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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. Polycystic kidney disease (PKD) is characterized by the formation of large fluid-filled cysts. Here Flowers and colleagues show that loss of Lkb1, downregulated in PKD, renders kidney cells dependent on glutamine for growth, and suggest that inhibition of glutamine metabolism may prevent cyst development in PKD.

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 GSK3β isoforms and has a crucial role in multiple cellular signaling pathways. We previously found that GSK3β, a regulator of cell proliferation, is also crucial for cAMP generation and vasopressin-mediated urine concentration by the kidneys. However, the role of GSK3β in the pathogenesis of PKDs is not known. Here we found that GSK3β 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 GSK3β 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. GSK3β inhibition also reduced pERK, c-Myc, and cyclin-D1, known mitogens in proliferation of cystic epithelial cells. Thus, GSK3β has a novel functional role in PKD pathophysiology, and its inhibition may be therapeutically useful to slow down cyst expansion and progression of PKD.

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.

MAP kinases transduce signals that are involved in a multitude of cellular pathways and functions in response to a variety of ligands and cell stimuli. Aberrant or inappropriate functions of MAPKs have now been identified in diseases ranging from cancer to inflammatory disease to obesity and diabetes. In many cell types, the MAPKs ERK1/2 are linked to cell proliferation. ERK1/2 are thought to play a role in some cancers, because mutations in Ras and B-Raf, which can activate the ERK1/2 cascade, are found in many human tumors. Abnormal ERK1/2 signaling has also been found in polycystic kidney disease, and serious developmental disorders such as cardio-facio-cutaneous syndrome arise from mutations in components of the ERK1/2 cascade. ERK1/2 are essential in well-differentiated cells and have been linked to long-term potentiation in neurons and in maintenance of epithelial polarity. Additionally, ERK1/2 are important for insulin gene transcription in pancreatic beta cells, which produce insulin in response to increases in circulating glucose to permit efficient glucose utilization and storage in the organism. Nutrients and hormones that induce or repress insulin secretion activate and/or inhibit ERK1/2 in a manner that reflects the secretory demand on beta cells. Disturbances in this and other regulatory pathways may result in the contribution of ERK1/2 to the etiology of certain human disorders.

Increased levels of 3′-5′-cyclic adenosine monophosphate (cAMP) stimulate cell proliferation and fluid secretion in polycystic kidney disease. Levels of this molecule are more sensitive to inhibition of phosphodiesterases (PDEs), whose activity far exceeds the rate of cAMP synthesis by adenylyl cyclase. Several PDEs exist, and here we measured the activity and expression of PDE families, their isoforms, and the expression of downstream effectors of cAMP signaling in the kidneys of rodents with polycystic kidney disease. We found a higher overall PDE activity in kidneys from mice as compared with rats, as well as a higher contribution of PDE1, relative to PDE4 and PDE3, to total PDE activity of kidney lysates and lower PDE1, PDE3, and PDE4 activities in the kidneys of cystic as compared with wild-type mice. There were reduced amounts of several PDE1, PDE3, and PDE4 proteins, possibly due to increased protein degradation despite an upregulation of their mRNA. Increased levels of cGMP were found in the kidneys of cystic animals, suggesting in vivo downregulation of PDE1 activity. We found an additive stimulatory effect of cAMP and cGMP on cystogenesis in vitro. Cyclic AMP–dependent protein kinase subunits Iα and IIβ, PKare, the transcription factor CREB-1 mRNA, and CREM, ATF-1, and ICER proteins were upregulated in the kidneys of cystic as compared with wild-type animals. Our study suggests that alterations in cyclic nucleotide catabolism may render cystic epithelium particularly susceptible to factors acting on Gs-coupled receptors. This may account, in part, for increased cyclic nucleotide signaling in polycystic kidney disease and contribute substantially to disease progression.

The basic biochemical mechanisms underlying many heritable human polycystic diseases are unknown despite evidence that most cases are caused by mutations in members of several protein families, the most prominent being the polycystin gene family, whose products are found on the primary cilia, or due to mutations in posttranslational processing and transport. Inherited polycystic kidney disease, the most prevalent polycystic disease, currently affects [almost equal to]500,000 people in the United States. Decreases in proteoglycans (PGs) have been found in tissues and cultured cells from patients who suffer from autosomal dominant polycystic kidney disease, and this PG decrease has been hypothesized to be responsible for cystogenesis. This is possible because alterations in PG concentrations would be predicted to disrupt many homeostatic mechanisms of growth, development, and metabolism. To test this hypothesis, we have generated mice lacking xylosyltransferase 2 (XylT2), an enzyme involved in PG biosynthesis. Here we show that inactivation of XylT2 results in a substantial reduction in PGs and a phenotype characteristic of many aspects of polycystic liver and kidney disease, including biliary epithelial cysts, renal tubule dilation, organ fibrosis, and basement membrane abnormalities. Our findings demonstrate that alterations in PG concentrations can occur due to loss of XylT2, and that reduced PGs can induce cyst development.

Mutations of the polycystic kidney and hepatic disease 1 (PKHD1) gene have been shown to cause autosomal recessive polycystic kidney disease (ARPKD), but the cellular functions of the gene product (PKHD1) remain uncharacterized. To illuminate its properties, the spatial and temporal expression patterns of PKHD1 were determined in mouse, rat, and human tissues by using polyclonal Abs and mAbs recognizing various specific regions of the gene product. During embryogenesis, PKHD1 is widely expressed in epithelial derivatives, including neural tubules, gut, pulmonary bronchi, and hepatic cells. In the kidneys of the pck rats, the rat model of which is genetically homologous to human ARPKD, the level of PKHD1 was significantly reduced but not completely absent. In cultured renal cells, the PKHD1 gene product colocalized with polycystin-2, the gene product of autosomal dominant polycystic disease type 2, at the basal bodies of primary cilia. Immunoreactive PKHD1 localized predominantly at the apical domain of polarized epithelial cells, suggesting it may be involved in the tubulogenesis and/or maintenance of duct-lumen architecture. Reduced PKHD1 levels in pck rat kidneys and its colocalization with polycystins may underlie the pathogenic basis for cystogenesis in polycystic kidney diseases.

Nephronophthisis belongs to a family of recessive cystic kidney diseases and may arise from mutations in multiple genes. In this report we have used a spontaneous mouse mutant of type 3 nephronophthisis to examine whether the doxycycline-inducible synthesis of Timp-2, a natural inhibitor of matrix metalloproteinases, can influence renal cyst growth in transgenic mice. Metalloproteinases may exert either a negative or a positive effect on the progression of cystic kidney disease, and we reasoned that this may be most effectively examined by using a natural inhibitor. Surprisingly, already the application of doxycycline, which also inhibits matrix metalloproteinases, accelerated renal cyst growth and led to increased renal fibrosis, an additional effect of Timp-2 was not detected. The positive effect of doxycycline on kidney size was not due to a non-specific “anabolic effect” but was specific for cystic kidneys because it was not observed in non-cystic kidneys. When looking for potential metabolic changes we noticed that the urine of control animals led to an increase in the calcium response of LLC-PK₁ cells, whereas the urine of doxycycline-treated mice showed the opposite effect and even antagonized the urine of control animals. Further experiments demonstrated that the urine of control animals contained a heat-labile, proteinase K-resistant substance which appears to be responsible for the induction of a calcium response in LLC-PK₁ cells. We conclude that doxycycline accelerates cyst growth possibly by the induction of a substance which lowers the intracellular calcium concentration. Our data also add a note of caution when interpreting phenotypes of animal models based upon the tet system.

Advances in the understanding of cystogenesis and availability of animal models orthologous to human autosomal dominant polycystic kidney disease (ADPKD) and recessive polycystic kidney disease (ARPKD) will likely facilitate the development of treatments for these diseases. Proteins mutated in ADPKD and ARPKD, as well as in several animal models, are localized to renal primary cilia. These are thought to have a sensory function and contribute to the regulation of the intracellular calcium ([Ca2+]i). It seems likely that the maintenance of a differentiated renal epithelial phenotype, characterized by controlled fluid secretion and cell proliferation, requires precise functional coordination of cAMP and Ras/Raf/MEK/ERK signaling by [Ca2+]i. [Ca2+]i alterations, linked to genetic defects causing polycystic kidney disease, may hinder negative feedback mechanisms that control cAMP and Ras/Raf/MEK/ERK signaling, and result in increased fluid secretion and cell proliferation. cAMP levels, Raf kinase activities and ERK phosphorylation are increased in polycystic kidneys. There is also evidence of abnormal cross-talk between cAMP and MAPK pathways, that can be reproduced in wild-type cells by altering [Ca2+]i. While cAMP inhibits Ras-Raf-1-stimulated phosphorylation of ERK in normal kidney cells, it markedly increases B-Raf kinase activity and ERK phosphorylation in polycystic kidney cells. Treatment strategies should probably be aimed at increasing [Ca2+]i, inhibiting Ras/Raf/MEK/ERK signaling or lowering cAMP in the distal nephron and collecting duct. Vasopressin is the major adenylyl cyclase agonist in the collecting duct principal cells via a V2 receptor. OPC31260, a V2 receptor antagonist, lowers renal cAMP and markedly inhibits cystogenesis in four animal models of polycystic kidney disease, three of which are orthologous to human diseases (PCK rat, ARPKD; pcy mouse, adolescent nephronophthisis; Pkd2WS25/- mouse, ADPKD). The renal selectivity and safety profile of this class of drugs make it an excellent candidate for clinical trials. Copyright © 2004 S. Karger AG, Basel

Decreased sulfotransferase SULT1C2 gene expression in DPT-induced polycystic kidney. The pathogenesis of polycystic kidney disease (PKD) remains unclear despite the identification of the genes responsible for hereditary PKD. In this study, we investigated the alteration of gene expressions in an acquired PKD model induced by 2-amino-4,5-diphenylthiazole (DPT) using the differential display method. Kidney mRNA from a Sprague-Dawley rat fed with 1% DPT for 4 days and from a control rat was compared by the RT-PCR differential display method. Differentially expressed bands were re-amplified and subcloned. Using these subclones as probes, the changes in gene expressions were confirmed by Northern blot analysis. Subsequently, mouse kidney cDNA library was screened. The isolated 1.5-kb cDNA contained an open reading frame encoding 296 amino acids, which shared 94.3% identity with rat SULT1C2 sulfotransferase, and was considered to be its mouse ortholog (GenBank Accession No. AY005469). Mouse SULT1C2 mRNA was abundant in the kidney and stomach among normal mouse tissues. The expression of SULT1C2 mRNA was decreased in the rat kidney after DPT feeding but not in the stomach. Mouse SULT1C2 was expressed successfully using pET plasmid vector and E. coli. The recombinant 34-kD protein was capable of catalyzing the sulfation of p-nitrophenol at a Km of 3.1 mmol/L, by utilizing 3′-phosphoadenosine 5′-phosphosulfate (PAPS) as the sulfate donor. Although the physiological substrate and function of SULT1C2 have yet to be elucidated, its down-regulation could be involved in the cystic changes of tubules by decreasing the sulfation of the tubular basement membrane components.