Illuminating a Dark Kinase: Structure-Guided Design, Synthesis, and Evaluation of a Potent Nek1 Inhibitor and Its Effects on the Embryonic Zebrafish Pronephros.

NIMA-related kinase 1 (Nek1) has lately garnered attention for its widespread function in ciliogenesis, apoptosis, and the DNA-damage response. Despite its involvement in various diseases and its potential as a cancer drug target, no directed medicinal chemistry efforts toward inhibitors against this dark kinase are published. Here, we report the structure-guided design of a potent small-molecule Nek1 inhibitor, starting from a scaffold identified by kinase cross-screening analysis. Seven lead compounds were identified in silico and evaluated for their inhibitory activity. The top compound, 10f, was further profiled for efficacy, toxicity, and bioavailability in a zebrafish polycystic kidney disease model. Administration of 10f caused the expansion of fluorescence-labeled proximal convoluted tubules, supporting our hypothesis that Nek1-inhibition causes cystic kidneys in zebrafish embryos. Compound 10f displayed insignificant inhibition in 48 of 50 kinases in a selectivity test panel. The findings provide a powerful tool to further elucidate the function and pharmacology of this neglected kinase.

Chronic activation of AMP-activated protein kinase leads to early-onset polycystic kidney phenotype.

AMP-activated protein kinase (AMPK) plays a key role in the cellular response to low energy stress and has emerged as an attractive therapeutic target for tackling metabolic diseases. Whilst significant progress has been made regarding the physiological role of AMPK, its function in the kidney remains only partially understood. We use a mouse model expressing a constitutively active mutant of AMPK to investigate the effect of AMPK activation on kidney function in vivo. Kidney morphology and changes in gene and protein expression were monitored and serum and urine markers were measured to assess kidney function in vivo. Global AMPK activation resulted in an early-onset polycystic kidney phenotype, featuring collecting duct cysts and compromised renal function in adult mice. Mechanistically, the cystic kidneys had increased cAMP levels and ERK activation, increased hexokinase I (Hk I) expression, glycogen accumulation and altered expression of proteins associated with autophagy. Kidney tubule-specific activation of AMPK also resulted in a polycystic phenotype, demonstrating that renal tubular AMPK activation caused the cystogenesis. Importantly, human autosomal dominant polycystic kidney disease (ADPKD) kidney sections revealed similar protein localisation patterns to that observed in the murine cystic kidneys. Our findings show that early-onset chronic AMPK activation leads to a polycystic kidney phenotype, suggesting dysregulated AMPK signalling is a contributing factor in cystogenesis.

Conserved role of ATP synthase in mammalian cilia.

We previously found that ATP synthases localize to male-specific sensory cilia and control the ciliary response by regulating polycystin signalling in Caenorhabditis elegans. Herein, we discovered that the ciliary localization of ATP synthase is evolutionarily conserved in mammals. We showed that the ATP synthase subunit F1ß is colocalized with the cilia marker acetylated α-tubulin in both mammalian renal epithelial cells (MDCK) and normal mouse cholangiocytes (NMCs). Treatment with ATP synthase inhibitor oligomycin impaired ciliogenesis in MDCK cells, and F1ß was co-immunoprecipitated with PKD2 in mammalian cells. Our study provides evidence for the evolutionarily conserved localization of ATP synthase in cilia from worm to mammals. Defects in ATP synthase can lead to ciliary dysfunction, which may be a potential mechanism of polycystic kidney disease.

Discoidin Domain Receptor 1 (DDR1) tyrosine kinase is upregulated in PKD kidneys but does not play a role in the pathogenesis of polycystic kidney disease.

Tolvaptan is the only drug approved to slow cyst growth and preserve kidney function in patients with autosomal dominant polycystic kidney disease (ADPKD). However, its limited efficacy combined with significant side effects underscores the need to identify new and safe therapeutic drug targets to slow progression to end stage kidney disease. We identified Discoidin Domain Receptor 1 (DDR1) as receptor tyrosine kinase upregulated in vivo in 3 mouse models of ADPKD using a novel mass spectrometry approach to identify kinases upregulated in ADPKD. Previous studies demonstrating critical roles for DDR1 to cancer progression, its potential role in the pathogenesis of a variety of other kidney disease, along with the possibility that DDR1 could provide new insight into how extracellular matrix impacts cyst growth led us to study the role of DDR1 in ADPKD pathogenesis. However, genetic deletion of DDR1 using CRISPR/Cas9 failed to slow cyst growth or preserve kidney function in both a rapid and slow mouse model of ADPKD demonstrating that DDR1 does not play a role in PKD pathogenesis and is thus a not viable drug target. In spite of the negative results, our studies will be of interest to the nephrology community as it will prevent others from potentially conducting similar experiments on DDR1 and reinforces the potential of performing unbiased screens coupled with in vivo gene editing using CRISPR/Cas9 to rapidly identify and confirm new potential drug targets for ADPKD.

ß-catenin ablation exacerbates polycystic kidney disease progression.

Polycystic kidney disease (PKD) results from excessive renal epithelial cell proliferation, leading to the formation of large fluid filled cysts which impair renal function and frequently lead to renal failure. Hyperactivation of numerous signaling pathways is hypothesized to promote renal epithelial cell hyperproliferation including mTORC1, extracellular signal-regulated kinase (ERK) and WNT signaling. ß-catenin and its target genes are overexpressed in some PKD models and expression of activated ß-catenin induces cysts in mice; however, ß-catenin murine knockout studies indicate it may also inhibit cystogenesis. Therefore, it remains unclear whether ß-catenin is pro- or anti-cystogenic and whether its role is canonical WNT signaling-dependent. Here, we investigate whether ß-catenin deletion in a PKD model with hyperactived ß-catenin signaling affects disease progression to address whether increased ß-catenin drives PKD. We used renal epithelial cell specific Inpp5e-null PKD mice which we report exhibit increased ß-catenin and target gene expression in the cystic kidneys. Surprisingly, co-deletion of ß-catenin with Inpp5e in renal epithelial cells exacerbated polycystic kidney disease and renal failure compared to Inpp5e deletion alone, but did not normalize ß-catenin target gene expression. ß-catenin/Inpp5e double-knockout kidneys exhibited increased cyst initiation, cell proliferation and MEK/ERK signaling compared to Inpp5e-null, associated with increased fibrosis, which may collectively contribute to accelerated disease. Therefore, increased ß-catenin and WNT target gene expression are not necessarily cyst promoting. Rather ß-catenin may play a dual and context-dependent role in PKD and in the presence of other cyst-inducing mutations (Inpp5e-deletion); ß-catenin loss may exacerbate disease in a WNT target gene-independent manner.

Expression of ILK in renal stroma is essential for multiple aspects of renal development.

Kidney development involves reciprocal and inductive interactions between the ureteric bud (UB) and surrounding metanephric mesenchyme. Signals from renal stromal lineages are essential for differentiation and patterning of renal epithelial and mesenchymal cell types and renal vasculogenesis; however, underlying mechanisms remain not fully understood. Integrin-linked kinase (ILK), a key component of integrin signaling pathway, plays an important role in kidney development. However, the role of ILK in renal stroma remains unknown. Here, we ablated ILK in renal stromal lineages using a platelet-derived growth factor receptor B ( Pdgfrb) -Cre mouse line, and the resulting Ilk mutant mice presented postnatal growth retardation and died within 3 wk of age with severe renal developmental defects. Pdgfrb-Cre;Ilk mutant kidneys exhibited a significant decrease in UB branching and disrupted collecting duct formation. From E16.5 onward, renal interstitium was disorganized, forming medullary interstitial pseudocysts. Pdgfrb-Cre;Ilk mutants exhibited renal vasculature mispatterning and impaired glomerular vascular differentiation. Impaired glial cell-derived neurotrophic factor/Ret and bone morphogenetic protein 7 signaling pathways were observed in Pdgfrb-Cre;Ilk mutant kidneys. Furthermore, phosphoproteomic and Western blot analyses revealed a significant dysregulation of a number of key signaling pathways required for kidney morphogenesis, including PI3K/AKT and MAPK/ERK in Pdgfrb-Cre;Ilk mutants. Our results revealed a critical requirement for ILK in renal-stromal and vascular development, as well as a noncell autonomous role of ILK in UB branching morphogenesis.

Triptolide delays disease progression in an adult rat model of polycystic kidney disease through the JAK2-STAT3 pathway.

The aim of our current study was to investigate the long-term effect and the mechanism of triptolide in an adult nonorthologous rat model of polycystic kidney disease (PKD). Male wild-type (+/+) and Cy/+ cystic Han:SPRD rats were treated with vehicle or triptolide from 4 to 16 wk of age. Rats were killed at 16 wk of age for blood, urine, and organ collection. Human-derived WT9-12 PKD cells were treated with triptolide with or without IL-6 pretreatment. Cell proliferation, apoptosis, and cytotoxicity were determined. Western blotting and immunohistochemistry analysis were performed to evaluate the activation of IL-6-JAK2-STAT3 pathway. Renal function was protected by 12 wk of triptolide treatment in cystic Han:SPRD rats as shown by reduced blood urea nitrogen, serum creatinine, and proteinuria levels. Cyst and kidney growth were also retarded by triptolide treatment in Cy/+ rats. We further found that the proliferation index was reduced by triptolide in cystic rats, which was correlated with the reduced expression of IL-6/IL-6 receptor, decreased phosphorylation of JAK2-STAT3, and increased expression of suppressor of cytokine signaling 3 (SOCS3). The inhibitory effect of triptolide was further studied in WT9-12 cells. Triptolide inhibited cell proliferation and the activation of JAK2-STAT3 pathway in PKD cells, but it increased the expression of SOCS3. Pretreatment with IL-6 attenuated the inhibitory effect of triptolide on STAT3 phosphorylation. Our study revealed a long-term beneficial effect of triptolide in PKD that was probably through inhibition of the JAK2-STAT3 pathway.

Casein kinase 1ε and 1α as novel players in polycystic kidney disease and mechanistic targets for (R)-roscovitine and (S)-CR8.

Following the discovery of (R)-roscovitine's beneficial effects in three polycystic kidney disease (PKD) mouse models, cyclin-dependent kinases (CDKs) inhibitors have been investigated as potential treatments. We have used various affinity chromatography approaches to identify the molecular targets of roscovitine and its more potent analog (S)-CR8 in human and murine polycystic kidneys. These methods revealed casein kinases 1 (CK1) as additional targets of the two drugs. CK1ε expression at the mRNA and protein levels is enhanced in polycystic kidneys of 11 different PKD mouse models as well as in human polycystic kidneys. A shift in the pattern of CK1α isoforms is observed in all PKD mouse models. Furthermore, the catalytic activities of both CK1ε and CK1α are increased in mouse polycystic kidneys. Inhibition of CK1ε and CK1α may thus contribute to the long-lasting attenuating effects of roscovitine and (S)-CR8 on cyst development. CDKs and CK1s may constitute a dual therapeutic target to develop kinase inhibitory PKD drug candidates.

Lkb1 deficiency confers glutamine dependency in polycystic kidney disease.

Polycystic kidney disease (PKD) is a common genetic disorder characterized by the growth of fluid-filled cysts in the kidneys. Several studies reported that the serine-threonine kinase Lkb1 is dysregulated in PKD. Here we show that genetic ablation of Lkb1 in the embryonic ureteric bud has no effects on tubule formation, maintenance, or growth. However, co-ablation of Lkb1 and Tsc1, an mTOR repressor, results in an early developing, aggressive form of PKD. We find that both loss of Lkb1 and loss of Pkd1 render cells dependent on glutamine for growth. Metabolomics analysis suggests that Lkb1 mutant kidneys require glutamine for non-essential amino acid and glutathione metabolism. Inhibition of glutamine metabolism in both Lkb1/Tsc1 and Pkd1 mutant mice significantly reduces cyst progression. Thus, we identify a role for Lkb1 in glutamine metabolism within the kidney epithelia and suggest that drugs targeting glutamine metabolism may help reduce cyst number and/or size in PKD.

Modulation of Polycystic Kidney Disease Severity by Phosphodiesterase 1 and 3 Subfamilies.

Aberrant intracellular calcium levels and increased cAMP signaling contribute to the development of polycystic kidney disease (PKD). cAMP can be hydrolyzed by various phosphodiesterases (PDEs). To examine the role of cAMP hydrolysis and the most relevant PDEs in the pathogenesis of PKD, we examined cyst development in Pde1- or Pde3-knockout mice on the Pkd2(-/WS25) background (WS25 is an unstable Pkd2 allele). These PDEs were selected because of their importance in cross-talk between calcium and cyclic nucleotide signaling (PDE1), control of cell proliferation and cystic fibrosis transmembrane conductance regulator (CFTR) -driven fluid secretion (PDE3), and response to vasopressin V2 receptor activation (both). In Pkd2(-/WS25) mice, knockout of Pde1a, Pde1c, or Pde3a but not of Pde1b or Pde3b aggravated the development of PKD and was associated with higher levels of protein kinase A-phosphorylated (Ser133) cAMP-responsive binding protein (P-CREB), activating transcription factor-1, and CREB-induced CRE modulator proteins in kidney nuclear preparations. Immunostaining also revealed higher expression of P-CREB in Pkd2(-/) (WS25);Pde1a(-/-), Pkd2(-) (/WS25);Pde1c(-/-), and Pkd2(-/) (WS25);Pde3a(-/-) kidneys. The cystogenic effect of desmopressin administration was markedly enhanced in Pkd2(-/WS25);Pde3a(-/-) mice, despite PDE3 accounting for only a small fraction of renal cAMP PDE activity. These observations show that calcium- and calmodulin-dependent PDEs (PDE1A and PDE1C) and PDE3A modulate the development of PKD, possibly through the regulation of compartmentalized cAMP pools that control cell proliferation and CFTR-driven fluid secretion. Treatments capable of increasing the expression or activity of these PDEs may, therefore, retard the development of PKD.

Glycogen synthase kinase-3ß promotes cyst expansion in polycystic kidney disease.

Polycystic kidney diseases (PKDs) are inherited disorders characterized by the formation of fluid filled renal cysts. Elevated cAMP levels in PKDs stimulate progressive cyst enlargement involving cell proliferation and transepithelial fluid secretion often leading to end-stage renal disease. The glycogen synthase kinase-3 (GSK3) family of protein kinases consists of GSK3α and GSK3ß isoforms and has a crucial role in multiple cellular signaling pathways. We previously found that GSK3ß, a regulator of cell proliferation, is also crucial for cAMP generation and vasopressin-mediated urine concentration by the kidneys. However, the role of GSK3ß in the pathogenesis of PKDs is not known. Here we found that GSK3ß expression and activity were markedly upregulated and associated with cyst-lining epithelia in the kidneys of mice and humans with PKD. Renal collecting duct-specific gene knockout of GSK3ß or pharmacological inhibition of GSK3 effectively slowed down the progression of PKD in mouse models of autosomal recessive or autosomal dominant PKD. GSK3 inactivation inhibited cAMP generation and cell proliferation resulting in reduced cyst expansion, improved renal function, and extended life span. GSK3ß inhibition also reduced pERK, c-Myc, and cyclin-D1, known mitogens in proliferation of cystic epithelial cells. Thus, GSK3ß has a novel functional role in PKD pathophysiology, and its inhibition may be therapeutically useful to slow down cyst expansion and progression of PKD.

An mTOR kinase inhibitor slows disease progression in a rat model of polycystic kidney disease.

BACKGROUND: The mTOR pathway, which consists of mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), is activated in polycystic kidney disease (PKD) kidneys. Sirolimus and everolimus indirectly bind and inhibit mTORC1. A novel group of drugs, the mTOR kinase inhibitors, directly bind to mTOR kinase, thus inhibiting both mTORC1 and 2. The aim of the study was to determine the therapeutic effect of an mTOR kinase inhibitor, PP242, in the Han:SPRD rat (Cy/+) model of PKD. METHODS: Male rats were treated with PP242 5 mg/kg/day IP or vehicle for 5 weeks. RESULTS: PP242 significantly reduced the kidney enlargement, the cyst density and the blood urea nitrogen in Cy/+ rats. On immunoblot of kidneys, PP242 resulted in a decrease in pS6, a marker of mTORC1 signaling and pAkt(Ser473), a marker of mTORC2 signaling. mTORC plays an important role in regulating cytokine production. There was an increase in IL-1, IL-6, CXCL1 and TNF-α in Cy/+ rat kidneys that was unaffected by PP242. Apoptosis or proliferation is known to play a causal role in cyst growth. PP242 had no effect on caspase-3 activity, TUNEL positive or active caspase-3-positive tubular cells in Cy/+ kidneys. PP242 reduced the number of proliferating cells per cyst and per non-cystic tubule in Cy/+ rats. CONCLUSIONS: In a rat model of autosomal dominant polycystic kidney disease, PP242 treatment (i) decreases proliferation in cystic and non-cystic tubules; (ii) inhibits renal enlargement and cystogenesis and (iii) significantly reduces the loss of kidney function.

Epidermal growth factor-induced proliferation of collecting duct cells from Oak Ridge polycystic kidney mice involves activation of Na+/H+ exchanger.

Epidermal growth factor (EGF) is linked to the pathogenesis of polycystic kidney disease (PKD). We explored signaling pathways activated by EGF in orpk cilia (-) collecting duct cell line derived from a mouse model of PKD (hypomorph of the Tg737/Ift88 gene) with severely stunted cilia, and in a control orpk cilia (+) cell line with normal cilia. RT-PCR demonstrated mRNAs for EGF receptor subunits ErbB1, ErbB2, ErbB3, ErbB4, and mRNAs for Na(+)/H(+) exchangers (NHE), NHE-1, NHE-2, NHE-3, NHE-4, and NHE-5 in both cell lines. EGF stimulated proton efflux in both cell lines. This effect was significantly attenuated by MIA, 5-(n-methyl-N-isobutyl) amiloride, a selective inhibitor of NHE-1 and NHE-2, and orpk cilia (-) cells were more sensitive to MIA than control cells (P < 0.01). EGF significantly induced extracellular signal-regulated kinase (ERK) phosphorylation in both cilia (+) and cilia (-) cells (63.3 and 123.6%, respectively), but the effect was more pronounced in orpk cilia (-) cells (P < 0.01). MIA significantly attenuated EGF-induced ERK phosphorylation only in orpk cilia (-) cells (P < 0.01). EGF increased proliferation of orpk cilia (+) cells and orpk cilia (-) cells, respectively, and MIA at 1-5 µM attenuated EGF-induced proliferation in orpk cilia (-) cells without affecting proliferation of orpk cilia (+) cells. EGF-induced proliferation of both cell lines was significantly decreased by the EGFR tyrosine kinase inhibitor AG1478 and MEK inhibitor PD98059. These results suggest that EGF exerts mitogenic effects in the orpk cilia (-) cells via activation of growth-associated amiloride-sensitive NHEs and ERK.

A protein kinase A-independent pathway controlling aquaporin 2 trafficking as a possible cause for the syndrome of inappropriate antidiuresis associated with polycystic kidney disease 1 haploinsufficiency.

Renal water reabsorption is controlled by arginine vasopressin (AVP), which binds to V2 receptors, resulting in protein kinase A (PKA) activation, phosphorylation of aquaporin 2 (AQP2) at serine 256, and translocation of AQP2 to the plasma membrane. However, AVP also causes dephosphorylation of AQP2 at S261. Recent studies showed that cyclin-dependent kinases (cdks) can phosphorylate AQP2 peptides at S261 in vitro. We investigated the possible role of cdks in the phosphorylation of AQP2 and identified a new PKA-independent pathway regulating AQP2 trafficking. In ex vivo kidney slices and MDCK-AQP2 cells, R-roscovitine, a specific inhibitor of cdks, increased pS256 levels and decreased pS261 levels. The changes in AQP2 phosphorylation status were paralleled by increases in cell surface expression of AQP2 and osmotic water permeability in the absence of forskolin stimulation. R-Roscovitine did not alter cAMP-dependent PKA activity but specifically reduced protein phosphatase 2A (PP2A) expression and activity in MDCK cells. Notably, we found reduced PP2A expression and activity and reduced pS261 levels in Pkd1(+/-) mice displaying a syndrome of inappropriate antidiuresis with high levels of pS256, despite unchanged AVP and cAMP. Similar to previous findings in Pkd1(+/-) mice, R-roscovitine treatment caused a significant decrease in intracellular calcium in MDCK cells. Our data indicate that reduced activity of PP2A, secondary to reduced intracellular Ca(2+) levels, promotes AQP2 trafficking independent of the AVP-PKA axis. This pathway may be relevant for explaining pathologic states characterized by inappropriate AVP secretion and positive water balance.

Phosphodiesterase 1A modulates cystogenesis in zebrafish.

Substantial evidence indicates the importance of elevated cAMP in polycystic kidney disease (PKD). Accumulation of cAMP in cystic tissues may be, in part, caused by enhanced adenylyl cyclase activity, but inhibition of cAMP degradation by phosphodiesterases (PDE) likely has an important role, because cAMP is inactivated much faster than it is synthesized. PDE1 is the only PDE family activated by Ca(2+), which is reduced in PKD cells. To assess the contribution of the PDE1A subfamily to renal cyst formation, we examined the expression and function of PDE1A in zebrafish. We identified two splice isoforms with alternative starts corresponding to human PDE1A1 and PDE1A4. Expression of the two isoforms varied in embryos and adult tissues, and both isoforms hydrolyzed cAMP with Ca(2+)/calmodulin dependence. Depletion of PDE1A in zebrafish embryos using splice- and translation-blocking morpholinos (MOs) caused pronephric cysts, hydrocephalus, and body curvature. Human PDE1A RNA and the PKA inhibitors, H89 and Rp-cAMPS, partially rescued phenotypes of pde1a morphants. Additionally, MO depletion of PDE1A aggravated phenotypes in pkd2 morphants, causing more severe body curvature, and human PDE1A RNA partially rescued pkd2 morphant phenotypes, pronephric cysts, hydrocephalus, and body curvature. Together, these data indicate the integral role of PDE1A and cAMP signaling in renal development and cystogenesis, imply that PDE1A activity is altered downstream of polycystin-2, and suggest that PDE1A is a viable drug target for PKD.

Novel transglutaminase-like peptidase and C2 domains elucidate the structure, biogenesis and evolution of the ciliary compartment.

In addition to their role in motility, eukaryotic cilia serve as a distinct compartment for signal transduction and regulatory sequestration of biomolecules. Recent genetic and biochemical studies have revealed an extraordinary diversity of protein complexes involved in the biogenesis of cilia during each cell cycle. Mutations in components of these complexes are at the heart of human ciliopathies such as Nephronophthisis (NPHP), Meckel-Gruber syndrome (MKS), Bardet-Biedl syndrome (BBS) and Joubert syndrome (JBTS). Despite intense studies, proteins in some of these complexes, such as the NPHP1-4-8 and the MKS, remain poorly understood. Using a combination of computational analyses we studied these complexes to identify novel domains in them which might throw new light on their functions and evolutionary origins. First, we identified both catalytically active and inactive versions of transglutaminase-like (TGL) peptidase domains in key ciliary/centrosomal proteins CC2D2A/MKS6, CC2D2B, CEP76 and CCDC135. These ciliary TGL domains appear to have originated from prokaryotic TGL domains that act as peptidases, either in a prokaryotic protein degradation system with the MoxR AAA+ ATPase, the precursor of eukaryotic dyneins and midasins, or in a peptide-ligase system with an ATP-grasp enzyme comparable to tubulin-modifying TTL proteins. We suggest that active ciliary TGL proteins are part of a cilia-specific peptidase system that might remove tubulin modifications or cleave cilia- localized proteins, while the inactive versions are likely to bind peptides and mediate key interactions during ciliogenesis. Second, we observe a vast radiation of C2 domains, which are key membrane-localization modules, in multiple ciliary proteins, including those from the NPHP1-4-8 and the MKS complexes, such as CC2D2A/MKS6, RPGRIP1, RPGRIP1L, NPHP1, NPHP4, C2CD3, AHI1/Jouberin and CEP76, most of which can be traced back to the last common eukaryotic ancestor. Identification of these TGL and C2 domains aid in the proper reconstruction of the Y-shaped linkers, which are key structures in the transitional zone of cilia, by allowing precise prediction of the multiple membrane-contacting and protein-protein interaction sites in these structures. These findings help decipher key events in the evolutionary separation of the ciliary and nuclear compartments in course of the emergence of the eukaryotic cell.

Loss of GM3 synthase gene, but not sphingosine kinase 1, is protective against murine nephronophthisis-related polycystic kidney disease.

Genetic forms of polycystic kidney diseases (PKDs), including nephronophthisis, are characterized by formation of fluid-filled cysts in the kidneys and progression to end-stage renal disease. No therapies are currently available to treat cystic diseases, making it imperative to dissect molecular mechanisms in search of therapeutic targets. Accumulating evidence suggests a pathogenic role for glucosylceramide (GlcCer) in multiple forms of PKD. It is not known, however, whether other structural glycosphingolipids (GSLs) or bioactive signaling sphingolipids (SLs) modulate cystogenesis. Therefore, we set out to address the role of a specific GSL (ganglioside GM3) and signaling SL (sphingosine-1-phosphate, S1P) in PKD progression, using the jck mouse model of nephronopthisis. To define the role of GM3 accumulation in cystogenesis, we crossed jck mice with mice carrying a targeted mutation in the GM3 synthase (St3gal5) gene. GM3-deficient jck mice displayed milder PKD, revealing a pivotal role for ganglioside GM3. Mechanistic changes in regulation of the cell-cycle machinery and Akt-mTOR signaling were consistent with reduced cystogenesis. Dramatic overexpression of sphingosine kinase 1 (Sphk1) mRNA in jck kidneys suggested a pathogenic role for S1P. Surprisingly, genetic loss of Sphk1 exacerbated cystogenesis and was associated with increased levels of GlcCer and GM3. On the other hand, increasing S1P accumulation through pharmacologic inhibition of S1P lyase had no effect on the progression of cystogenesis or kidney GSL levels. Together, these data suggest that genes involved in the SL metabolism may be modifiers of cystogenesis, and suggest GM3 synthase as a new anti-cystic therapeutic target.

The Raf kinase inhibitor PLX5568 slows cyst proliferation in rat polycystic kidney disease but promotes renal and hepatic fibrosis.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a common cause of renal failure. Aberrant epithelial cell proliferation is a major cause of progressive cyst enlargement in ADPKD. Since activation of the Ras/Raf signaling system has been detected in cyst-lining epithelia, inhibition of Raf kinase has been proposed as an approach to retard the progression of ADPKD. Methods and results. PLX5568, a novel selective small molecule inhibitor of Raf kinases, attenuated proliferation of human ADPKD cyst epithelial cells. It reduced in vitro cyst growth of Madin-Darby Canine Kidney cells and of human ADPKD cells within a collagen gel. In male cy/+ rats with polycystic kidneys, PLX5568 inhibited renal cyst growth along with a significant reduction in the number of proliferating cell nuclear antigen- and phosphorylated extracellular signal-regulated kinase-positive cyst-lining epithelial cells. Furthermore, treated animals showed increased capacity to concentrate urine. However, PLX5568 did not lead to a consistent improvement of renal function. Moreover, although relative cyst volume was decreased, total kidney-to-body weight ratio was not significantly reduced by PLX5568. Further analyses revealed a 2-fold increase of renal and hepatic fibrosis in animals treated with PLX5568. CONCLUSIONS: PLX5568 attenuated cyst enlargement in vitro and in a rat model of ADPKD without improving kidney function, presumably due to increased renal fibrosis. These data suggest that effective therapies for the treatment of ADPKD will need to target fibrosis as well as the growth of cysts.

CaMK-II is a PKD2 target that promotes pronephric kidney development and stabilizes cilia.

Intracellular Ca²âº signals influence gastrulation, neurogenesis and organogenesis through pathways that are still being defined. One potential Ca²âº mediator of many of these morphogenic processes is CaMK-II, a conserved calmodulin-dependent protein kinase. Prolonged Ca²âº stimulation converts CaMK-II into an activated state that, in the zebrafish, is detected in the forebrain, ear and kidney. Autosomal dominant polycystic kidney disease has been linked to mutations in the Ca²âº-conducting TRP family member PKD2, the suppression of which in vertebrate model organisms results in kidney cysts. Both PKD2-deficient and CaMK-II-deficient zebrafish embryos fail to form pronephric ducts properly, and exhibit anterior cysts and destabilized cloacal cilia. PKD2 suppression inactivates CaMK-II in pronephric cells and cilia, whereas constitutively active CaMK-II restores pronephric duct formation in pkd2 morphants. PKD2 and CaMK-II deficiencies are synergistic, supporting their existence in the same genetic pathway. We conclude that CaMK-II is a crucial effector of PKD2 Ca²âº that both promotes morphogenesis of the pronephric kidney and stabilizes primary cloacal cilia.

Prospects for mTOR inhibitor use in patients with polycystic kidney disease and hamartomatous diseases.

Mammalian target of rapamycin (mTOR) is the core component of two complexes, mTORC1 and mTORC2. mTORC1 is inhibited by rapamycin and analogues. mTORC2 is impeded only in some cell types by prolonged exposure to these compounds. mTOR activation is linked to tubular cell proliferation in animal models and human autosomal dominant polycystic kidney disease (ADPKD). mTOR inhibitors impede cell proliferation and cyst growth in polycystic kidney disease (PKD) models. After renal transplantation, two small retrospective studies suggested that mTOR was more effective than calcineurin inhibitor-based immunosuppression in limiting kidney and/or liver enlargement. By inhibiting vascular remodeling, angiogenesis, and fibrogenesis, mTOR inhibitors may attenuate nephroangiosclerosis, cyst growth, and interstitial fibrosis. Thus, they may benefit ADPKD at multiple levels. However, mTOR inhibition is not without risks and side effects, mostly dose-dependent. Under certain conditions, mTOR inhibition interferes with adaptive increases in renal proliferation necessary for recovery from injury. They restrict Akt activation, nitric oxide synthesis, and endothelial cell survival (downstream from mTORC2) and potentially increase the risk for glomerular and peritubular capillary loss, vasospasm, and hypertension. They impair podocyte integrity pathways and may predispose to glomerular injury. Administration of mTOR inhibitors is discontinued because of side effects in up to 40% of transplant recipients. Currently, treatment with mTOR inhibitors should not be recommended to treat ADPKD. Results of ongoing studies must be awaited and patients informed accordingly. If effective, lower dosages than those used to prevent rejection would minimize side effects. Combination therapy with other effective drugs could improve tolerability and results.