INPP5E regulates phosphoinositide-dependent cilia transition zone function.

Human ciliopathies, including Joubert syndrome (JBTS), arise from cilia dysfunction. The inositol polyphosphate 5-phosphatase INPP5E localizes to cilia and is mutated in JBTS. Murine Inpp5e ablation is embryonically lethal and recapitulates JBTS, including neural tube defects and polydactyly; however, the underlying defects in cilia signaling and the function of INPP5E at cilia are still emerging. We report Inpp5e-/- embryos exhibit aberrant Hedgehog-dependent patterning with reduced Hedgehog signaling. Using mouse genetics, we show increasing Hedgehog signaling via Smoothened M2 expression rescues some Inpp5e-/- ciliopathy phenotypes and "normalizes" Hedgehog signaling. INPP5E's phosphoinositide substrates PI(4,5)P2 and PI(3,4,5)P3 accumulated at the transition zone (TZ) in Hedgehog-stimulated Inpp5e-/- cells, which was associated with reduced recruitment of TZ scaffolding proteins and reduced Smoothened levels at cilia. Expression of wild-type, but not 5-phosphatase-dead, INPP5E restored TZ molecular organization and Smoothened accumulation at cilia. Therefore, we identify INPP5E as an essential point of convergence between Hedgehog and phosphoinositide signaling at cilia that maintains TZ function and Hedgehog-dependent embryonic development.

Bi-allelic CLPB mutations cause cataract, renal cysts, nephrocalcinosis and 3-methylglutaconic aciduria, a novel disorder of mitochondrial protein disaggregation.

Whole exome sequencing was used to investigate the genetic cause of mitochondrial disease in two siblings with a syndrome of congenital lamellar cataracts associated with nephrocalcinosis, medullary cysts and 3-methylglutaconic aciduria. Autosomal recessive inheritance in a gene encoding a mitochondrially targeted protein was assumed; the only variants which satisfied these criteria were c.1882C>T (p.Arg628Cys) and c.1915G>A (p.Glu639Lys) in the CLPB gene, encoding a heat shock protein/chaperonin responsible for disaggregating mitochondrial and cytosolic proteins. Functional studies, including quantitative PCR (qPCR) and Western blot, support pathogenicity of these mutations. Furthermore, molecular modelling suggests that the mutations disrupt interactions between subunits so that the CLPB hexamer cannot form or is unstable, thus impairing its role as a protein disaggregase. We conclude that accumulation of protein aggregates underlies the development of cataracts and nephrocalcinosis in CLPB deficiency, which is a novel genetic cause of 3-methylglutaconic aciduria. A common mitochondrial cause for 3-methylglutaconic aciduria appears to be disruption of the architecture of the mitochondrial membranes, as in Barth syndrome (tafazzin deficiency), Sengers syndrome (acylglycerol kinase deficiency) and MEGDEL syndrome (impaired remodelling of the mitochondrial membrane lipids because of SERAC1 mutations). We now propose that perturbation of the mitochondrial membranes by abnormal protein aggregates leads to 3-methylglutaconic aciduria in CLPB deficiency.

Development of renal cysts after crizotinib treatment in advanced ALK-positive non-small-cell lung cancer.

INTRODUCTION: The development of complex renal cysts after crizotinib treatment for non-small-cell lung cancer (NSCLC) is a reported side effect. However, its occurrence and characteristics have not been reported. METHODS: Medical records and computed tomography images of crizotinib-treated patients in three prospective clinical trials were reviewed. The size and Bosniak category of the renal cysts before and after crizotinib treatment were determined. Patients' clinical characteristics, tumor stage, treatment response, renal function, and outcomes were analyzed. RESULTS: During December 2010 to March 2013, we enrolled 32 patients who received crizotinib. There were 23 patients who had renal cysts before crizotinib. The median follow-up time was 493 days. Seven patients (22%, six with baseline renal cyst and one without baseline renal cyst) had significant renal cyst change. Four (13% of all) had new complex renal cysts. The median time from crizotinib treatment to first recognization of significant renal cyst change was 77 days. After stopping crizotinib, complex renal cysts regressed significantly. Patients with significant renal cyst change received more previous anticancer therapy (median, 5 lines versus 3 lines, p = 0.04) and received crizotinib for longer duration (median, 956 days versus 248 days, p = 0.007) compared with those without significant renal cyst change. CONCLUSIONS: Change of renal cysts after crizotinib treatment is not uncommon. Development of complex renal cysts reverses after stopping crizotinib.

Arl13b-regulated cilia activities are essential for polarized radial glial scaffold formation.

The construction of cerebral cortex begins with the formation of radial glia. Once formed, polarized radial glial cells divide either symmetrically or asymmetrically to balance appropriate production of progenitor cells and neurons. Following birth, neurons use the processes of radial glia as scaffolding for oriented migration. Radial glia therefore provide an instructive structural matrix to coordinate the generation and placement of distinct groups of cortical neurons in the developing cerebral cortex. We found that Arl13b, a cilia-enriched small GTPase that is mutated in Joubert syndrome, was critical for the initial formation of the polarized radial progenitor scaffold. Using developmental stage-specific deletion of Arl13b in mouse cortical progenitors, we found that early neuroepithelial deletion of ciliary Arl13b led to a reversal of the apical-basal polarity of radial progenitors and aberrant neuronal placement. Arl13b modulated ciliary signaling necessary for radial glial polarity. Our findings indicate that Arl13b signaling in primary cilia is crucial for the initial formation of a polarized radial glial scaffold and suggest that disruption of this process may contribute to aberrant neurodevelopment and brain abnormalities in Joubert syndrome-related ciliopathies.

CDK inhibitors R-roscovitine and S-CR8 effectively block renal and hepatic cystogenesis in an orthologous model of ADPKD.

Autosomal dominant polycystic kidney disease (ADPKD) and other forms of PKD are associated with dysregulated cell cycle and proliferation. Although no effective therapy for the treatment of PKD is currently available, possible mechanism-based approaches are beginning to emerge. A therapeutic intervention targeting aberrant cilia-cell cycle connection using CDK-inhibitor R-roscovitine showed effective arrest of PKD in jck and cpk models that are not orthologous to human ADPKD. To evaluate whether CDK inhibition approach will translate into efficacy in an orthologous model of ADPKD, we tested R-roscovitine and its derivative S-CR8 in a model with a conditionally inactivated Pkd1 gene (Pkd1 cKO). Similar to ADPKD, Pkd1 cKO mice developed renal and hepatic cysts. Treatment of Pkd1 cKO mice with R-roscovitine and its more potent and selective analog S-CR8 significantly reduced renal and hepatic cystogenesis and attenuated kidney function decline. Mechanism of action studies demonstrated effective blockade of cell cycle and proliferation and reduction of apoptosis. Together, these data validate CDK inhibition as a novel and effective approach for the treatment of ADPKD.

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.

CA9 as a molecular marker for differential diagnosis of cystic renal tumors.

OBJECTIVE: CA9 is proven to be a powerful marker for clear cell renal cell carcinoma. The studies on CA9 have been limited to solid renal cell carcinomas (RCC). We have conducted a study of CA9 expression in renal cystic tumors. The purpose of the present study was to extend the utility of CA9 for cystic renal tumors. MATERIALS AND METHODS: Immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) were used to detect CA9 expression in cystic renal tumors. Forty-three cystic renal tumors (22 benign and 21 malignant) were included for the immunohistochemical staining. Thirty-six patients with a cystic renal mass (20 malignant and 16 benign cystic tumors) were studied to measure CA9 level in the fluid by ELISA. Sixteen cysts (9 malignant and 7 benign cysts) were subjected both to immunohistochemistry and CA9 measurement in the fluid. RESULTS: Using immunohistochemical staining, all the benign cystic renal tumors including the 18 simple cyst and 4 benign multilocular cystic nephromas did not express CA9. All 13 cystic clear cell RCC were scored as strong staining for CA9. For 8 multilocular clear cell RCC, 7 were scored as strong staining for CA9 and the other one was negative. There was a significant difference in positive percentage (P < 0.001) between the 2 groups of malignant and benign cysts. For the 16 benign cysts, the mean concentration of CA9 in the fluid of cyst was 162 ± 133 pg/ml (median: 0 pg/ml; range: 0-2140 pg/ml). For the 20 malignant renal cystic tumors, the mean concentration of CA9 in the fluid of cyst was 2043 ± 62 pg/ml (median: 2,140 pg/ml; range: 1,112-2,140 pg/ml). There was a significant difference in mean concentration of CA9 between the two groups of malignant and benign cysts (P < 0.001). The presence or absence of CA9 expression measured by immunohistochemistry and ELISA test was concordant in 14 out of 16 cases (88%). CONCLUSIONS: Malignant cystic renal tumors expressed strongly CA9 while the benign renal cysts did not express CA9. CA9 can be detected in the fluid of malignant cystic renal tumors. CA9 is a promising molecular marker to differentiate the malignant cystic renal tumors from the benign cysts.

The ciliopathy-associated protein homologs RPGRIP1 and RPGRIP1L are linked to cilium integrity through interaction with Nek4 serine/threonine kinase.

Recent studies have established ciliary dysfunction as the underlying cause of a broad range of multi-organ phenotypes, known as 'ciliopathies'. Ciliopathy-associated proteins have a common site of action in the cilium, however, their overall importance for ciliary function differs, as implied by the extreme variability in ciliopathy phenotypes. The aim of this study was to gain more insight in the function of two ciliopathy-associated protein homologs, RPGR interacting protein 1 (RPGRIP1) and RPGRIP1-like protein (RPGRIP1L). Mutations in RPGRIP1 lead to the eye-restricted disease Leber congenital amaurosis, while mutations in RPGRIP1L are causative for Joubert and Meckel syndrome, which affect multiple organs and are at the severe end of the ciliopathy spectrum. Using tandem affinity purification in combination with mass spectrometry, we identified Nek4 serine/threonine kinase as a prominent component of both the RPGRIP1- as well as the RPGRIP1L-associated protein complex. In ciliated cells, this kinase localized to basal bodies, while in ciliated organs, the kinase was predominantly detected at the ciliary rootlet. Down-regulation of NEK4 in ciliated cells led to a significant decrease in cilium assembly, pointing to a role for Nek4 in cilium dynamics. We now hypothesize that RPGRIP1 and RPGRIP1L function as cilium-specific scaffolds that recruit a Nek4 signaling network which regulates cilium stability. Our data are in line with previously established roles in the cilium of other members of the Nek protein family and define NEK4 as a ciliopathy candidate gene.

The NIMA-related kinase NEK1 cycles through the nucleus.

Mutations in NEK1 in mice are causal for cystic kidneys, and model the ciliopathy polycystic kidney disease caused by abnormal ciliary structure or signaling. NEK1 has previously been shown to localize near centrosomes and to play a role in centrosomal stability and ciliogenesis. Recent data suggest that the etiology of kidney cysts involves aberrant signaling from the primary cilium to the nucleus. Here we demonstrate that NEK1 contains functional nuclear localization signals, is exported from the nucleus via a nuclear export signal-dependent pathway and that the protein cycles through the nucleus. Our data suggest that NEK1 is a candidate to transduce messages from the ciliary-basal body region to the regulation of nuclear gene expression.

CA9 level in renal cyst fluid: a possible molecular diagnosis of malignant tumours.

AIMS: The preoperative differentiation of malignant renal cystic tumours from benign lesions is critical, and it remains a common diagnostic problem. The aim was to examine if the Carbonic anhydrase 9 (CA9) level in cyst fluid can provide a molecular diagnosis of malignant cyst. METHODS AND RESULTS: Twenty-eight patients with a cystic renal mass were included. Fine-needle aspiration was performed to obtain the fluid. Postoperative pathology confirmed that there were 16 cystic renal cell carcinomas. Twelve benign cystic tumours were used as controls. One hundred microlitres of supernatant of cyst fluid was used to measure the CA9 protein level, which was measured by an enzyme-linked immunosorbent assay technique. CA9 was strongly detected and considered as positive in the cyst fluid of all 16 cystic malignant tumours (>1000 pg/ml), whereas its expression was negative in 11/12 benign cystic tumours (<300 pg/ml). The difference in percentages of positive CA9 between malignant and benign renal cystic tumours was significant (P < 0.001). CONCLUSION: The fluid of malignant cystic renal tumours contains a high level of CA9 protein. The measurement of CA9 level in cyst fluid may be used as a molecular diagnosis for differentiation between malignant and benign renal cystic masses.

pVHL and PTEN tumour suppressor proteins cooperatively suppress kidney cyst formation.

In patients with von Hippel-Lindau (VHL) disease, renal cysts and clear cell renal cell carcinoma (ccRCC) arise from renal tubular epithelial cells containing biallelic inactivation of the VHL tumour suppressor gene. However, it is presumed that formation of renal cysts and their conversion to ccRCC involve additional genetic changes at other loci. Here, we show that cystic lesions in the kidneys of patients with VHL disease also demonstrate activation of the phosphatidylinositol-3-kinase (PI3K) pathway. Strikingly, combined conditional inactivation of Vhlh and the Pten tumour suppressor gene, which normally antagonises PI3K signalling, in the mouse kidney, elicits cyst formation after short latency, whereas inactivation of either tumour suppressor gene alone failed to produce such a phenotype. Interestingly, cells lining these cysts frequently lack a primary cilium, a microtubule-based cellular antenna important for suppression of uncontrolled kidney epithelial cell proliferation and cyst formation. Our results support a model in which the PTEN tumour suppressor protein cooperates with pVHL to suppress cyst development in the kidney.

Understanding eNOS for pharmacological modulation of endothelial function: a translational view.

Knowledge about the function of endothelial nitric oxide synthase (eNOS), and its regulation in pathophysiological states has tremendously increased. It is now clear that diminished activity of nitric oxide (NO) contributes to endothelial dysfunction, which is a characteristic of impeding atherosclerosis. This review aims to summarize the available knowledge about the impact of important cardiovascular risk factors on NO production by eNOS. There are 4 principle causes of diminished NO bio-activity: decreased expression and/or activity of the eNOS enzyme, eNOS uncoupling, enhanced breakdown or scavenging of NO and impaired transmission of NO-mediated signaling events (failure of the effector mechanisms). From the analysis, it becomes clear, that several aspects of eNOS functionality have only scarcely been tested under conditions of increased (experimental) cardiovascular risk. These aspects include palmitoylation, myristoylation and phosphorylation of the eNOS enzyme. Clear is that enhanced production of reactive oxygen species (ROS) and eNOS uncoupling are relatively important causes of reduced NO-bioactivity in cardiovascular disease states. Ideally, eNOS is sufficiently expressed, produces NO sufficiently and not abundantly, does not produce superoxide and is not scavenged by ROS; the produced NO then reaches its signaling target, mainly soluble guanylyl cyclase (sGC) and elicits a cellular response. Considering which aspects of eNOS are now assessable in a clinical setting and which therapeutic measures are available, there is a great challenge ahead.

Altered regulation of SHP-2 and PTP 1B tyrosine phosphatases in cystic kidneys from bcl-2 -/- mice.

Protein tyrosine phosphorylation is a dynamic reversible process in which the level of phosphorylation, at any time, is the result of phosphatase and/or kinase activity. This balance is critical for control of growth and differentiation. The role of tyrosine phosphatases during nephrogenesis and in kidney disease requires delineation. Appropriate regulation of focal adhesion proteins such as focal adhesion kinase (FAK) and paxillin are important in cell adhesion, migration, and differentiation. We have previously shown that B cell lymphoma/leukemia-2 (bcl-2) -/- mice develop cystic kidneys and exhibit sustained phosphorylation of FAK and paxillin. We have examined the expression and activity of focal adhesion tyrosine phosphatases [Src homology-2 domain phosphatase (SHP-2), protein tyrosine phosphatase (PTP 1B), and PTP-proline, glutamate, serine, and threonine sequences (PEST)] during normal nephrogenesis and in cystic kidneys from bcl-2 -/- mice. Cystic kidneys from postnatal day 20 bcl-2 -/- mice demonstrate a reduced expression, sixfold decrease in activity, and altered distribution of SHP-2 and PTP 1B. PTP-PEST expression and distribution were similar in both bcl-2 +/+ and bcl-2 -/- mice. The altered regulation of PTP 1B and SHP-2 in kidneys from bcl-2 -/- mice correlates with sustained phosphorylation of FAK and paxillin. Thus renal cyst formation in the bcl-2 -/- mice may be the result of an inability of complete differentiation due to continued activation of growth processes, including activation of FAK and paxillin.

Matrix metalloproteinases and TIMPS in cultured C57BL/6J-cpk kidney tubules.

Restructuring of basement membranes is a hallmark of the pathology of renal cystic disorders. Here, we present findings consistent with the view that basement membrane degradation by matrix metallo-proteinases (MMPs) may contribute to abnormal basement membrane structure in polycystic kidney disease. Cells from cystic kidney tubules embedded in collagen gels appeared to migrate through the gel. This migration through collagen indicated that these cells could degrade the matrix. To examine this activity, we cultured cystic kidney tubules derived from the C57BL/6J cpk/cpk mouse, a hereditary model of polycystic kidney disease, and assayed conditioned medium for the presence of MMPs and tissue inhibitors of metalloproteinases (TIMPs). The conditioned medium from the cystic tubules contained higher than normal levels of MMP-9, MMP-2, and MMP-3 as well as TIMP-1 and TIMP-2. A 101 kDa protease was present equally in cystic and control cultures and although inhibited by EDTA, it was not inhibited by TIMPs, nor activated by the mercurial compound APMA. These data suggest that cystic kidney tubules synthesize and secrete high levels of MMPs which may then participate in the restructuring of the tubular basement membrane.

Immunocytochemical studies of the distribution of alpha and pi isoforms of glutathione S-transferase in cystic renal diseases.

We describe immunohistochemical studies of the expression of alpha and pi class glutathione S-transferases (GSTs) in normal fetal kidneys. These define, in greater detail, changes in expression of alpha isoforms in the proximal tubule. At about 36 weeks of gestation expression of alpha isoforms was down-regulated in the distal tubules and collecting ducts while pi was expressed throughout the nephron. Tubular expression of alpha isoforms was restricted to the part adjacent to the glomerulus; cells farthest from the glomerulus were negative. After 40 weeks of gestation, alpha isoforms were expressed along the entire proximal tubule, while pi was restricted to the distal tubule and collecting ducts. GST expression was also studied in multicystic renal dysplasia, autosomal recessive polycystic kidney disease, and autosomal dominant polycystic kidney disease to determine whether the patterns of expression of alpha and pi isoforms allow identification of the origin of the cysts that characterize these diseases. Cysts were lined by epithelia that were strongly positive for alpha and pi isoforms. The epithelia of noncystic nephrons in renal cystic dysplasia demonstrated delayed maturity, suggesting that GST expression was dependent on the stage of development and not length of gestation.

The urine urokinase concentration in end stage renal disease with acquired renal cyst.

To see whether there was any difference in the urine urokinase concentration between acquired cystic kidney disease (ACKD) group and control (non cyst) group in end stage renal disease patients (ESRD), we evaluated fifty ESRD patients who had been maintained on chronic hemodialysis for various period. The urine urokinase concentration was higher in the ACKD group (17.5 +/- 14.7 unit/ml, range 13.5-47.0 unit/ml, n = 9) than the control group (4.1 +/- 3.4 unit/ml, range 0.5-12.0 unit/ml, n = 36) (p less than 0.001), and polycyst group (2.6 +/- 1.8 unit/ml, range 1.0-5.1 unit/ml, n = 5) (p less than 0.01). But there was no difference between the control group and polycyst group. In the control group and the ACKD group, there was a direct relation between the dialysis duration and the urokinase concentration and the longer the dialysis duration, the higher the urine urokinase concentration (r squared = 0.424, p = 0.0001). The hemodialysis duration was longer in the ACKD group (42 +/- 17.0 months) than the control group (20.0 +/- 12.5 months) (p less than 0.005). These findings suggest that urokinase may be responsible for cystogenic degeneration in ESRD.

Triiodothyronine-induced cyst formation in metanephric organ culture: the role of increased Na-K-adenosine triphosphatase activity.

Previous organ culture investigations into the pathogenesis of renal cyst formation have demonstrated that glucocorticoid-induced proximal tubular cyst formation is associated with increases in renal sodium-potassium adenosine triphosphatase (Na-K-ATPase) activity. To explore the relationship between cyst production and transport enzyme induction, we examined the effects of the potent inducer of Na-K-ATPase activity, L-3,5,3'-triiodothyronine (T3), on renal tubular morphologic and enzymatic development in murine metanephric organ culture. The addition of T3 (2 X 10(-8) mol/L) to completely characterized, serum-free growth medium produced striking proximal tubular cystic abnormalities. Frank cyst development was preceded by ultrastructural alterations consisting of basolateral intercellular spreading, which increased with progressive tubular dilation. Ultrastructural analysis demonstrated no abnormalities of tubular cyst wall basal laminae, and immunohistologic staining with affinity-purified antibodies to the basal lamina glycoproteins fibronectin, laminin, and entactin, revealed no differences between cystic and control tissue. With use of an enzyme-linked kinetic microassay, T3-induced cystic organ culture explants (CY) showed significant increases in Na-K-ATPase when compared with controls from 72 to 120 hours of organ culture incubation. The initial differences in CY Na-K-ATPase occurred contemporaneously with the earliest ultrastructural evidence of cyst formation, and subsequent increases paralleled progressive tubular cyst formation. Tubular cyst formation in CY could be largely prevented by daily incubation of explants with ouabain, 0.2 mmol/L (final concentration) for 120 minutes without deleterious effects on overall metanephric development. We conclude that T3 induces proximal tubular cyst formation in metanephric organ culture, and that T3-induced increases in Na-K-ATPase have a primary role in the pathogenesis of tubular cyst formation in this model system.

Sodium-potassium ATPase activity mediates cyst formation in metanephric organ culture.

To study the possible role of altered transtubular transport in renal tubular cyst formation, the ontogeny of renal Na-K ATPase was studied during glucocorticoid-induced cystic metanephric tubular development in serum-free, murine organ culture (SFMOC). Utilizing an enzyme-linked kinetic microassay, a developmental profile of total ATPase and specific Na-K ATPase activity was established for control (CON) and glucocorticoid-induced cystic organ culture (CY) explants. During 120 hr of CON and CY organ culture nephrogenesis total Na-K ATPase activity, specific Na-K ATPase activity, and the Na-K ATPase: total ATPase ratio progressively increased, simulating normal in vivo murine enzyme development. However, from 48 to 120 hr of organ culture, CY showed significant increases in Na-K ATPase activity when compared to CON at similar stages of development. Na-K ATPase activity (expressed as nmoles . min-1 . mg protein -1, mean +/- SD) was, at: 48 hr, CY 13.1 +/- 0.7 vs. CON 11.0 +/- 0.9 (P less than 0.01); 72 hr, CY 16.4 +/- 1.1 vs. CON 12.2 +/- 0.7 (P less than 0.001); 96 hr, CY 35.4 +/- 4.9 vs. CON 13.7 +/- 0.4 (P less than 0.001); and 120 hr, CY 26.1 +/- 1.4 vs. CON 16.3 +/- 0.9 (P less than 0.001). The initial differences in CY enzyme activity preceded the earliest ultrastructural evidence of cyst formation by 18 to 24 hr, while subsequent increases in Na-K ATPase activity in CY paralleled progressive tubular cyst formation. Tubular cyst formation in CY could be largely prevented by daily incubation of explants with ouabain, 0.2 mM (final concentration) X 120 min, without deleterious effects on overall metanephric development.(ABSTRACT TRUNCATED AT 250 WORDS)