptor	1. In human ADPKD cells: Inhibition of cyst growth 2. In knocked‐out Pkd2: Caused an unexpected increase in liver cyst area and cell proliferation	Yamaguchi, Reif, Calvet, and Wallace (2010)
PD184352	MEK inhibitor	1. pcy mouse: Inhibited cyst growth 2. inv mutant mice: Decrease in cystogenesis and improvement of kidney function	Calvet (2006); Okumura et al. (2009)
UO126	MEK inhibitor	Failed	Shibazaki et al. (2008)
FR167653	P38 inhibitor	Reduced renal fibrosis	Sugiyama, Kohno, and Yokoyama (2012)
NVP‐BEZ235	Dual mTOR/PI3K inhibitor	In heterozygous (Cy/+) Han: SPRD rats, Pkd1 conditional knockout mouse and in vitro culture of primary cell: Reduced cell proliferation, cyst growth, interstitial fibrosis, kidneys weight, and improved BUN, SCr, urine albumin/creatinine ratio	Liu et al. (2018)

Note. ADPKD: autosomal dominant polycystic kidney disease; BUN: blood urea nitrogen; PKD: polycystic kidney disease; MAPK: mitogen‐activated protein kinase; SCr: serum creatinine concentration.

As mentioned, MEK is an upstream activator of ERK kinase. MEK‐targeted therapies reduce the formation of cysts, decreases blood pressure, and improves kidney function (Okumura et al., 2009). For example, PD184352, a MEK inhibitor (now named CI‐1040), was shown to effectively blocked cyst growth and kidney enlargement and preserved renal function recessive PKD models.

The ERK activation can be induced by stretching cyst‐lining cells through fluid pressure (Omori et al., 2006) or cAMP (as discussed above) or epidermal growth factor (EGF; City, 2003). ERK activation was significantly abolished by a dual mTOR/PI3K inhibitor (NVP‐BEZ235), resulting in reduced cell proliferation and cyst growth (Liu et al., 2018), due to extensive regulatory crosstalk exists between PI3K/MEK1/2/ERK and PI3K/AKt/mTOR signaling pathways (Carracedo et al., 2008; Liu et al., 2018). Detailed explanation of MAPK pathways‐related therapies is shown in Table 3.

2.2.2. Results of increased cAMP in fluid secretion

Similar to all secretory membranes, release of fluid into the cysts in ADPKD depends on the presence of channels and ion transporters in different parts of the membrane.

Arginine vasopressin (AVP) is an antidiuretic hormone that stimulates cAMP production in the sites for cyst formation in PKD (Verani, 1998). AVP binding to vasopressin V2 receptors (V2R) increases intracellular cAMP and phosphorylation of aquaporin‐2 channels (by PKA), leading to AQP‐2 activation and insertion into the apical membrane (Clark, Devuyst, & Roussel, 2017; Yamaguchi et al., 2000). Several studies have shown that V2R are overexpressed in cystic kidneys of PKD animals. On the other hand, patients with ADPKD has a defect in the concentrating ability of the cystic kidney so they have increased levels of circulating AVP. The combination of increased V2R expression and increased circulating levels of AVP may give rise to persistent cAMP production in cystic epithelial cells of PKD kidneys (Wallace, 2011). Blocking the vasopressin V2 receptors (V2R) with receptor antagonists inhibit production of cAMP. Preclinical and clinical studies supported investigation of V2R antagonists (like Tolvaptan, Mozavaptan, and Satavaptan) as a potential PKD therapy through hormonally modulated cAMP (Table 4). However, V2R antagonists have side effects (such as thirst, polydipsia, polyuria, and nycturia which can cause sleep disturbance) that may limit widespread clinical use. Moreover, suppression of AVP by forced water intake (3.5‐fold increase in urine output) attenuated PKD progression in the recessive PKD model.

Another important result of cAMP increase in ADPKD is activation of CFTR channels in lumen (apical membrane). In this situation, the outflow stream for chloride is provided and the chloride ions enter the lumen space (Rajagopal & Wallace, 2015). On the other hand, there are lots of potassium channels in the collecting ducts such as Kir6.2 and Kca3.1 (suppressed and activated by ATP and cAMP, respectively), which are active in ADPKD cells (Brill et al., 1996; Welling & Ho, 2009). Increasing apical chloride and potassium levels create a transepithelial negative electrical potential, which induces inactive and specific conductivity of sodium to the lumen space (Hanaoka, Devuyst, Schwiebert, Wilson, & Guggino, 1996). Following the transition of sodium and chloride into the lumens, due to osmotic pressure, water penetrates into the cavity through the aquaporins. It should be noted that subsequent activation of potassium channels is necessary to maintain the negative potential of the membrane for transporting chloride into the lumen (Chebib, Sussman, Wang, Harris, & Torres, 2015; Torres, 2015; Wallace, Grantham, & Sullivan, 1996).

Pharmacological inhibitors of KCa3.1 and CFTR channels such as TRAM‐34 and Senicapoc provide an effective therapy to delay progression to kidney failure in patients with ADPKD. Senicapoc has shown little or no toxicity in a Phase 3 trial for sickle cell disease (Table 4). The Na+, K+‐ATPase, acting in concert with K+ channels in the basolateral membrane, establishes and maintains the chemical and electrical gradients that are utilized by secondary active transporters. Ouabain, an inhibitor of the Na+, K+‐ATPase, blocked cAMP‐dependent fluid secretion by cysts and anion secretion by polarized ADPKD cell monolayers (Nguyen, Wallace, & Blanco, 2007).

Treatments that target cAMP signaling pathway are shown in Table 4.

3. OTHER THERAPEUTIC APPROACHES

3.1. Treatments associated with apoptosis pathway

Increased rate of apoptosis and hyperproliferation are characteristics of the cyst‐lining epithelial cells and the association between enhanced apoptosis rate and creation of cystic lumens has been proven in PKD models (Ibrahim, 2007). Both the intrinsic and extrinsic apoptotic pathways are responsible for cell death in cyst‐lining epithelial cells (Tao et al., 2005). Caspases as the major mediators of apoptosis are increased in polycystic kidneys (Tao et al., 2005). Studies have shown that mice with a defective BCL‐2 antiapoptotic gene, exhibit greater apoptosis rate in the kidney and have higher chance of development of renal cysts (Ucia, Chrier, & Delstein, 2008). Many factors and mechanisms can influence apoptosis pathways in cystic cells. Also PC‐1 is a regulator of programmed cell death in renal cells, and its expression causes activation of the PI3‐K/Akt signaling pathway so leads to resistance to apoptosis (Boca et al., 2006; Venugopal & Blanco, 2016). The role of apoptosis in the PKD therapy could be challenging. IDN‐8050, a caspase inhibitor, causes cystogenesis inhibition in Han:SPRD rats through reduction of renal epithelial cell apoptosis and proliferation. It also improves cystic disease and lives longer in cpk mice (Tao et al., 2005). Many studies indicated that inhibition of apoptotic pathways promote therapeutic reductions in cystogenesis of PKD models (Jia et al., 2010; Tao, Zafar, Kim, Schrier, & Edelstein, 2008). On the other hand, several studies suggested that induction of renal cystic cell apoptosis may delay renal cyst growth in PKD models (Fan et al., 2013; Liu, Dai, Fu, Jia, & Mei, 2010; Ostrom, Tang, Gruss, & Dressler, 2000).

GT13072, a Smac‐Mimetic, induces TNF‐a–dependent extrinsic apoptotic pathway and slows cyst growth in Pkd1 mutant mice (Fan et al., 2013). Moreover, rosiglitazone induces apoptosis of cystic cells via altering expression of Bcl‐2/Bax that result in amelioration of renal cyst growth (Liu et al., 2010). Together, these studies suggest that apoptosis play complex role in cystogenesis.

3.2. Targeting the AMP‐activated protein kinase for treatment of PKD

The adenosine monophosphate‐activated protein kinase (AMPK) molecule is a cellular energy sensor that reduces energy consumption when cellular levels of AMP are higher than those of ATP. The activation of AMPK prevents cellular growth due to inhibition of mTOR (Takiar et al., 2011) and activation of P53/P21 (He et al., 2014). It also reduces fluid secretion by directly inhibition of the CFTR activity (Takiar et al., 2011). Hence, agents that activate this molecule can be useful treatment modalities for ADPKD. Metformin is an AMPK activator which has been used in preclinical PKD research. Studies showed that Metformin inhibited the growth of MDCK cysts in collagen gels, also decreased renal cystic index in metanephric organ cultures and inducible Pkd1 knockout mice (Takiar et al., 2011).

3.3. Therapies associated with histone deacetylases

Histone deacetylases (HDACs) alter the expression of genes by affecting chromatin. These enzymes also cause deacetylation of proteins (Liu & Zhuang, 2015). For example, HDAC1 suppresses the transcription of PKD1 gene and deacetylation of P53 (Zhou et al., 2013). HDAC6 causes clamping of cilia structure throughout the cell cycle by tubulin deacetylation. HDAC6 expression and activity increases in certain cancers, neurodegenerative diseases, and in Pkd1‐mutant renal epithelial cells so could play a role in cyst formation and serves as a therapeutic approach in PKD (Chun, 2017). In this regard, Tubacin and ACY‐1215 are specific HDAC6 inhibitors studied on PKD models. Treatment with tubacin and ACY‐1215 prevented the cyst formation in an in vitro model of cystogenesis (MDCK cells; Cebotaru et al., 2016). ACY‐1215 slowed cyst growth in the PKD mouse model and slowed cyst growth and size in PN cells (an ADPKD cell line derived from the proximal tubules from a pkd1^−/− mouse). ACY‐1215 also lowered cAMP levels and protein expression of AC6 (Yanda, Liu, & Cebotaru, 2017). Moreover, Valproic acid, an inhibitor of class I HADCs (HDAC1, HDAC2, HDAC3, and HDAC8) and trichostatin A, an inhibitor of class II HDACs (HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10), suppress cyst formation in PKD models. Histone deacetylases‐related therapies are described in Table 5.

Table 5.

Current therapies involving HDAC inhibitors for PKD

Name	Mechanism	Effect on PKD model	Reference
Valproic acid	Inhibitor of class I HDACs	In Pkd1 and Pkd2 knockout mice: Inhibition of cyst growth	Cao et al. (2009)
Trichostatin A	Pan‐HDACs Inhibitor	In Pkd2 knockout mice: Suppress cyst formation by regulating cell proliferation	Fan, Li, Magenheimer, Calvet, and Li (2012)
Tubacin	HDAC6 inhibitor	In MDCK cells and Pkd1‐conditional mouse model of ADPKD: Downregulated cyclic AMP levels, inhibited cell proliferation, thereby prevented cystogenesis	Cebotaru et al. (2016)
ACY‐1215	HDAC6 inhibitor	1. In MDCK cells and PKD1‐null (PN) and heterozygous (PH) cells: Reduced HDAC6 activity and thereby downregulation of cAMP levels 2. In Pkd1fl/fl; Pax8rtTA; TetO‐cre mice: Slow renal cyst growth	Yanda et al. (2017)

Note. HDAC: histone deacetylase; PKD: polycystic kidney disease.

3.4. Growth factors as targets for PKD treatment modalities

Growth factors and their tyrosine kinase receptors play multiple roles during both kidney development and regeneration. Many studies indicated the pivotal role of these factors in the pathogenesis of PKD (Huang et al., 2015; Qin, Taglienti, Cai, Zhou, & Kreidberg, 2012; Torres et al., 2003; Zheleznova, Wilson, & Staruschenko, 2011). These include members of the EGF family, VEGF, HGF, and IGF1 and their receptors. Therefore, agents that target these molecules or their receptors can ameliorate PKD (Table 6).

Table 6.

Summary of growth factor targeted therapy in polycystic kidney disease

Name	Mechanism	Effect on PKD model	Reference
EKI‐785	EGFR tyrosine kinase and Erb‐B2 inhibitors	1. In bpk and orpk mice and Han:SPRD rats: Inhibits cystogenesis. 1. In PCK rat: no effect	Sweeney, Chen, Nakanishi, Frost, and Avner (2000); Torres et al. (2003); Torres et al. (2004)
EKB‐569
SKI‐606	Src inhibitor	In PCK rats and Bpk rat model of recessive PKD	Sweeney, von Vigier, Frost, and Avner (2008)
		In PCK rats and Bpk, Pkd1 mice: Reduced cell proliferation and cystogenesis
		In BPK kidneys correlates with decreased EGFR (ErbB1) and in PCK kidneys decreases ErbB2 activity
Bosutinib	Src/Abl inhibitor	A Phase II clinical trial in patients with ADPKD (NCT01233869)	Tesar et al. (2017)
			NCT01233869
SU‐5416	VEGF receptor inhibitor	In Pkd2WS25/− mice: Reduced cystic development of the liver but did not affect renal cysts	Amura et al. (2007)
B20.4.1	Anti‐VEGF antibody	In heterozygous (Cy/+) Han:SPRD rats and in vitro: Increased PTEC proliferation, cystogenesis, proteinuria, severe renal failure, and glomerular damage	Raina et al. (2011)
Exogenous VEGFC	VEGF‐C expression	In Cys1cpk/cpk and Pkd1nl/nl mouse: Reduction in cyst size, BUN and serum creatinine levels, and inflammation	Franchi et al. (2016)
SU11274	HGF receptor (c‐met) inhibitor	In mouse Pkd1‐null cystic kidneys: Inhibition of mTOR activity and blocked cystogenesis	Qin et al. (2010)
PHA665752
Soy bean diet	Dietary lowering of kidney IGF‐1 concentrations	In Han:SPRD‐cy rats: Reduction in kidney weight, water content and cyst size, lower serum urea and creatinine, and higher creatinine clearance	Aukema and Housini (2001)
Tesavatinib	Multikinase inhibitor	1. In bpk and PCK models of ARPKD: Reduction of renal and biliary disease 2. A Phase II clinical trial (NCT03203642)	Sweeney, Frost, and Avner (2017)
			NCT03203642

Note. ADPKD: autosomal dominant polycystic kidney disease; BUN: blood urea nitrogen; EGFR: epidermal growth factor receptor; PTEC: Tubular epithelial cell; SCr: serum creatinine concentration; VEGF: vascular endothelial growth factor.

3.4.1. EGFR hyperactivation and mislocalization

EGF is one of the key signaling molecules in cyst growth. EGF/ErbB signaling is critically involved in cell proliferation, and regulation of several ion channels in renal epithelial cells. EGFR expression in apical membrane of cystic epithelial cells is increased, and its concentration in cystic fluid is elevated, which results in prolonged stimulation of proliferation in these cells (Zheleznova et al., 2011).

As a therapeutic approach inhibition of EGF receptor and downstream molecules slow the progression of PKD. EKI‐785 is a specific inhibitor for EGFR tyrosine kinase and Erb‐B2 activity. This compound binds to the ATP binding site of EGFR, inhibits the kinase activity of this protein, blocks EGFR autophosphorylation, and the result is inhibition of cell proliferation. EKB‐ 569 is a derivative of EKI‐785 with improved pharmacokinetics and enteral bioavailability compared with the EKI‐785 and this compound also has the same effects (Torres et al., 2003; Table 6).

Src is identified as a key mediator in the activation and amplification of the EGFR axis, as well as a common intermediary in multiple cellular pathways specially those which are disrupted in PKD. Src is overexpressed and activated in several epithelial cancers, whereby Src inhibition results in decreased proliferation, adhesion, and migration. Src activation and PKD progression has been demonstrated in animal models of PKD. Studies showed that pharmacological inhibitor of Src, SKI‐606, resulted in amelioration of renal cyst formation and biliary ductal abnormalities in PKD models. Furthermore, the effects of Src inhibition in PCK kidneys suggest that the ErbB2 and MEK/ERK pathways are involved in Src‐mediated signaling in PKD and this occurs without reducing elevated cAMP. So, these data suggest that reduction of elevated cAMP is not an absolute requirement for amelioration of cystic disease and Src inhibition may provide therapeutic target in PKD. Bosutinib, an oral dual Src/Bcr‐Abl tyrosine kinase inhibitor, reduced kidney growth in a Phase II clinical trial in patients with ADPKD (Sweeney, von Vigier, Frost, & Avner, 2008; Tesar et al., 2017; Table 6).

3.4.2. Dysregulation of the vascular endothelial growth factors system

From the early stages of PKD, a pericystic network of vessels is formed and it becomes more amorphous as cystogenesis progresses. These vessels are responsible for delivering oxygen and nutrients to cystic cells (Huang, Woolf, & Long, 2013). VEGF‐A is a pivotal stimulator of physiological and pathological angiogenesis. VEGF‐C and VEGF‐D mostly function as regulators of lymphangiogenesis (Shibuya, 2011).

Studies have shown that exogenous VEGF‐A may improve kidney function. For instance, as shown in Table 6, VEGF‐A‐targeted therapy using SU‐5416 and celecoxib are somewhat effective in improving cystic disease (Xu et al., 2012). Delivery of exogenous mesenchymal stem cells (MSCs) improved vascular density and kidney function in PCK rats by releasing VEGF‐A (Franchi et al., 2016). Although the above‐mentioned studies indicated that VEGF‐A inhibition may improve cyst growth, one study reported that B20.4.1, as an anti‐VEGF‐A antibody increased cell proliferation and cyst growth in heterozygous (Cy/+) Han:SPRD rats; therefore, utilization of anti‐VEGF agents exacerbate glomerular damage and renal failure (Raina et al., 2011).

In the early stages of cystogenesis, VEGF‐C expression in cyst‐lining cells is reduced. VEGF‐C administration was shown to improve cystic disease in Cys1cpk/cpk mice, as a model of recessive PKD (ARPKD), leading to a modest but significant increase in lifespan (Huang et al., 2015).

VEGF receptors are also remarkably expressed in cystic cells. VEGF/VEGFR signaling may play a role in cell proliferation (Tao et al., 2007) and fluid secretion in cyst‐lining epithelial cell (Huang et al., 2013). Agents that target these molecules or their receptors (such as SU‐5416, B20.4.1) are shown in Table 6.

3.4.3. Upregulation of hepatocyte growth factor and its receptor

c‐Met is expressed in renal epithelial cells and binds HGF. HGF/c‐Met plays important roles in branching morphogenesis within the kidney development. HGF/c‐Met systems are overexpressed in cyst‐lining cells in kidneys of individuals with PKD or acquired cystic disease (Qin et al., 2012). Treatment with c‐Met pharmacological inhibitors, like SU11274 and PHA665752, affects mTOR, NF‐ƙB, and Wnt signaling pathways and improves cystic disease (Qin et al., 2012; Table 6).

3.4.4. Insulin‐like growth factor‐1 overexpression

Insulin‐like growth factor‐1 (IGF‐1) is involved in the control of renal growth, as well as glomerular filtration rate and ion transportation (Hirschberg & Kopple, 1988). IGF‐1 is found in cystic fluids and the expression of its receptor is increased in cystic tissues of patients with ADPKD (Aukema & Housini, 2001; Parker et al., 2007). Reducing this factor by a diet containing soy protein can improve renal function (Aukema & Housini, 2001).

3.4.5. Multitargeted tyrosine kinase inhibitor

Tesevatinib as a multispecific inhibitor of several tyrosine kinases can simultaneously reduce phosphorylation of key mediators of cystogenesis: EGFR, ErbB2, c‐Src, and KDR; as shown in Table 6, this reduction of kinase activity results in significant improvement of cystic disease in both bpk and PCK rat models of ARPKD (Sweeney, Frost, & Avner, 2017). Tesevatinib is currently being examined in Phase II clinical trials for treatment of patients with ADPKD (NCT03203642).

3.5. Treatments targeting interstitial inflammation

One of the PKD characteristics and causes of cyst progression is interstitial inflammation (Chen et al., 2015). Inflammatory cells such as macrophages have been reported in both human and animal models of PKD, with the degree of macrophage infiltrate associated with disease progression in humans. Studies show a direct role of PKD genes in regulating expression of some of the pro‐inflammatory chemoattractants such as monocyte chemoattractant protein‐1 (MCP‐1) and other cytokines. Experimental evidence suggested that MCP‐1 had a role in the development of interstitial inflammation and renal failure in PKD. It implicated in the inflammatory cell recruitment. Bindarit is an inhibitor of MCP‐1/CCL2 synthesis which can influence the evolution of PKD in PCK rats (Table 7).

Table 7.

Therapeutic effects of anti‐inflammatory drugs on PKD

Name	Mechanism	Effect on PKD model	Reference
Celecoxib	COX‐2 inhibitor	1. In cyst‐lining epithelial cells from patients with ADPKD: Inhibited expression of VEGFR‐2 and Raf‐1, thereby, reduced inflammation and fibrosis	Xu et al. (2012)
NS‐398	COX‐2 inhibitor	In Han:SPRD‐cy rats with inherited kidney disease: Slows disease progression and attenuates altered prostanoid production	Sankaran, Bankovic‐Calic, Ogborn, Crow, and Aukema (2007)
Acetylsalicylic acid	COX‐2 inhibitor	In Han:SPRD‐cy rats: Reduced COX products, cyst growth, and kidney water content	Ibrahim et al. (2015)
Etanercept	TNF‐α inhibitor	In Pkd2+/− mice: Inhibit cyst formation	Li et al. (2008)
WTACE2	Inhibitor of TNF‐α‐converting enzyme	In bpk mouse: Ameliorate the polycystic disease	Dell et al. (2001)
Bindarit	MCP1 inhibitor	In PCK rats: Reduction of interstitial inflammation and renal failure	Zoja et al. (2015)
Pyrimethamin	STAT3 inhibitor	In Pkd1 knockout mice: Inhibit cyst formation	Takakura et al. (2011)
		In human ADPKD cells: decreases cell proliferation
S3I‐201	STAT3 inhibitor	In neonatal PKD mouse model: Reduces cyst formation and growth	Takakura et al. (2011)

Note. ADPKD: autosomal dominant polycystic kidney disease; COX: cyclooxygenase; PKD, polycystic kidney disease; VEGFR: vascular endothelial growth factor receptor.

Tumor necrosis factor‐alpha (TNF‐α) is an inflammatory cytokine presents in ADPKD cystic fluid and disrupts the localization of PC2 to the plasma membrane and primary cilia (through a scaffold protein, FIP2). So, TNF‐α inhibitors could be therapeutic approach to control PKD (Table 7).

Fluid flow, calcium level changes and activation of cytokines have been shown to be associated with the activation of the Janus kinase/signal transducer (JAK/STAT) pathway, which is critical for developmental regulation, growth control, and homeostasis in organs (Weimbs, Olsan, & Talbot, 2013). The nuclear factor NF‐κB and JAK/STAT pathway have been considered two pro‐inflammatory signaling pathways in microenvironment of polycystic kidneys (Karihaloo, 2015). Phosphorylation of STAT3 occurs in response to a variety of cytokines and growth factors associated with PKD progression (such as interleukin‐6, EGF, and HGF) and is activated in mouse and human PKD1 disease. So, blocking STAT3 signaling may be an attractive target for the PKD treatment. The antiparasitic drug pyrimethamine and another compound STAT3 inhibitor, S3I‐201, significantly inhibited the cyst formation and growth in adult and neonatal PKD mouse model, respectively (Takakura et al., 2011).

Moreover, it was reported that cyclooxygenase 2 (COX‐2) had high expression in the kidney of PKD animal models, suggesting that COX‐2 might be implicated in the pathophysiology of ADPKD. Pharmaceutical inhibition of cyclooxygenase (COX) can provide relief from the symptoms of inflammation and attenuated renal injury (Ibrahim et al., 2015). Celecoxib (CXB), a highly selective COX‐2 inhibitor, is a sulfa nonsteroidal anti‐inflammatory drug. Inhibition of COX‐2 with CXB prevented growth of human ADPKD cyst‐lining epithelial cells (Xu et al., 2012). Also, NS‐398, a selective COX‐2 inhibitor, markedly slowed disease progression and attenuated altered prostanoid production in a rat model (Sankaran, Bankovic‐Calic, Ogborn, Crow, & Aukema, 2007). Drugs that intervene with interstitial inflammation and their impact on PKD is shown in Table 7.

3.6. Therapies interfering with renin–angiotensin–aldosterone system

Hypertension is a hallmark of ADPKD, and activation of intrarenal renin–angiotensin–aldosterone system (RAAS) is implicated in the pathogenesis of the disease. In polycystic kidney, renin, angiotensinogen, and angiotensin II are produced by cystic epithelial and tubular cells, and these molecules are secreted into the cystic fluid. Angiotensin II plays a key role in the increment of cAMP (Saigusa et al., 2015) and promotes infiltration of macrophages/monocytes, proinflammatory cytokines synthesis, and fibrosis (Belibi & Edelstein, 2010). Therefore, effective control of blood pressure may delay the onset of end stage renal disease by approximately 15 years. This finding has particular clinical importance because cardiovascular complications are the most common cause of death in patients with ADPKD (Ecder et al., 2000). The antihypertensive drugs tested for the PKD treatment are listed in Table 8.

Table 8.

Therapies targeting the renin–angiotensin system in PKD

Name	Mechanism	Effect on PKD model	Reference
Lisinopril	ACE inhibitors	HALT PKD study: Reduced cystogenesis and left ventricular mass index in the patients with PKD	NCT00283686
Ramipril	ACE inhibitors	ADPKD patients: Effective BP control and regressed renal progression	Ulusoy, Ozkan, Kosucu, Kaynar, and Eyuboglu (2012)
Enalapril	ACE inhibitors	ADPKD patients: BP control, urinary albumin, and left ventricular hypertrophy decrease	Schrier (2002); Ecder et al. (2000)
Telmisartan	ARB	HALT PKD study: Reduced cystogenesis and left ventricular mass index in the patients with PKD	NCT00283686
Losartan	ARB	ADPKD patients: Effective BP control and regressed renal progression	Ulusoy et al. (2012)
Aliskiren	Renin inhibitor	ADPKD patient: Excellent blood pressure control and reduction of edema, with aldosterone levels normalizing within 2 months	Amico, Kalbermatter, and Kiss (2009)

Note. ADPKD: autosomal dominant polycystic kidney disease; ACE: angiotensin‐converting enzyme; ARB: angiotensin receptor blockers; BP: blood pressure; PKD: polycystic kidney disease.

3.7. mTOR activation in PKD

PKD1 and TSC2 genes lie immediately adjacent to each other on chromosome 16 and the protein products of them are PC1 and tuberin. Tuberin (through a small GTPase, Rheb) regulates the kinase activity of mTOR. Activated mTOR phosphorylates and causes activation of its downstream effectors and leads to the stimulation of protein synthesis and proliferation. C‐terminal tail of PC1 interacts with tuberin. PC1 physically interacts and retains tuberin at the plasma membrane thus preventing it phosphorylation by AKT (protein kinase B) and its ability to inhibit the mTOR pathway. So, in the most cases of ADPKD due to absence of a functional PC1, the mTOR pathway is activated. Actually, increased mTOR activity was reported in epithelial cells lining ADPKD cysts (Dere, Wilson, Sandford, & Walker, 2010; Shillingford et al., 2006). Administration of mTOR inhibitors (such as rapamycin, sirolimus, and everolimus) could considered as a therapeutic approach because of a role in suppression of cell proliferation. They were investigated in rodent models of PKD. Administration of mTOR inhibitors ameliorated PKD in the Han:SPRD rat, orpk, and bpk mice (Tao, 2004; Wahl et al., 2006). Unfortunately, studies of mTOR inhibitors in human ADPKD have been disappointing. Sirolimus has side effects, like oral mucositis, diarrhea, acne, peripheral edema, amenorrhea, and ovarian cysts, and everolimus has oral ulcer, leukopenia, acne, and peripheral edema. It is suggested that combining low dose of mTOR inhibitors with non‐mTOR based treatments could maximize efficacy, whereas minimizing their many potential side effects (Serra et al., 2010; Walz et al., 2010; Watnick & Germino, 2010).

4. CELL THERAPY IN ADPKD

Mesenchymal stem cells (MSCs) are adult stem cells that can be often isolated from bone marrow (Ullah, Subbarao, & Rho, 2015). Transplantation of MSCs is a recent therapeutic approach for renal disease (Suzuki, 2016). Using animal nephropathy models, MSCs have been shown to be effective in improving kidney function. Nevertheless, their beneficial effects are mediated through their paracrine effects instead of their direct differentiation into renal cells (Ullah et al., 2015). Recent experimental studies reported that MSCs predominantly improve kidney tubular regeneration by secretion of growth factors, cytokines, and chemokines, which decrease apoptosis, inflammation, and fibrosis (Morigi, Rota, & Remuzzi, 2016). These properties subsequently ameliorate kidney function and structure (including creatinine clearance, GFR, and hypertension) in CKD (chronic kidney disease) models (Hickson, Eirin, Lerman, & Clinic, 2017; Morigi et al., 2016).

We showed that MSC‐derived conditioned medium induces regeneration of renal tubular cell after nephrotoxicity induction (Moghadasali et al., 2013); also, we approved the BM‐MSCs lowered serum creatinine and urea levels and increased regulatory T cells in a monkey AKI model (Moghadasali et al., 2014). In a recent study, our results indicated that MSCs suppress CKD progression in a monkey model (Moghadasali et al., 2015). Also, we indicated safety and tolerability of a single‐dose infusion of autologous BM‐MSCs in patients with CKD which provides an important foundation for future clinical trials to assess the efficacy of autologous MSCs in CKD (Makhlough et al., 2018).

These capabilities of MSCs (Patel et al., 2013) are not limited to antifibrotic and anti‐inflammatory effects on CKD and AKI model but also justify their use to control the progression of PKD. As the only preclinical study done on PKD, Franchi et al. used allogeneic MSCs in a PKD rat model (Franchi et al., 2016). They showed the beneficial effect of a single intravenous infusion in terms of improvements in systolic hypertension and fibrosis. Infusion significantly improved cortical and parenchymal vasculature density (however, it did not affect cyst size and number), which enhanced tubular function and creatinine clearance in an ARPKD model. Their findings approved the safety of MSC infusion as no mortality was recorded. In sum, Franchi et al. reported that transplanted MSCs improve kidney function and damaged vasculature in PKD (Franchi et al., 2016). In this regard, recently we performed a single‐arm Phase I clinical trial with a 12‐month follow‐up to evaluate effects of MCSs in patients with ADPKD. Safety and tolerability of an intravenous transplantation of autologous BM‐MSCs were also approved in this study (Makhlough et al., 2017). We concluded that BM‐MSCs efficacy in patients with ADPKD should be investigated in a randomized placebo‐controlled trial with a larger population, which we intend to perform. From another perspective, Carvalhosa et al. reported that CD133 + progenitor cells exist in ADPKD lining cysts (Carvalhosa et al., 2011). Also, our unpublished data demonstrated that in vitro coculture of MSCs and tubular CD133 + progenitor cells obtained from patients with ADPKD, resulted in reduced cyst formation and proliferation potential.

As discussed above, the ADPKD progression is associated with several processes and pathways such as apoptosis (Riella, Czarnecki, & Steinman, 2014), fibrosis (Grantham, Chapman, and Torres, 2006), inflammation (Nagao et al., 2003; Okumura et al., 2009), cyst proliferation, renin–angiotensin system (RAS) activation (Saigusa et al., 2015; Torres et al., 1992), and kidney vasculature impairment (Franchi et al., 2016). MSCs may affect these pathways through different mechanisms that are briefly discussed below.

As stated before, the MAPK signaling plays an important role in the pathogenesis of ADPKD through increment of proliferation. Studies showed that MSC‐derived conditioned medium inhibits p38 MAPK and ERK induces antiapoptotic effects in an AKI rat model (Wu, 2013). MSCs are capable of decreasing the activity of the NF‐ĸB pathway (Wu et al., 2014) and pro‐inflammatory cytokines, consequently suppressing inflammation in a CKD model (Semedo et al., 2009). Human MSCs are a source of VEGF so they could be appropriate for angiogenesis (Franchi et al., 2016). Studies showed that RAS inhibition improves blood pressure (BP) and total kidney volume (TKV), and prevents cyst development in animal and human models (Schrier et al., 2014; Zafar et al., 2007). MSCs reduce renin, angiotensin‐converting enzyme (ACE), and angiotensin II type 1 (AT1) receptor expression, and decrease BP, inflammation, and fibrosis (Gregorini et al., 2016; Oliveira‐Sales et al., 2013). The MSC inhibitory effect on RAS is more stable than ACE inhibitors effect (Gregorini et al., 2016). Hence, these capabilities of MSCs make them useful for the PKD treatment (Figure 1).

Figure 1.

Mesenchymal stem cells affect some of ADPKD disrupted pathways through their paracrine effects with different mechanisms. MSCs predominantly improve kidney tubular regeneration by secretion of growth factors, cytokines, and chemokines which decrease apoptosis, inflammation, and fibrosis. ADPKD: autosomal dominant polycystic kidney disease; MSCs: mesenchymal stem cells [Color figure can be viewed at wileyonlinelibrary.com]

5. SUMMARY AND CONCLUSION

Generally, ADPKD occurs following a germ‐line mutation in one of the polycystin gene alleles, a somatic second hit which leads to the loss of the normal allele, and a third hit, which can be anything that triggers cell proliferation. This event leads to the dilation of the tubules and continued dilation of the tubules (through increased cell proliferation, fluid secretion, and separation from the parental tubule) will lead to cyst formation. Studies on cystic cells and animal models of ADPKD depicted the important role of calcium and cAMP in disease pathogenicity. Initially, intracellular calcium decreases due to the mutations in PKD genes which cause intracellular phenotype changes confronting with cAMP. cAMP blocks proliferation of normal kidney epithelial cells through inhibition of the Ras/Raf/MEK/ERK pathway. Although in sharp contrast, cAMP induces proliferation through activation of the B‐Raf/MEK/ERK pathway in ADPKD cells because of decreased intracellular calcium levels. Increased levels of cAMP also affect chloride channels and induce fluid secretion into cysts. Other disrupted pathways that are involved in cystogenesis and cyst progression include defective planar cell polarity, extracellular matrix abnormalities, inflammation, increased apoptosis, modifying genes, and environmental factors. Based on recent knowledge of molecular mechanisms underlying PKD pathogenicity, new therapeutic approaches are being examined for treatment of this disease. Paraclinical studies have shown that treatments like decreasing cAMP, increasing intracellular calcium, inhibiting cellular proliferation, and suppression of fluid secretion in cysts, reduce cyst growth (Figure 2).

Figure 2.

Schematic representation of pathways those are upregulated or downregulated in polycystic kidney disease and potential therapeutic interventions. Dysregulation of Ca²⁺ levels and increased concentrations of cAMP are two important characteristics of ADPKD cells. PKD mutations cause defects in polycystins functions resulting in reduction of intracellular Ca²⁺ concentration. Stimulation of Ca²⁺ inhibitable AC6 and/or inhibition of Ca²⁺‐dependent PDE1 (by reducing Ca²⁺) and increased levels of circulating vasopressin and upregulation of vasopressin V2 receptors, increase cAMP concentrations. Increased cAMP levels contribute to stimulation of chloride channels and fluid secretion. Increased cAMP also stimulates MAPK/ERK signaling in a Src‐ and Ras‐dependent manner. Upregulation of EGF, IGF1, and VEGF in ADPKD cells activates tyrosine kinase receptors and stimulates MAPK/ERK signaling and cell proliferation. Another ADPKD characteristic is interstitial inflammation through nuclear factor NF‐κB and JAK‐STAT pathways. Furthermore, upregulation of TNF‐α or downregulation of AMPK signaling may also stimulate mTOR signaling. Activation of AMPK may also blunt cystogenesis through inhibition of CFTR and ERK. ADPKD: autosomal dominant polycystic kidney disease; AMPK: adenosine monophosphate‐activated protein kinase; cAMP: Cyclic adenosine monophosphate; EGF: epidermal growth factor; PDE1: phosphodiesterase; MAPK: mitogen‐activated protein kinase [Color figure can be viewed at wileyonlinelibrary.com]

FUNDING

This study was supported by a grant from Royan Institute and the Royan Charity Association for Health Research (Tehran, Iran) (Grant no: 91000687).

CONFLICTS OF INTEREST

The authors declare no financial or commercial conflicts of interest.

AUTHORS’ CONTRIBUTIONS

R.M., T.M., N.K., and A.S.: Manuscript writing. R.M.: Final approval of the manuscript.