Renal ciliopathies: promising drug targets and prospects for clinical trials

ABSTRACT

Introduction

Renal ciliopathies represent a collection of genetic disorders characterized by deficiencies in the biogenesis, maintenance, or functioning of the ciliary complex. These disorders, which encompass autosomal dominant polycystic kidney disease (ADPKD), autosomal recessive polycystic kidney disease (ARPKD), and nephronophthisis (NPHP), typically result in cystic kidney disease, renal fibrosis, and a gradual deterioration of kidney function, culminating in kidney failure.

Areas covered

Here we review the advances in basic science and clinical research into renal ciliopathies which have yielded promising small compounds and drug targets, within both preclinical studies and clinical trials.

Expert opinion

Tolvaptan is currently the sole approved treatment option available for ADPKD patients, while no approved treatment alternatives exist for ARPKD or NPHP patients. Clinical trials are presently underway to evaluate additional medications in ADPKD and ARPKD patients. Based on preclinical models, other potential therapeutic targets for ADPKD, ARPKD, and NPHP look promising. These include molecules targeting fluid transport, cellular metabolism, ciliary signaling and cell-cycle regulation. There is a real and urgent clinical need for translational research to bring novel treatments to clinical use for all forms of renal ciliopathies to reduce kidney disease progression and prevent kidney failure.

KEYWORDS:

• renal ciliopathies
• therapeutics
• ciliary signaling
• clinical trials
• pre-clinical studies
• fibrosis
• nephronophthisis
• polycystic kidney disease

1. Introduction

1.1. Overall aims

Within the last 20 years, research of ciliopathies has expanded exponentially. Ciliopathies, a group of genetic diseases that were once isolated or unknown, are now being characterized as a spectrum of pleiotropic inherited disorders with shared defects in the biogenesis, maintenance, or functioning of the ciliary complex. One subset of primary ciliopathy syndromes are renal ciliopathies, which all share a kidney defect, often leading to kidney failure (KF). Notably, there are currently no curative treatments for renal ciliopathies.

The increased understanding of the molecular composition, structure, and function of the primary cilium, as well as developments in ciliopathy disease models, have uncovered shared pathogenic mechanisms across the spectrum of renal ciliopathies. Together, these scientific advances have driven the identification of potential therapeutic targets. This review will explore promising small molecular compounds and drug targets within preclinical studies and clinical trials, discussing the prospects for potential and future treatments of renal ciliopathies.

1.2. An overview of the primary cilium

The primary cilium is a microtubule-based cellular appendage, found as a single organelle on nearly every mammalian cell, including renal tubular epithelial cells [1]. It is a sensory organelle and functions as a signaling hub, which is vital in numerous biological processes such as left-right patterning in development, organogenesis, tubulogenesis, cell cycle regulation, and homeostasis.

During quiescent stages of the cell cycle (G0) the primary cilium is formed, which can also be mimicked by serum starvation in vitro. Upon initiation of ciliogenesis, the mature centriole, a cylindrical 9-fold triplet microtubule structure (150–200 nm diameter, 400–450 nm length) with distal and subdistal appendages, forms the basal body (BB) of the primary cilium [2–4]. The BB anchors to the plasma membrane via distal appendage proteins (DAPs) that project radially from the BB, marking the transition zone region [5,6]. The core BB extends to form the 9-fold doublet microtubule-based axoneme, lacking a central axonemal pair (9+0), rendering it immotile. The primary cilium itself is encased in a ciliary membrane contiguous with the plasma membrane. A discrete ciliary compartment is formed using the transition zone (TZ) of the cilium which cross-links the ciliary membrane with the proximal axonemal microtubules, utilizing Y-links and ciliary necklace proteins and works with DAPs, septin ring barrier, nucleoporins, and other ciliary protein complexes to regulate selective targeting, sorting and localization of molecular cargo [7–10]. Protein complexes integral to ciliary function include the BBSome, MKS complex, NPHP complex, Inversin, and CEP290 complexes [6,11–13]. Intraflagellar transport (IFT) is used to transport protein cargo within the ciliary compartment and is also required for formation of the central axoneme and numerous ciliary signaling pathways [1]. Anterograde movement (up) and retrograde movement (down) is exchanged at the ciliary tip and at other points along the ciliary axoneme [1,14]. Upon entry into the cell cycle, due to the requirement of the mature centriole within the centrosome, the cilium is disassembled and resorbed [15].

The primary cilium acts a vital transducer in numerous signaling pathways. Patched 1 (PTCH1), the Sonic hedgehog (Shh) receptor, localizes to the ciliary membrane and prevents entry of Smoothened (SMO) [16]. Upon Shh binding to PTCH1, PTCH1 is trafficked out of the cilium allowing SMO to accumulate in the primary cilium, which activates Gli transcription factors. Shh signaling pathway is vital in processes such as cell determination and patterning, and is involved in proper kidney development and functioning, with defective signaling implicated in various renal ciliopathies [16–19]. Components of the canonical Wnt signaling pathway, which is involved in cell proliferation, differentiation and survival, are located at the primary cilium [20]. Components of the non-canonical Wnt planar cell polarity pathway (PCP), regulating cell morphology, migration, and orientated cell division, are also present at the primary cilium [20]. PCP signaling is required for proper nephron tubular formation and maintenance, defects of which can lead to cystogenesis within the kidney tubules [18,21,22]. There are other ciliary signaling pathways which are vital in cell cycle regulation and proliferation, that have been implicated in proper renal tubular formation and kidney function. These includes cyclic AMP (cAMP) signaling, mammalian target of rapamycin (mTOR) signaling, epidermal growth factor (EGF) signaling, AMP-activated protein kinase (AMPK) signaling and insulin-like growth factor (IGF) signaling, although this list is not exhaustive [23,24].

The primary cilia are also transducers of mechanical stimuli. In the renal tubules, primary cilia project from the apical surface of renal tubular epithelial cells into the lumen and are stimulated by urinary flow. Polycystin-1 (PC1) and polycystin-2 (PC2) act as mechanotransducers, which mediate numerous downstream signaling pathways such as PCP, mTOR, and Shh, which can be dysregulated in cystic kidney models [25–28]. Primary cilia also sense morphogens, hormones, and odorants [29]. Emerging studies have also indicated that primary cilia may have roles in releasing ‘ectosomes’ as signaling messengers [23].

As proteins encoded by primary cilium-related genes are closely linked with the function of centrioles, it is logical that some ciliary genes, such as CEP290 and CEP164, have also been implicated in DNA damage response (DDR) pathways and cell cycle regulation, which effect renal cell proliferation, apoptosis and fibrosis [23,30,31].

There are also other specialized primary ciliary structures, including the connecting cilium within the photoreceptor cells of the retina and olfactory sensory neurons [32–34]. The other main subtype of cilia are motile cilia, which are found as groups of organelles on specialized cells such as the respiratory epithelium and reproductive tract [35]. Sperm flagella is also a specialized motile cilium. Additionally, nodal cilia found at the embryonic node have overlapping structures with both primary and motile cilia and are vital for left-right patterning during development [36]. However, the cilia type present on kidney epithelial cells are primary cilia, and therefore this will be focus of this renal ciliopathy review.

2. Renal ciliopathies

2.1. Renal ciliopathies overview

Primary ciliopathies are a group of rare genetic multi-system syndromes, caused by defects in the maintenance, biogenesis or functioning of the primary cilium. Primary ciliopathies are classified further into disease syndromes with defined clinical presentations, but they often have genetic and phenotypic overlaps. There are over 35 ciliopathy syndromes with an estimated grouped incidence of 1 in 1,000 people, with over 180 known primary ciliopathy genes, but approximately 50% of ciliopathy cases remain genetically unsolved [1,35,37–39]. Also, primary ciliopathy genes have high pleiotropy, making it difficult for clear diagnosis, prognosis, and appropriate disease management based upon genotype alone. It might be that as the number of ciliary genes increases and syndromes overlap further, ciliopathies will become a continuum of disorders rather than the current system of discrete syndromes [38].

Renal ciliopathy syndromes often manifest in the form of cystic kidney disease, but can also manifest as tubulointerstitial fibrosis, both can lead to KF (Figure 1) [40]. The primary cilium is found on the apical surface of renal epithelial cells and projects into the lumen of the nephron, acting as both a chemical and mechanical signal transducer. Archetypal renal ciliopathy syndromes include autosomal dominant polycystic kidney disease (ADPKD), autosomal recessive polycystic kidney disease (ARPKD), and nephronophthisis (NPHP) [40]. There are other renal primary ciliopathies with extra-renal manifestations affecting organs such as the retina, brain, skeletal system, metabolism, liver, auditory perception, fertility, and development. These include Bardet-Biedl syndrome (BBS), Joubert syndrome (JBTS), Senior-Løken syndrome (SLS), Jeune asphyxiating thoracic syndrome (JATD), and the perinatally lethal Meckel-Gruber syndrome (MKS). There are no curative treatments for renal ciliopathies, and renal manifestations are typically life limiting [24,41]. Current therapeutics involve symptom management and supportive treatments, some including dialysis, treatment of hypertension and anemia, and dietary restrictions including salt restriction, but they do not target the underlying cause of disease or prevent KF [40].

Figure 1. Clinical phenotypes and molecular genetics of renal ciliopathies.

Note:Variability in the age of onset of KF and the median age of onset of clinical symptoms are shown as extended diamond bars. ADPKD mostly presents in adulthood (30-40 years of age) although a small proportion of cases reported in early life (1-10 years of age) due to biallelic or digenic or severe loss of function PKD1/PKD2 variants. The age of presentation for ARPKD varies with approximately one third of patients presenting before 1 year of age, one third between 1 and 20 years of age and one third after 20 years with mutations in PKHD1 being the most common cause. In juvenile NPHP, KF develops at a median age of 13 years whereas in adult form, which is clinically and histologically similar to the juvenile NPHP, KF develops after 15-20 years of age. Infantile NPHP is rare with KF appearing during first year of life. The current clinical trials with name of the drug, ClinicalTrials.gov identifier number and the age range of patients the study is recruiting is shown for ADPKD and ARPKD. There are currently no clinical trials in progress for NPHP. (Created using BioRender.com).

2.2. ADPKD

The most common inherited renal ciliopathy is ADPKD, frequently leading to KF, requiring renal replacement therapy [38,42]. ADPKD is estimated to effect 1 in 1000 people, and it typically presents in adulthood with hypertension and bilateral cystic kidneys, although age-dependent penetrance and disease variability means prevalence is likely underestimated [43,44]. Patients with typical ADPKD have numerous large fluid-filled bilateral kidney cysts, which cause kidney enlargement. The cysts themselves may arise from all nephron segments, but predominantly originate from the collecting duct [24]. ADPKD is often characterized by misorientated hyperproliferation of renal epithelial cells, which causes the formation of renal tubular cysts [45]. Subsequent cyst enlargement causes obstruction of neighboring nephrons, displacing renal parenchyma, and compressing microvasculature, causing urine drainage to be hindered, glomerular filtration to be gradually impaired, following which renal tubules become atrophic. The kidney injury causes inflammation and fibrosis pathways to be triggered [45,46]. ADPKD often results in KF.

Heterozygous mutations in PKD1 and PKD2, encoding polycystin-1 (PC1) and polycystin-2 (PC2) respectively, account for over 90% of ADPKD patients. PKD1 mutations account for 75–85% of identified mutations, and PKD2 mutations are less common, accounting for 15% of identified mutations and milder phenotypes with KF developing at a later age [41,47]. PC1 and PC2 are located in the primary cilium. PC1 acts as an ion channel and g-protein coupled receptor, and PC2 acts as an ion channel that forms a heteromeric complex with PC1 with fibrocystin, regulating PC1’s localization in kidney epithelia [48,49]. It has traditionally been proposed that the PC1/PC2 complex mediates Ca²⁺ signaling in the primary cilium, in response to fluid flow; however, more recently, this has been refuted, with the exact pathomechanism unclear [41,45,50,51]. Nevertheless, it is thought that loss or malfunction of PC1/PC2 has implications downstream for multiple pathways, including cAMP, mTOR, EGF, AMPK, and cMyc signaling, which effect orientated cell proliferation [52].

The majority of patients present with ADPKD in adulthood (30–40 years old) with a typical radiological pattern of cystic kidney disease, with bilateral and diffuse distribution of kidney cysts and mild, moderate or severe replacement of kidney tissue by cysts, where all cysts contribute similarly to an increase in total kidney volume [53]. There is a large variability in the age of presentation and disease progression can be measured using total kidney volume (TKV) following kidney magnetic resonance imaging to predict risk of KF [54]. TKV has been used as a surrogate end point in interventional clinical trials [55]. Pathogenic mutations in PKD1 and PKD2 can include frame-shift, nonsense, splice-site, missense and synonymous SNV changes, with more recent evidence for deep intronic alleles [41]. In general patients with loss of function mutations in PKD1 and PKD2 present with a more severe, earlier-onset cystic kidney phenotype (Figure 1). Prenatal and antenatal presentations of ADPKD are often associated with a PKD1 or PKD2 loss of function allele and the presence of an additional second allele, often hypomorphic, in PKD1 or PKD2 acting as a genetic modifier [56]. Digenic heterozygous alleles in both PKD1 and PKD2 could also contribute to earlier onset ADPKD [56]. Occasionally, severe early onset PKD phenotypes may also be caused by biallelic loss of function mutations in PKD1 or PKD2 (Figure 1). The “somatic second hit” hypothesis of PKD1/PKD2 mutations also may affect phenotypic variability [57]. On the other end of the spectrum, mild ADPKD phenotypes with preserved renal function may be due to single heterozygous hypomorphic mutations in PKD1 and PKD2 [56].

Radiologically, there are other forms of atypical ADPKD, where there may be unilateral cystic kidney disease, segmental cystic kidney disease involving one pole of one or both kidneys, asymmetric cystic kidney disease and lopsided kidney disease, where less than or equal to 5 kidney cysts account for greater than or equal to 50% of total kidney volume [53]. Such patients present with cystic kidney disease phenotypes, as well as the other renal and extra-renal phenotypes, such as polycystic liver disease. These are caused by mutations in genes encoding proteins not typically associated with primary cilia but are predicted to effect the PC1 or PC2 complex [41]. An example is heterozygous mutations in the transcription factor HNF1B which causes atypical ADPKD with a wide range of renal phenotypes, such as congenital abnormalities of the kidney and urinary tract (CAKUT) or autosomal dominant tubulointerstitial kidney disease (ADTKD)-like presentations [58]. Other genetic causes of atypical ADPKD include heterozygous variants in; IFT140, DNAJB11, GANAB, ALG8, ALG9, SEC61A1 and PRKCSH [58–67]. Atypical ADPKD is characterized clinically with a milder disease progression phenotype with some mutations in genes such as GANAB and ALG8 having no instances of KF reported to date; however, some patients reach KF in later adulthood [41,61–64].

2.3. ARPKD

ARPKD is a rare renal ciliopathy than ADPKD, with an estimated incidence of 1 in 20,000 live births [68]. Patients with ARPKD have numerous fluid-filled bilateral kidney cysts and poor corticomedullary differentiation, which causes kidney enlargement, subsequent kidney injury, and KF. ARPKD is also often associated with liver abnormalities. Unlike ADPKD, patients with ARPKD often present with symptoms in early childhood with 50% suffering with KF by 10 years of age. However, there are also later childhood and adult presentations of ARPKD which predominately have liver phenotypes including congenital hepatic fibrosis and ductal plate malformations, alongside milder cystic kidney disease (Figure 1) [41,69].

ARPKD is most commonly caused by mutations in PKHD1 that encodes fibrocystin, which interacts with PC1 and PC2 within the primary cilia [50,70–72]. Fibrocystin is a key regulator of cell proliferation, apoptosis and polarization [73]. More recently, mutations in DZIP1L have been associated with ARPKD which develop KF later in life, although this accounts for less than 1% of individuals [74]. DZIP1L is believed to be involved in proper localization of PC1 and PC2. Biallelic mutations in TULP3 have also been shown to give a phenotype resembling ARPKD, with progressive fibrocystic liver and kidney disease, as well as polycystic kidney disease [75]. The encoded protein TULP3 acts as a critical adaptor protein for ciliary trafficking.

2.4. NPHP

NPHP patients present with a progressive decline in kidney function, often with a loss of urinary concentrating ability, resulting in polyuria and polydipsia. Histological features following renal biopsy reveal tubulointerstitial disease associated with progressive tubular interstitial fibrosis, inflammation, and tubular atrophy, and sometimes corticomedullary cysts [19,76]. NPHP is not typically associated with kidney enlargement other than in infantile cases [23]. Notably, NPHP leads to a progressive loss of renal tubular function and represents the most common pediatric inherited cause of KF.

NPHP has a disease spectrum; there are traditionally three clinical subtypes of NPHP, infantile, juvenile, and adolescent [41,77]. Infantile NPHP has a severe phenotype, with a median age of onset of 1 year old and KF typically before 3 years old. Infantile NPHP presents with enlarged kidneys with cortical cysts, moderate fibrosis, and rapid disease progression, similar to ARPKD, and lacks the tubular basement membrane changes present in other NPHP subtypes [23,76]. Juvenile NPHP, the most common, has median age of onset of 13 years old with KF typically in the second decade of life, and patients present with small to normal sized kidneys with tubulointerstitial fibrosis, thickened tubular membranes and in less than 50% patients, corticomedullary cysts. There may also be adult-onset NPHP, which clinically presents as juvenile NPHP, albeit later [41,77]. NPHP gene mutations such as NPHP1 whole gene deletions may be detected in adult KF patients who have previously been labelled as KF of uncertain cause (Figure 1) [78].

NPHP has an estimated incidence of 1 in 50,000–1 in 1,000,000 individuals [76]. To date, there are over 20 NPHP causative genes identified, encoding protein products known as nephrocystins, inherited in an autosomal recessive pattern, accounting for less than 50% of NPHP ciliopathies [40]. Mutations in the NPHP complex, a distinct module found in the transition zone of the primary cilium, causes NPHP. The NPHP complex is involved in transition zone and ciliary biogenesis, as well as cargo trafficking. The NPHP complex includes protein products from RPGRIP1L, NPHP4, NPHP1, NPHP5, NPHP3, and NEK8. Mutations in NPHP1, encoding nephrocystin-1, is the most common genetic cause of NPHP (approximately 20% of cases) [77,79]. Nephrocystin-1 is an adaptor protein that localizes to the primary cilium and apical membrane of kidney epithelial cells, with functions in cell signaling, adhesion and cell maintenance, interacting with PC1 and PC2 and nephrocystins [80]. Other genetic causes of NPHP include genes encoding proteins which are localized to the primary cilium or centriole, including CEP290 and CEP164 [41,76,81]. Specifically, infantile NPHP is commonly caused by mutations in INVS, NPHP3, NEK8 and CEP83 [82–85]. Adults presenting with later onset NPHP have been shown to have disease causing variants in NPHP1 and NPHP3 [79,86,87]. However, there are several NPHP genes that are not currently directly linked to primary ciliary function, including ZNF423, MAPKBP1 and GLIS2 [38,81,88,89].

2.5. Other renal ciliopathies with associated clinical manifestations

At least 15% of NPHP cases are associated with extra-renal manifestations [23], which can be grouped together as other multisystem ciliopathy syndromes. If genes that cause syndromic NPHP-related ciliopathies are accounted for, there are over 90 implicated genes [23,90]. A well-described NPHP-related ciliopathy is JBTS, a primary ciliopathy syndrome characterized by brain axial magnetic resonance imaging (MRI) ‘molar tooth sign’, caused by cerebellar vermis aplasia (CVA), thickened and horizontalized cerebellar peduncles and deepened interpeduncular fossa. This neurological development disorder can lead to many problems beginning in early life including developmental delay, hypotonia leading to ataxia, breathing problems and intellectual disability [35,91]. JBTS may characterized by a neurological phenotype but can be a renal-retinal-cerebellar syndrome with the NPHP-associated kidney disease leading to KF, and retinal degeneration leading to blindness. Liver and skeletal phenotypes may also be present in patients with JBTS. It is estimated that 1 in 80,000–1 in 100,000 people have JBTS; however, the heterogenous nature of the disease means it is likely underdiagnosed [92]. Mutations in over 35 genes have been identified as genetic causes of JBTS, most of which are autosomal recessive, apart from OFD1 which has X-linked recessive inheritance [93]. Mutations in CEP290 are the most common cause of JBTS accounting for more than 38% of cases [94].

BBS is an autosomal recessive ciliopathy where patients present with retinal degeneration usually leading to blindness as well as at least two of the following primary/major symptoms; renal dysfunction (which is frequent and often presents on prenatal ultrasound with renal and urinary tract malformation, such as cystic kidney, pelvic dilation, and ureter obstruction), intellectual disability, obesity, polydactyly, and hypogonadism [95]. Patients can also present with secondary/minor symptoms, some of which include growth retardation, delayed development, speech disorders, thyroid problems, anosmia, and dental anomalies [96]. BBS clinical diagnosis is based upon the presence of 4 primary symptoms, or 3 primary symptoms and at least two secondary symptoms (Beales criteria) [97]. Renal biopsy in patients with chronic kidney disease (CKD) and BBS typically shows renal dysplasia and chronic tubulointerstitial nephropathy [98,99]. Overall BBS is estimated to occur in 1 in 100,000 live births, although there is a higher prevalence in non-European Union countries with high consanguinity, specifically Pakistan [96,100]. There are over 20 known BBS genes accounting for approximately 85% of cases. Many of the protein products of genes which cause BBS are found within the BBSome protein complex, which mediates the binding of IFT particles (IFT-A, IFT-B) to protein cargo at the base of the cilium and ciliary tip, enabling anterograde and retrograde ciliary transport [11,12]. The BBSome also interacts with both PC1, PC2 and NPHP-associated proteins [101]. The BBSome complex contains; BBS2, BBS7, BBS9, BBS1, BBS4, BBS18, BBS8 and BBS5, which interacts with ADP ribosylation factor (ARF)-like GTPase ARL6/BBS3 required for membrane recruitment of the BBSome. The most prevalent causes of BBS are BBS1 mutations whose encoded protein is found within the BBSome and BBS10 mutations, whose encoded protein is found within the BBSome chaperonin-like complex to mediate BBSome assembly [102,103].

NPHP-like ciliopathies also include SLS where patients present with NPHP associated with retinitis pigmentosa. The most common cause of SLS is mutations in NPHP5 alias IQCB1 [104,105].

Meckel Gruber Syndrome (MKS) is a rare ciliopathy syndrome characterized by NPHP associated with skeletal defects and developmental abnormalities, often resulting in embryonic and postnatal lethality [106]. Causative genes have been identified for 60% of MKS cases, mutations of which are present in the MKS complex located at the transition zone. It contains the core proteins; B9D1, B9D2, TCTN1, TCTN2, TCTN3, CC2D2A, TMEM216, TMEM67, MKS1, and TMEM237. The MKS complex is thought to be involved in transition zone biogenesis and regulation of trafficking in and out of the primary cilium [6,13,107].

JATD is an autosomal recessive syndrome characterized by short-rib thoracic skeletal dystrophies, with short stature, narrow thorax with short ribs, short limbs, and polydactyly. JATD is also linked with cystic renal disease [93]. Genes associated with JATD include CEP120, CSPP1, KIAA0586, and PIBF1. Oral-facial-digital syndromes (OFD) are ciliopathy syndromes with characteristic facial abnormalities, oral abnormalities, and polydactyly. The gene OFD1 causes X-linked dominant OFD with polycystic kidney disease [108].

Exploration of all the known and potential genes underlying renal and multisystem primary ciliopathies is beyond the scope of this review; however, other reviews can be recommended [40,41].

3. Modelling renal ciliopathies

Conventionally, primary ciliopathies have been studied using 2D cell line cultures that can form primary cilia upon entry into G0, mimicked by serum starvation. Examples of immortalized renal cell lines include HEK293 (Human embryonic kidney), mIMCD-3 (murine kidney inner medullary collecting duct), MDCK (canine kidney distal tubule/collecting duct), LLC-PK1 (pig kidney proximal tubule) and A6 (frog kidney) [109–114]. These cells can be utilized to study the normal functioning of renal primary cilia, be genetically modified to understand gene and variant-specific pathogenic mechanisms of disease and be used to examine the effects of potential therapeutics. Developed more recently, human urine-derived renal epithelial cells (hURECs) from healthy donors and patients can be used as a renal ‘liquid biopsy’, which is a powerful tool for understanding patient-specific molecular mechanisms of disease, working toward personalized therapies [40,115,116].

More recently, 3D kidney tubuloids and organoids from hURECs and stem cells have been utilized to model kidney development and disease. Organoids have the advantage of containing different parts of the nephron and interstitial cells, which is especially useful for modelling the 3D renal tubule structure with cilia projecting into the lumen [116–123]. Kidney tubuloids and organoids are also utilized for personalized medicine approaches, by culturing patient-derived stem cells or genetically engineering patient mutations [23,38,109,124]. Other more recent scientific advances include kidney-on-a-chip platforms which integrate living kidney cells and biophysical systems such as fluid flow, pressure, and chemical gradients, creating a model more accurate to the human in vivo system [45,125].

A large insight into renal ciliopathies has been gained by using animal models, typically zebrafish and rodents in which cilia form in the same tissues as humans, but Xenopus, Chick, and C. elegans have also been utilized [109]. Zebrafish are useful tools for studying embryonic consequences of renal ciliopathies as they are easily genetically manipulated, the embryos and larva (24–72 hours post fertilization) are transparent allowing non-invasive imaging during development, and classic zebrafish ‘ciliopathy’ defects such as pericardiac edema, hydrocephalus, abnormal body curvature and pronephric cysts present rapidly [45,81,126]. Additionally, zebrafish are low cost and have fast development, meaning they allow efficient functional studies and large-scale screening analysis [23].

Rodents have been invaluable in ciliopathy research, especially as they can be utilized to study post-embryonic phenotypes including adult histology and fibrosis [23]. Prior to the discovery of genetic causes of renal ciliopathies rodent strains which develop spontaneous renal cysts were utilized as disease models; an example is cpk mice as a murine model of ARPKD [38,127–129]. With improvement in gene sequencing and development of genetic modification techniques numerous transgenic animals with gene knockouts, knockins, and patient-specific point mutations have been designed. The use of inbred transgenic mouse models also helps to remove confounding influences such as oligogenecity and triallelism, to understand the exact pathogenic mechanisms driven by specific genetic mutations [127]. Biological parameters such as kidney volume, kidney weight, and urine/plasma biomarkers [130] can be measured, histopathology examined, and molecular pathways investigated by transcriptomics and proteomics, to uncover disease mechanisms and determine the efficacy of potential therapeutics.

4. Disease mechanisms of renal ciliopathies

Although renal ciliopathies can be classified into distinct syndromes, causative mutations in genes encoding proteins involved in the primary cilium or centrosome mean they may share overlapping mechanisms of disease, which may be amenable for therapeutic intervention (Figure 2). Abnormal functioning of proteins involved in ciliogenesis, such as CEP164, can prevent proper cilia formation, which will effect a myriad of downstream ciliary signaling pathways. Additionally, mutations in genes encoding for proteins involved in cargo trafficking or regulation, such as CEP290, will have implications for signal pathway transduction, as well as mutations in components of signaling pathways themselves, such as PKD1. In regard to renal ciliopathies, abnormalities in signaling pathways such as cAMP, Shh, Wnt, mTOR, and AMPK, likely cause misoriented cellular divisions, increased proliferation, increased fluid secretion and subsequent cystogenesis, consequently leading to further kidney damage. Ciliary and centriolar proteins which have roles in DDR and cell cycle regulation may also be driving a renal cystogenesis phenotype alongside increased fibrosis and apoptosis. Increased inflammation and dysfunctional mitochondria are also by-products of dysregulated signaling pathways have been shown to contribute to the progression of renal ciliopathies. Extensive reviews of mechanisms of renal ciliopathy diseases have recently been performed [23,24]. Importantly, due to the wide range of cellular processes that primary cilia regulate, it is likely that in each syndrome there are multiple pathogenic drivers of disease. In some ways, this is advantageous as it offers many points for potential therapeutic targets. However, the cross talk between pathways and feedback loops introduces complications of changing one pathway without negatively affecting another. Further challenges arise with core biological pathways, such as Shh signaling, in which modification in vitro may be beneficial, but systemic treatment is unrealistic due to the expected severe side effects [18,24,116].

Figure 2. Renal ciliopathies and potential drug targets and prospects for clinical trials.

Note: Summary diagram showing the molecular pathways and the therapeutic targets studied in cell based and rodent models (highlighted in green) and those studied in human clinical trials (highlighted in blue) in renal ciliopathies. The drug names are shown in red. (Created using BioRender.com).

5. Preclinical drug targets and clinical trials in renal ciliopathies

Preclinical research on drug targets for renal ciliopathies has focused on understanding the molecular mechanisms underlying these disorders. This has led to ongoing clinical trials for renal ciliopathies, with several drugs in various stages of development (Table 1). Potential therapeutics are being evaluated for their ability to slow the progression of cystic kidney disease. Here we provide a brief overview of the current landscape.

Table 1. Molecular targets, drug compounds, preclinical trials, and clinical trials for renal ciliopathies.

Molecular target	Pathway	Renal Ciliopathy Disease	Drug Compound	Preclinical Studies	Clinical trials	References
Transmembrane Channels
Vasopressin receptor 2 (VR2)	VR2 activation increases cAMP and PKA, promoting AQP2 localisation to membrane and increased water reabsorption and fluid secretion.	Upregulated in ADPKD, ARPKD and NPHP, increasing epithelial cell proliferation and fluid secretion promoting cystogenesis.	Tolvaptan (AV2R inhibitor)	PCK rat (ARPKD), pcy mouse (NPHP), Pkd1 mouse (ADPKD)	ADPKD, ARPKD	[45,131–135]
			Lixivaptan (AV2R inhibitor)	MCD4 cells, PCK rat (ARPKD).	ADPKD	[136,137]
Cystic fibrosis transmembrane conductance regulator (CFTR)	Channels contribute to increased chloride secretion in renal tubules.	Upregulated in ADPKD, contributing to increased chloride transport and fluid secretion, promoting cystogenesis.	CFTR inhibitors (CFTR inhibitor) (Tetrazolo-CFTR-172, Ph-GlyH-101)	Pkd1 mouse (ADPKD)	N/A	[138]
			Lumacaftor (CFTR corrector and potentiator)	Pkd1 mouse (ADPKD)	N/A	[139]
Transmembrane protein 16 A (TMEM16A)			Niclosamide (CFTR inhibitor)	Pkd1 (ADPKD)	N/A	[140]
			Benzbromarone (CFTR inhibitor)	Pkd1 (ADPKD)	N/A	[140]
			Ani9 (CFTR inhibitor)	Pkd1 (ADPKD)	N/A	[140]
Peroxisome proliferator-activator receptor gamma (PPARγ)	PPARγ inhibits the synthesis of CFTR and ERK signaling.		Pioglitazone (PPARγ inhibitors)	MDCK-C7, PCK rat (ARPKD), Pkd1 (ADPKD)	ADPKD	[141–146]
			Rosiglitazone (PPARγ inhibitors)	MDCK-C7, Han:SPRD rat (NPHP)	N/A	[142,145]
Lipids
Hydroxy-3-methylglutarylCoA reductase	Statins are Hydroxy-3-methylglutarylCoA reductase inhibitors which modulate lipids and effect AMPK activation.		Lovastatin (HMG-CoA inhibitors)	Han:SPRD rat (NPHP)	N/A	[147]
			Simvastatin (HMG-CoA inhibitors)		ADPKD	[148]
			Pravastatin (HMG-CoA inhibitors)		ADPKD	[149]
Glycosphingolipids	Components of ciliary membrane and effectors of ciliary signaling.	Glycosphingolipids accumulate in PKD models.	Genz-123346	Pkd1 mice (ADPKD), jck mice, pcy mice (NPHP)	N/A	[150]
			Venglustat (Glucosylceramide synthase inhibitor)		ADPKD	[151]
Inflammation
Cyclooxygenase 2 (COX2)	COX2 is a prostaglandin metaboliser, and contributes to inflammation.	COX2 is upregulated in PKD and NPHP disease models.	Celecoxib (COX2 inhibitors)	Pkd2 mouse (ADPKD), Han:SPRD rat (NPHP)	N/A	[152–154]
			Flax-oil diet	pcy mouse (NPHP)	N/A	[155]
Nuclear factor erythroid 2 like-2 (NFR2)	NFR2 promotes antioxidant activity, and negatively regulates primary ciliogenesis and Hh signaling.		Bardoxolone (promote NFR2 activation)		ADPKD, PKD patents	[156–159]
Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-KB)	NF-KB is a pro-inflammatory cytokine		Triptolide (NF-KB inhibitor)	Pkd1 mouse (ADPKD)	N/A	[160]
Cellular Metabolism
Mammalian target of Rapamycin (mTOR) complex 1	mTOR pathway is a central regulator of cellular metabolism, protein translation, growth and cell cycle regulation, integrating other pathways including IGF and AMPK.	mTOR pathway is aberrantly activated in renal ciliopathies, stimulating uncontrolled cell growth and proliferation, driving cystogenesis.	Rapamycin/Sirolimus (mTORC1 inhibitor)	Orpk mouse (ADPKD), Bpk mouse (ARPKD), Pkd1 mouse (ADPKD) Pkd2 (ADPKD), pcy mouse (NPHP), Han:SPRD rat (NPHP), NPHP zebrafish models, Lpk rat (NPHP)	ADPKD	[27,28,161–170]
			TORKi (mTORC1 inhibitor)	Pkd1 mouse (ADPKD)	N/A	[171]
			Everolimus	Han:SPRD rat (NPHP)	ADPKD	[172,173]
c-Src	Epidermal growth factor (EGF) signaling activates RAF/MEK pathways to drive cellular proliferation and cystogenesis. C-Src phosphorylates and activates the EGF receptor.	EGF is upregulated in PKD/	Tesevatinib (multikinease inhibitor of c-Src)	Bpk mice (ARPKD)	ADPKD, ARPKD	[132,174,175]
			Bosutinib (multikinease inhibitor of c-Src)	Bpk mice (ARPKD), PCK rat (ARPKD), Pkd1 mouse (ADPKD)	ADPKD	[176–178]
			EGF inhibitors	Bpk mice (ARPKD), Han:SPRD rat (NPHP)	N/A	[174,179]
			Sorafenib (multi-kinase inhibitor of c-Src)	Human ADPKD cells	N/A	[180]
MAPK/ERK	MAPK/ERK pathways stimulate increased cellular proliferation and promote cystogenesis.		PD184352 (MAPK/ERK inhibitor)	pcy mice (NPHP), and inv mice (NPHP)	N/A	[181,182]
Somatostatin	Somatostatin is a growth factor negative regulator, and inhibitor of insulin and glucagon secretion, whilst lowering cAMP.		Lanreotide (somatostatin analogue)		ADPKD	[183–186]
			Octreotide (somatostatin analogue)	PCK rat (ARPKD) Nphp 2, 6, 8 KO spheroids	ADPKD	[183,187–190]
			Pasireotide (somatostatin analogue)	PCK rat (ARPKD), Pkd2 mouse (ADPKD)	ADPKD	[187,191]
AMP-activated protein kinase (AMPK)	AMPK signaling senses low ATP and mitochondrial function. It is negatively regulated by CFTR and mTOR.	PKD have impaired mitochondrial function and are prone to ER stress and increased ROS.	Metformin (AMPK activator)	Pkd1 (ADPKD), Zebrafish Pkd2 (ADPKD)	ADPKD	[149,192–196]
			Salsalate (AMPK inhibitor)	Pkd1 mouse (ADPKD)	N/A	[197]
Aerobic glycolysis	Conversion of glucose into pyruvate.	PKD models show shift in energy production from oxidative phosphorpylation to glycolysis.	2-deoxyglucose (glycolysis inhibitor)	Pkd1 mouse	N/A	[198]
Calmodulin-dependent protein kinase II (CaMKII)	Transducer of Ca2+ signaling, vital in mitochondrial function.	PKD have impaired mitochondrial function and are prone to ER stress and increased ROS.	CAMKII inhibitors	jck mice	N/A	[199]
Prostaglandin receptors	Prostaglandin signaling increases cAMP.	NPHP models sometimes show reduction in cAMP signaling.	PGE2 inhibitors	Nphp1 mice	N/A	[200]
Angiogenesis
VEGF Receptors	VEGF signaling promotes angiogenesis.	Increased angiogenesis is thought to promote cystogenesis.	VEGFR inhibitors	Han:SPRD rat (NPHP)	N/A	[201]
Ciliary signalling
Hedgehog (Hh)	Hh signaling is required for renal development, renal cell proliferation and has roles in fibrosis.	Hh signaling is dysregulated in renal ciliopathies.	Purmorphamine (Hh agonist)	Cep290 mouse model and human cells (NPHP)	N/A	[116]
			Gantb1 (Gli inhibitor)	Pkd1, jck, Ttc21b/Ift39 mouse (ADPKD, NPHP)	N/A	[202]
			Sant2 (Smo inhibitor)	Pkd1, jck, Ttc21b/Ift39 mouse (ADPKD, NPHP)	N/A	[202]
Cilium disassembly
Histone deacetylase 6 (HDAC6)	HDAC6 mediates cilium disassembly and is associated with heat shock protein 90 (HSP90).	In preclinical ADPKD models, HDAC6 and HSP90 are upregulated.	ACY-1215 (HDAC6 inhibitor)	PC1 (cells), PC1 (mouse)	N/A	[203]
			Tubacin (HDAC6 inhibitor)	MDCK cells, Pkd1 mouse (ADPKD)	N/A	[204]
			Trichostatin A (HDAC6 inhibitor)	Pkd2 zebrafish (ADPKD), Pkd2 mouse	N/A	[205,206]
			Valproic acid (VPA) (HDAC6 inhibitor)	Pkd2 zebrafish (ADPKD), Pkd1;Pkhd1 mouse (PKD)	N/A	[205]
			Nicotinamide (HDAC6 inhibitor)	Pkd1 mouse	ADPKD [175]	[175,207]
Heat shock protein 90 (HSP90)		HSP90 is increased in ADPKD models of disease	Ganetespib	Pkd1 mouse and Pkd2 mouse	N/A	[208]
			STA-2842	Pkd1 mouse (ADPKD)	N/A	[209]
Cell cycle regulation/DNA damage response
Cyclin dependent kinases (CDK)	CDK regulate the cell cycle and is used in DNA damage response (DDR) to arrest the cell cycle to remove damaged cells or allow for repair.	Cystogenesis is often driven by hyperproliferation of epithelial cells lining the cysts.	Roscovitine (CDK 1,2,5,7,9 inhibitor)	Pkd1 mouse (ADPKD), jck mouse (NPHP), Cpk mouse (ARPKD), Cep164 mouse (NPHP)	N/A	[30,210–213]
			CDK inhibitors	Cep290 mice and human cells	N/A	[214]
Cell division cycle 25 A (Cdc25A)	Cdc25A is involved in cell cycle regulation.	Cdc25A overexpressed in PKD mouse models.	Menadione (vitamin K3) (Cdc25A inhibitor)	Pkhd1 (ARPKD)	N/A	[215]
Genetic Therapies
MicroRNAs 17	Short-coding miRNA17 decreases PC1 and PC2 expression and affects mitochondrial function.	MicroRNA17s are upregulated in ADPKD murine models.	miR-17 inhibitors	Pkd1 (ADPKD)		[216]
			RGLS4326	Pkd2 (ADPKD)	ADPKD	[217,218]
The causative genetic mutation, targeted by ASOs.		The aim is to correct the causative genetic defect or modifier alleles, by binding to target mRNA to alter mRNA expression or splicing.	Cep290 ASOs	Cep290 mouse and human cells (NPHP)	N/A	[219]
			BSND ASOs	Cep290 mouse (NPHP)	N/A	[220]

5.1. Targeting transmembrane channels

ADPKD is currently the only renal ciliopathy where an approved treatment option called tolvaptan is available to patients which slows kidney disease progression. The arginine vasopressin receptor 2 (AV2R) is a G-protein coupled receptor (GPCR) that localizes to the basolateral membrane of renal tubular cells and the ciliary membrane, and is upregulated in ADPKD patients. AV2R upregulation causes increased cAMP and PKA activation, which in turn leads to aquaporin 2 (AQP2) mediated and chloride channel dependent fluid secretion, promoting cystogenesis [221,222]. Tolvaptan, is an arginine vasopressin type receptor (AVPR2) antagonist and has been used with success in selected ADPKD patients with progressive forms of the disease [45,131]. As tolvaptan is a very expensive drug, and the associated side effects of polyuria are significant, its use is restricted to only selected patients with rapidly progressive disease, as evidenced by declining kidney function and large kidney volumes. Reports of acute liver toxicity within clinical trials mean regular monitoring is necessary [131,223,224]. A newer AV2R antagonist lixivaptan has been shown promise in in vitro preclinical trials, and shown to have less hepatotoxic effects in rat models, although in humans this drug was terminated early by the sponsor, due to reassessment of commercial potential of the drug (ClinicalTrials.gov, NCT04064346) [136,225,226].

Although there are currently no approved treatment options for ARPKD, there are 2 clinical studies currently underway using tolvaptan in the treatment of ARPKD. The first study is a phase 3b multicenter, open label, non-randomized trial to evaluate the safety, tolerability, and efficacy of tolvaptan in infants and children 28 days to less than 18 years of age with ARPKD. The participants receiving the tolvaptan treatment will be evaluated for 18 months over the course of the trial (ClinicalTrials.gov, NCT04782258) [132]. The second study is a phase 3 clinical trial currently recruiting patients from different locations across the globe. It will evaluate the effect of tolvaptan on the need for renal replacement therapy in pediatric patients 28 days to less than 12 weeks of age with ARPKD (ClinicalTrials.gov, NCT04786574) [132].

In ADPKD, transepithelial chloride secretion through cystic fibrosis transmembrane conductance regulator (CFTR) and a non -CFTR activated chlorine channel, Transmembrane protein 16A (TMEM16A) are known to drive the cyst enlargement [140]. These channels contribute to increased chloride transport into the lumen of the renal tubular cyst, which drives fluid secretion, promoting cyst growth [227,228]. In vivo studies have shown that small molecular CFTR modulator drugs can slow the cystic kidney phenotype and preserve kidney function in PKD1 murine models of ADPKD [138]. Interestingly, repurposing of the currently approved cystic fibrosis drug VX-809 (lumacaftor), which acts as a CFTR misfolding corrector and potentiator, cause reduced cyst growth in PKD1 mouse models, alongside decreased intracellular calcium, cell proliferation and endoplasmic reticulum (ER) stress markers [139]. The beneficial actions of lumacaftor and its analogues are due to the translocalization of CFTR to the basolateral membrane along with stimulation of apical sodium reabsorption via the epithelial sodium channel to reduce cyst growth [229]. There are other approved CFTR corrector drug treatments including ivacaftor, tezacaftor, and elexacaftor. It would therefore be interesting to see whether these also have an effect on renal ciliopathy models; however, none of these are currently in clinical trials for PKD. Also, Tmem16a knockdown in mouse models of ADPKD using the FDA approved drugs niclosamide and benzbromarone, and also by using ani9, a specific small molecule inhibitor of Tmem16a [140,230,231] have shown to strongly suppress the cystic phenotype in vivo, this is yet to make it to clinical trials. Additionally, peroxisome proliferator-activator receptor gamma (PPARγ) inhibits the synthesis of CFTR protein and inhibits ERK signaling, which can be exploited by agonists pioglitazone and rosiglitazone to ameliorate cystic kidney disease as shown in vitro and in vivo using PCK rat, Han:SPRD rat, and PKD1 mouse models [141–145,232]. However, high doses of rosiglitazone led to adverse effects including cardiac enlargement [145]. Interestingly, pioglitazone in combination with tolvaptan showed improved renal survival and slowed cystic kidney disease progression in an adult-onset PKD mouse model [146]. A recently concluded phase 2 clinical study showed using a low-dose pioglitazone slowed down the kidney cyst progression in humans with ADPKD (ClinicalTrials.gov, NCT02697617) [146]. PPARγ agonists could also be explored for ARPKD and NPHP diseases.

5.2. Targeting lipid metabolism

More recently, statins have been explored as treatments of renal ciliopathies. Statins are hydroxy-3-methylglutarylCoA reductase inhibitors which modulate lipids, such as cholesterol, and effect AMPK activation [233]. Cholesterol is also enriched in the ciliary membrane and has important roles in ciliary membrane signaling. Statins, such as lovastatin, have shown to reduce kidney weight and cyst volume in models of NPHP [147]. Simvastatin has also showed promising results of ameliorating kidney function in phase 1 clinical trials in ADPKD patients, but there were disadvantages to the study, with too small patient numbers and a very short follow up time. Hence a bigger patient cohort with longer follow up duration is required to assess the effects of Simvastatin [148]. Pravastatin is in phase 4 clinical trial for ADPKD designed to assess the efficacy and benefits of statin therapy in adults with ADPKD recruiting patients from 25 to 60 years of age. This clinical study evaluates parameters such as kidney volume and function, renal blood flow, plasma and urine protein markers and blood vessel stiffness at baseline and 2 years after pravastatin treatment in 150 patients with ADPKD (ClinicalTrials.gov, NCT03273413) [149]. As statins have shown to improve renal function in the rat models of PKD, it would be interesting to see if statins have a beneficial effect on NPHP in humans [23]. Glycosphingolipids also accumulate in PKD models which disrupts ciliary signaling. Treatments with glucosylceramide inhibitors in ADPKD and NPHP murine models have shown reduced cystogenesis [150,234]. Venglustat, a glucosylceramide synthase inhibitor is currently being used in clinical trials for ADPKD (ClinicalTrials.gov, NCT03523728) [151].

5.3. Targeting inflammation

Inflammatory cells, such as macrophages, drives the cyst epithelial cell proliferation, growth, and kidney fibrosis in the early stages of ADPKD and ARPKD [235]. Therefore, preventing inflammation could be another approach in treatment of PKD [236]. Literature evidence shows that cyclooxygenase 2 (COX2), a prostaglandin metabolizer, is upregulated in cystic kidney diseases, including PKD and NPHP disease models, and hence could be considered a potential target for therapy [152,237,238]. Celecoxib, a specific (COX2) inhibitor ameliorated the ADPKD disease progression and improved renal function in mouse model of ADPKD [153], which has been replicated for other COX inhibitors in the Han:SPRD Cy rat model [152,154]. Diets enriched in flax-oil have also demonstrated reduced COX-derived oxylipin levels, with reduced kidney cyst progression and kidney fibrosis in Nphp3 mice models [155]. Potentially treatment with COX inhibitors, or dietary supplementation may be useful for reducing inflammation in renal ciliopathy diseases. Another possible inflammatory target is the transcription factor nuclear factor erythroid 2-like 2 (NFR2), which increases the presence of antioxidants and detoxification proteins. Recent studies also indicated that NRF2 negatively regulated the primary ciliogenesis process and hedgehog signaling (Hh) [239,240]. Bardoxolone, a semisynthetic derivative of triterpenoids exerts antioxidant activity by promoting the activation of Nrf2 and the downregulation of the proinflammatory nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) signaling, and is considered a potential therapeutic option for patients with ADPKD [156–158]. Bardoxolone methyl oral capsules used for treatment of patients with ADPKD is currently in phase 3 clinical trial to study the safety, tolerability and efficacy of the compound (ClinicalTrials.gov, NCT03918447) [159]. Triptolide, an NF-kB inhibitor with anti-proliferative and pro-apoptotic properties was used in ADPKD mouse models to reduce cystic burden; however, there were adverse effects including infertility and immunosuppression [160]. Inflammation could also be a driver of NPHP kidney phenotypes. NPHP1 regulates CCL2 chemokine expression alongside LKB1, with LKB1 loss causing increased CCL2 levels and subsequent macrophage activation, and thus CKD disease progression in vivo [241]. Modulation of inflammatory pathways should be explored as a therapeutic option for NPHP.

5.4. Targeting cellular metabolism

One pathway thought to have a core role in renal ciliopathies is the mTOR pathway, which acts as a central regulator of cellular metabolism, protein translation, growth, cell cycle regulation and cell survival, integrating effectors from other pathways including IGF and AMPK signaling [161]. Multiple in vitro and in vivo studies have demonstrated that the mTOR pathway is aberrantly activated in renal ciliopathy models and human cells, especially in the cystic renal epithelial cells, which stimulates uncontrolled cell growth and proliferation, driving cystogenesis [26,161]. It is thought that in a ‘normal’ environment, mTOR receptor 1 (mTORC1) is negatively regulated by ciliary flow sensing, with aberrant flow sensing leading to mTOR activation, which further downregulates PC1 [26,242,243]. Rapamycin (alias sirolimus) and everolimus are mTOR complex 1 inhibitors. Rapamycin has shown to decrease cystogenesis and cell proliferation in models of renal ciliopathies, including NPHP zebrafish knockdown models and ADPKD, ARPKD, and NPHP murine models [28,161–167,172,244]. In murine models of ADPKD both rapamycin and mTOR kinase inhibitor (TORKi) caused a similar decreased cystic kidney burden and improved kidney function, associated with decrease mTOR and cell proliferation [171]. In Nphp3 mouse models, rapamycin did not affect initial kidney cyst development, but reduced kidney cyst size and kidney fibrosis, indicating that it might be useful in later stages of NPHP [27]. mTOR inhibitors have been studied in human PKD patients. ADPKD patients that have received a renal transplant, but retain the cystic kidney, are often given rapamycin as an immunosuppressant, to prevent transplant rejection. It was shown that rapamycin treatment showed a trend toward reduced kidney volumes of the remaining polycystic kidney compared to patients on other immunosuppressant treatments [161,168]. However, other clinical trials have not shown as much promise as disease models. Treatment of ADPKD patients with everolimus slowed total increase in kidney volume but not overall renal failure (ClinicalTrials.gov, NCT00414440), and treatment of ADPKD patients with sirolimus did not have an ameliorative effect (ClinicalTrials.gov, NCT00346918) [169,173]. A meta-analysis of 9 clinical trials from 2009–2016 of mTOR inhibitor treatment in ADPKD patients demonstrated no significant renal benefit with an increased risk of associated adverse effects [245]. Use of folate-conjugated forms of rapamycin have been tested in preclinical models, to try and overcome the lack of efficiency in human studies [170]. However, this has not been explored in human studies.

AMPK which localizes to the basal body of cilia functions as a sensor of low ATP levels and subsequently mitochondrion function, and is negatively regulated by CFTR and mTOR [246]. Polycystic disease models have also shown impaired mitochondrial function and are more prone to ER stress and reactive oxygen species due to a shift in energy production from oxidative phosphorylation to glycolysis through inhibition of AMPK [247–249]. Activation of AMPK by metformin reduced cyst growth in several models of disease, and investigated in clinical trials; however, there were concerns that the therapeutic AMPK activation in human kidneys might require a higher oral dose [149,192–196]. Hence, to overcome the non-sufficient efficacy of metformin alone, another drug salsalate, a pro drug dimer of acetyl salicylic acid was identified [197,250]. Salsalate was highly effective in attenuating the renal cysts in the PKD1 mouse models. It had a strong inhibitory effect on NF-κB, activated AMPK, and also down regulated cAMP, and stimulated mitochondrial biogenesis and function [197]. With an excellent oral bioavailability and safety profile in humans, salsalate which has long been approved for use in treatment of arthritis is yet to be tested in clinical trials for ADPKD. Preclinical studies with AMPK agonists may also be useful for ARPKD and NPHP. Mitochondrial dysfunction itself could also be a potential target for treatment of cystic kidney disease. In vivo studies have shown that 2-deoxyglucose can inhibit aerobic glycolysis and subsequently reduce cystic epithelial proliferation, slowing cyst expansion and reducing kidney volume [198]. Inhibition of Ca2+/Calmodulin-dependent protein kinase II (CaMKII) has also been shown to improve ER and oxidative stress, as well as mitochondrial function in jck mice [199].

Setmelanotide, a hypothalamic melanocortin 4 receptor (MC4R) agonist showed potential in a phase 2 clinical study in decreasing the hunger and weight related issues in patients with Bardet-Biedl Syndrome (BBS). A phase 3 clinical trial is currently underway to test the long-term safety and efficacy of Setmelanotide for the treatment of obesity and hyperphagia in individuals with BBS (CliniclaTrials.gov, NCT03746522) [251].

EGF signaling is upregulated in polycystic kidney disease, with EGF receptors (EGFR) overexpressed and mislocalized to the apical surface of renal epithelial cells [252]. EGF activates ERK and RAF/MEK pathways, which drives cystic kidney disease. Tesevatinib, a multikinase inhibitor functions as an inhibitor of c-Src which attenuates the EGFR axis and cAMP pathways, as well as being a potent inhibitor of KDR which is a key mediator of cyst growth [174]. Moreover, Tesevatinib ameliorated the renal cyst progression rodent models of ARPKD and significantly reduced the phosphorylation levels of c-Src, EGFR and ERK1/2 which led to a reduction of MAPK activity and the resultant cellular proliferation. Tesevatinib, is already in phase 2 clinical trial for ADPKD (ClinicalTrials.gov, NCT01559363). It is also currently in phase 1 clinical trial for ARPKD recruiting children aged 5–12 years whose kidney function is at least 50% normal (ClinicalTrials.gov, NCT03096080). Tesevatinib has been selected on the basis that both ADPKD and ARPKD patients share similar pathophysiological features and clinical manifestations in new-borns, children, and young adults. Another tyrosine kinase inhibitor, Bosutinib, has been utilized in clinical trials in ADPKD patients, showing some success in reducing kidney growth but glomerular filtration rate still declined, and gastrointestinal and liver toxicity was observed in patients recruited [176]. Preclinical trials have shown the EGFR inhibitors reduced progression of renal cysts, cyst size and improved renal function in Bpk mouse model and Han:SPRD rat model of NPHP, but not in ADPKD PCK rat model [174,179]. Treatment was associated with increased cAMP, which might actually be beneficial in NPHP diseases, but might explain poor results with ADPKD models. Sorafenib is another multikinase inhibitor which can suppress cystogenesis in vitro in models of ADPKD; although it has not been tested in clinical trials for PKD [180]. MAPK/ERK pathways can also be inhibited by PD184352, which has prevented cyst growth and protect kidney functions in pcy mice and Inv mice, which are models NPHP [181,182].

Somatostatin is a growth hormone inhibitor essential for regulation of growth factors, and inhibition of insulin and glucagon secretion, whilst lowering cAMP availability. Somatostatin G-coupled receptors are expressed in renal tubule cells and cholangiocytes [253–257]. Somatostatin analogs such as lanreotide, octreotide, and pasireotide act in a similar pharmacokinetic action to somatostatin, and have been shown to slow cell proliferation in PKD models [187,258,259]. In vitro spheroid models of NPHP disease treated with octreotide rescued ciliary defects and abnormal spheroid formation, they were also associated with a decrease in cAMP [260]. Treatment of ADPKD patients with octreotide show some potential toward slowing disease progression, particularly in late-stage ADPKD; however, studies have been limited by small cohort size and patients show some adverse gastrointestinal effects [183,188] (ClinicalTrials.gov, NCT01377246, NCT00309283). Treatment with pasireotide in patients with severe polycystic liver disease, associated with polycystic kidney disease, slowed progression of liver and kidney growth, but did not improve glomerular filtration rate (GFR) decline and participants experienced increased frequency of adverse effects including diabetes and hyperglycemia (ClinicalTrials.gov, NCT01670110) [191]. However, there is conflicting evidence in the effectiveness in using lanreotide treatment to slow declining kidney function and kidney enlargement in ADPKD (ClinicalTrials.gov, NCT01354405, NCT01616927) [183–186]. Recently, a pilot clinical trial with tolvaptan and octreotide was shown to reduce GFR more effectively than octreotide alone, and significantly reduced cystic kidney volume and ameliorate the aquaretic effect of tolvaptan (ClinicalTrials.gov, NCT03541447) [189].

Recently, prostaglandin receptor 2 (PGE2) agonists rescued the NPHP1-associated ciliary and kidney phenotype in vitro and in vivo, mediated by increased downstream cAMP signaling which is often reduced in NPHP ciliopathy models, and decreased fibrosis [200]. This is an exciting potential therapeutic target for NPHP.

5.5. Targeting angiogenesis

Increased angiogenesis in early disease progression of ADPKD, with increased expression of vascular endothelial growth factor receptor (VEGFR) in endothelial cells is thought to drive cystogenesis [261]. VEGFR mRNA inhibition in vivo models of NPHP led to reduced tubule cell proliferation and subsequently lower cystogenesis and recovered renal function [201].

5.6. Targeting cilium disassembly

Histone deacetylase 6 (HDAC6) is an enzyme which mediates cilium disassembly, it is also associated with the heat shock protein 90 (HSP90) [262–264]. In PC1 deficient cells, HDAC6 and HSP90 are upregulated; inhibition of HDAC6 restores the ciliary phenotype [203]. Preclinical studies have shown that inhibition of HDAC6 in PKD models with ACY-1215 slows cyst growth and reduces cystic volume, associated with reduction in cAMP and CFTR activity. Other HDAC6 inhibitors including; Tubacin, Trichostatin A (TSA), Valproic acid (VPA) and Nicotinamide ameliorates cystic kidney disease, reduces cAMP expression and improve kidney function in ADPKD preclinical studies [204–207,265]. There have been small pilot studies of nicotinamide treatment in ADPKD patients, which did not show any difference between drug and placebo (ClinicalTrials.gov, NCT02558595). Another potential target is HSP90; inhibition of HSP90 with ganetespib or STA-2842 in ADPKD mouse models also slowed cyst growth, alongside reduced ciliation [208,209].

5.7. Targeting ciliary signaling

The primary cilium is a vital transducer of Hh signaling, which is required for proper kidney development and renal epithelial cell proliferation [266,267]. Hh signaling is also involved in proliferation of myofibroblasts which secrete ECM components, increasing fibrosis [19]. Hh signaling is dysregulated in numerous ciliopathies [268,269], with lots of evidence pointing toward dysregulation in NPHP [17,18,116]. CEP290 mutated spheroids show decreased Hh signaling; treatment with Purmorphamine, a Hh agonist, restores 3D spheroid structure [116]. However, Pkd1, Nphp9 and Ttc21b/Ift39 murine models have increased Hh signaling, and inhibition of GLI and SMO with small molecular antagonists Gantb1 and Sant2 respectively, prevented renal cyst formation [202]. It is likely that the direction of Hh dysregulation will be dependent on the genetic cause of disease, which will change the type of treatment required. However, as Hh signaling is a core developmental and homeostatic pathway, any manipulation of Hh signaling is thought to be likely to lead to the risk of serious side effects [24].

There are other ciliary-mediated signaling pathways that are dysregulated in renal ciliopathies some including cAMP, calcium-sensing receptor activation, canonical Wnt and Hippo [21,270–285]. Direct modulation of these pathways in preclinical studies have shown promise at ameliorating the disease phenotype, but they have not yet progressed to clinical trials [274,277,278,284,286,287].

5.8. Targeting cell cycle regulation/DNA damage response

A major phenotype driving cystogenesis in renal ciliopathies is hyperproliferation of epithelial cells lining the cysts. Cyclin dependent kinases (CDK) regulate the cell cycle. Preclinical studies with murine models of ADPKD, ARPKD and NPHP, have used treatment with roscovitine, a reversible inhibitor of CDK1,2,5,7 and 9, to attenuate hyperproliferation by inducing G1 AND G2-M cell cycle arrest. This causes slower kidney functional decline and cystogenesis [30,210–213,288]. Other CDK inhibitors have rescued ciliation in Cep290 KO cells [214]. CDK inhibitors are currently being used in clinical trials for numerous cancer treatments, and other diseases such as cystic fibrosis, inflammatory bowel disease and ulcerative colitis, but have not been used in renal ciliopathy trials. Cell division cycle 25 A (Cdc25A) is overexpressed in PKD animal models; inhibition of Cdc25A with menadione (vitamin K3) slows cystogenesis in rodent models of PKD [215].

DNA damage repair (DDR) pathways detect DNA damage and activates either repair mechanisms, senescence, or apoptosis. This is vital for removal of severely damaged cells and regulation of tissue repair. Multiple ciliary proteins have been implicated in DDR including CEP290, CEP164, ZNF243, NEK8, SDCCAG8 and MAPKBP1 knockout [81,88,214,288,289]. Elevated DNA damage and replicative stress are found in models of renal ciliopathies. It is hypothesized that aberrant DDR leads to leads to increased DNA damage and impaired cell cycle regulation, driving increased proliferation and misregulated apoptosis [81,237]. Specifically, Cep290 murine models show primary mouse kidney cells with increased double-strand breaks, supernumerary centrioles and replication fork asymmetry, indicative of replicative stress; CDK inhibitors can rescue these phenotypes in vitro [214]. Cep164 KD in HeLa and IMCD3 cells is also thought to cause increased DNA damage, genomic instability and replicative stress, although results have been conflicting with some studies showing no different in the cell cycle, checkpoint activation or DNA damage [30,31,81,290–292]. It is possible that ciliopathy proteins effect DDR in a cell specific manner. Abnormal DDR also contributes to increased fibrosis, which is a characteristic of NPHP. Cep164 KD in IMCD3 cells induces DNA damage, with s-phase block and anaphase lag, as well as increased epithelial-mesenchymal transition [81]. More research is needed to understand the role of ciliopathy proteins in DDR, and how this may be exploited for therapeutic purposes.

5.9. Utilizing gene therapy

Aberrant activation of microRNAs, short non-coding RNAs, has been shown to promote the progression of ADPKD [216,293]. MicroRNA17 (miR-17) induces kidney cysts in PKD murine models via decreased PC1 and PC2 expression, which also has effects on mitochondrial function to promote cyst growth [294,295]. miR-17 is upregulated in ADPKD murine models, deletion of which reduces cyst growth, fibrosis, and inflammation, and improves mitochondrial function in a PKD mouse model [216]. In a recent study Lee et al. reported that RGLS4326, a short oligonucleotide inhibitor of miR-17 significantly attenuated the cyst growth in vitro in cell culture models, and also in vivo in the PKD mouse models by upregulating Pkd1 and Pkd2 expression [217]. A phase 1 clinical study of RGLS4326 in patients with ADPKD is currently under way (ClinicalTrials.gov, NCT04536688) [218]. Other miRNAs are known to be involved in kidney cyst growth regulation, including miR-21, miR-193 and miR-214. The role of miRNA in ARPKD and NPHP is yet to be explored.

Another potential personalized therapeutic technique is to ‘correct’ the causative genetic defect. Antisense oligonucleotides (ASOs) can be used to bind to target mRNA to alter mRNA expression or splicing; ASOs have been shown to distribute to the kidney tubule [24,41]. ASOs have been utilized in models of Cep290 ciliopathies to skip over the effected in-frame exon, subsequently rescuing the ciliary and renal cystogenesis phenotype [219]. This has also been completed for murine models of Alport nephropathy [296,297]. ASO therapies are currently showing promise in clinical trials for retinal ciliopathies, although they have not been utilized in clinical trials for renal ciliopathies [298–301]. ASOs have also been utilized to regulate genetic modifiers of renal ciliopathies, such as BSND to slow the cystogenesis phenotype models of Cep290 ciliopathy disease [220]. Modifier alleles are likely contribute to the heterogeneity of renal ciliopathies, and could offer a personalized treatment approach.

6. Expert opinion

Currently, for renal ciliopathies, the only approved treatment for slowing disease progression is tolvaptan for ADPKD, utilizing a very specific target, the AVPR2, expressed in the renal collecting duct. All other forms of treatment at present are merely supportive, aimed at treatment of anemia, hypertension, growth retardation, and other symptoms related to CKD. Currently kidney transplantation is the only potential long-term cure [302]. There are, however, promising clinical trials for both adults and children with ADPKD and ARPKD including tolvaptan, pioglitazone, pravastatin, simvastatin, RGLS4326, bardoxolone methyl, metformin, lanreotide, octreotide, pasireotide, tesevatinib, and bosutinib. These drugs are clearly targeting many different aspects of the disease pathogenesis including cyst expansion and cell proliferation. The discovery of tolvaptan as an effective agent for ADPKD by using preclinical models including the pcy mouse (which has a Nphp3 mutation) gives hope that disease mechanisms and therefore therapeutic interventions may be shared across different ciliopathies, and from dominant to recessive forms of cystic kidney disease and nephronophthisis.

A common final pathway associated with many forms of CKD is renal fibrosis. Therefore, there remains the possibility of using drugs directed at this disease course, rather than a ciliopathy specific pathway/mechanism for the treatment of NPHP and other related renal ciliopathies. Recently, treatment of CKD related to diabetes has been revolutionized by two classes of drugs: the sodium-glucose transporter-2 (SGLT2) inhibitors [303–305] and mineralocorticoid receptor antagonists (MRA) [306].

SGLT2i drugs now have a proven track record in CKD patients with and without diabetes in reducing glomerular hypertension mediated through tubuloglomerular feedback independent of their effect on glycemic control. They remain the most exciting therapeutic advance in the treatment of kidney disease and as nephroprotective agents since the discovery of angiotensin converting enzyme (ACE) inhibitors. They remain, however, untested in ADPKD patients as such patients were excluded from most clinical trials exploring kidney protection [307]. Intriguingly, SGLT2i may have anti-inflammatory role as well as impacting autophagy and improving mitochondrial dysfunction [308,309]. We propose that establishing the potential benefit of SGLT2i in patients with ADPKD is a research priority.

MRA drugs such as spironolactone have been available as antihypertensive agents for many years. However, recently attention has been focused on finerenone. This is relevant as finerenone has been studied for its potential to reduce fibrosis, particularly in the context of CKD. As a selective MRA, finerenone works by blocking the activity of the mineralocorticoid receptor, which is involved in regulating salt and water balance as well as promoting inflammation and fibrosis. By inhibiting this receptor, finerenone may reduce the harmful effects associated with excessive fibrosis in CKD. Clinical studies, such as the FIDELIO-DKD trials, have evaluated the effects of finerenone on kidney outcomes in patients with CKD and type 2 diabetes [310]. This trial showed that finerenone treatment reduced the risk of kidney failure, including the need for dialysis or transplantation, in these patients. Additionally, it demonstrated a decrease in the progression of albuminuria and a reduction in cardiovascular events. While the precise mechanisms through which finerenone reduces fibrosis are not yet fully understood, it is believed that by targeting the mineralocorticoid receptor, the drug can mitigate the inflammation and fibrotic processes in the kidneys, potentially slowing down the progression of fibrosis and preserving kidney function. It therefore becomes a very attractive drug to slow down the tubulointerstitial fibrosis associated with renal ciliopathies, independent of the ciliary defect. Further work in relevant pre-clinical models is required as well as designing future clinical trials for testing this drug in relevant phenotypic groups. The use of biomarkers for renal fibrosis may aid the assessment of these agents [130].

We have already discussed briefly the use of anti-miR-17 oligonucleotides for the treatment of PKD in preclinical studies. Again, following the theme of agents to prevent renal fibrosis, anti-miR-21 oligonucleotide therapies have been applied to animal models of renal fibrosis, specifically the Col4a3-/- Alport syndrome mice [311]. Use of anti-miR-21 drugs provided enough evidence for a clinical trial in Alport syndrome patients (called HERA) to assess the efficacy of lademirsen (an anti miR-21 agent) in reducing the decline in renal function [312]. This clinical study was unfortunately terminated early, after 24 weeks, as it did not lead to a meaningful improvement in kidney function when compared with placebo. Targeting miR-21 may still represent a promising therapy for other kidney diseases, aside from Alport syndrome, but pharmaceutical companies may be wary of this agent given the disappointment of the HERA trial. Targeting kidney fibrosis remains a promising approach [313]. But, any future drug targeting fibrosis of the kidneys will now need to demonstrate added benefit to a standard of care that combines renin-angiotensin system with MRA or SGLT2 inhibitors.

Notably, there are currently no clinical trials underway for NPHP using small molecules, such as rapamycin in spite of the in vitro and in vivo evidence. This could be explained by the fact that there is severe phenotypic and genotypic heterogeneity in NPHP patients and relatively shorter therapeutic window compared to ADPKD [23]. There is a need, however, to define better disease progression in this group of patients so that clinical trial ready cohorts can be identified. For NPHP, targeting cilia-related signaling pathways seems to be the most promising approach. Manipulating ciliary signaling and downstream pathways may represent druggable targets for the treatment of renal ciliopathies.

Overall, the development of new treatments for renal ciliopathies is an active area of research, and advances in gene therapy, stem cell therapy, and drug development hold promise for improving outcomes for patients with these disorders. There is a recognized need for the development of longitudinal cohorts of well-phenotyped and genotyped renal ciliopathy patients with enriched clinical and genomic datasets to underpin future clinical trials of precision therapies and personalized medicine approaches. This will allow cohorts of patients to be well characterized and to be “trial-ready” for novel therapies and other disease interventions. As an example, in the UK, longitudinal data is already captured by the well-established UK Renal Registry and National Registry of Rare Kidney Diseases (RaDaR) platform. RaDaR currently has 242 participants registered with NPHP or ARPKD and 8006 with ADPKD, constituting among the largest cohorts in these disorders reported anywhere in the world. We also believe that by the renal ciliopathy research community interacting with well-organized patient networks this will empower patients, utilize patient and public involvement into design of clinical trials (industry), and allow patient focused research to become truly translational.

7. Conclusions

In conclusion, renal ciliopathies pose a significant challenge to healthcare professionals, as current treatments are largely supportive, and a long-term cure is only possible through kidney transplantation. However, recent clinical trials have shown promising results for drugs such as tolvaptan, pioglitazone, and bardoxolone methyl and clinical indications for use of these agents will hopefully broaden to pediatric disease settings for ADPKD and ARPKD. Although there are no ongoing trials for NPHP, there is recognition that patient cohorts need to be identified and characterized so that they will be trial ready. Across the spectrum of renal ciliopathies, preclinical models have provided insight into the understanding the function of PKD- and NPHP-related proteins, highlighting their ciliary and extraciliary functions. Dissecting further their role in cellular biology is a prerequisite to developing novel specific therapies for these complex disorders.

Article highlights

• Renal ciliopathies include a range of inherited kidney diseases, including polycystic kidney disease and nephronophthisis, which lead to progressive chronic kidney disease and kidney failure.

• There is a need for improved treatments for all renal ciliopathies.

• Targeting kidney cyst growth has proved to be useful in autosomal dominant polycystic kidney disease in adults.

• New treatments for childhood onset diseases including autosomal recessive polycystic kidney disease and nephronophthisis are urgently needed as there are no current therapies.

• Pre-clinical models of renal ciliopathies provide a diverse set of disease mechanisms to target and these insights need to be urgently translated into clinical care.

• Harmonizing diagnostic, investigative, and management approaches for renal ciliopathy patients will allow patient cohorts to become clinical trial ready and allow new therapeutic agents to enter clinical practice.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants, or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

This manuscript was funded by Kidney Research UK (support to LAD) (Paed_RP_001_20180925) and the Northern Counties Kidney Research Fund. PDS is supported by Kidney Research UK (RP_007_20210729). JAS is supported by the Northern Counties Kidney Research Fund (22/01), Kidney Research UK (Paed_RP_001_20180925), LifeArc, and the Medical Research Council.