Pkd1 Knockout Mice Research Articles

Histone deacetylases 6 increases the cyclic adenosine monophosphate level and promotes renal cyst growth

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by abnormal enhanced cell proliferation and fluid secretion, which are triggered by increased levels of cyclic adenosine monophosphate (cAMP). Cebotaru etal. showed that a HDAC6 inhibitor reduced the cAMP level and inhibited cyst formation in Pkd1 knockout mice, which may become a new potential therapeutic agent for ADPKD. This study also raised several intriguing questions that might advance our understanding of the molecular pathogenesis of ADPKD.

Abstract 19664: Molecular Mechanisms of Gα12 Regulated E-cadherin Shedding in Epithelial Cells

Introduction and hypothesis: E-cadherin plays an important role in maintaining the integrity of cell polarity and cell junctions. It is one of the major proteins that forms part of the polycystin-1 (PC1) complex in renal epithelial cells. We previously showed that deletion of the PC1-regulated protein, Gα12, protected kidneys from development of kidney cysts induced by Pkd1 inactivation and activation of Gα12 increased the shedding of E-cadherin in autosomal dominant polycystic kidney disease (ADPKD). The objective of this study was to determine the molecular mechanisms by which Gα12 regulates E-cadherin shedding. Methods: We analyzed polycystic kidneys from human and from Pkd1 knock-out mice. Using Madin-Darby canine kidney (MDCK) cells, we developed a Pkd1 deletion in vitro model using Pkd1 siRNA. Results: Our data shows that Pkd1 deletion caused accumulation of cleaved E-cadherin fragment in renal cystic fluid in both human polycystic kidneys and in our mice model. In addition, we found that activation of Gα12 increased the active form of ADAM10, the cleavage protein for E-cadherin in both human and mice polycystic kidneys, in vivo. In our MDCK cellular three-dimensional culture system, Gα12 activation promoted cyst formation and this phenomenon was inhibited by ADAM10 knockdown. Furthermore, knockdown of ADAM10 abolished the shedding of E-cadherin caused by Gα12 activation. These data indicate that ADAM10 is the major sheddase for cleavage of E-cadherin caused by Gα12 activation. Conclusion: Our results demonstrate that Gα12 activation increases ADAM10 activity and promotes the ectodomain shedding of E-cadherin, which is an important mechanism in the development of kidney cysts induced by Pkd1 deletion in ADPKD.

Renal expression of FGF23 and peripheral resistance to elevated FGF23 in rodent models of polycystic kidney disease

Fibroblast growth factor 23 (FGF23) regulates phosphate homeostasis and is linked to cardiovascular disease and all-cause mortality in chronic kidney disease. FGF23 rises in patients with CKD stages 2-3, but in patients with autosomal dominant polycystic kidney disease, the increase of FGF23 precedes the first measurable decline in renal function. The mechanisms governing FGF23 production and effects in kidney disease are largely unknown. Here we studied the relation between FGF23 and mineral homeostasis in two animal models of PKD. Plasma FGF23 levels were increased 10-fold in 4-week-old cy/+ Han:SPRD rats, whereas plasma urea and creatinine concentrations were similar to controls. Plasma calcium and phosphate levels as well as TmP/GFR were similar in PKD and control rats at all time points examined. Expression and activity of renal phosphate transporters, the vitamin D3-metabolizing enzymes, and the FGF23 co-ligand Klotho in the kidney were similar in PKD and control rats through 8 weeks of age, indicating resistance to FGF23, although phosphorylation of the FGF receptor substrate 2α protein was enhanced. In the kidneys of rats with PKD, FGF23 mRNA was highly expressed and FGF23 protein was detected in cells lining renal cysts. FGF23 expression in bone and spleen was similar in control rats and rats with PKD. Similarly, in an inducible Pkd1 knockout mouse model, plasma FGF23 levels were elevated, FGF23 was expressed in kidneys, but renal phosphate excretion was normal. Thus, the polycystic kidney produces FGF23 but is resistant to its action.

Activation of Gα12 regulates ectodomain shedding of cadherins in renal epithelial cells (1124.1)

Disruption of cell‐cell and cell‐matrix adhesions in kidney epithelial cells is involved in the progression of autosomal dominant polycystic kidney disease (ADPKD). We previously showed that polycystin‐1 (PC1) inhibits activation of the G‐protein, which results in downregulation of focal adhesions. The aim of this study was to investigate whether adheren junction proteins are involved in the progression of renal cyst formation in ADPKD after Gα12 activation. Using mass spectral analysis, we first showed expression of E‐, N‐ and K‐cadherins are altered in kidney epithelial cells after Gα12 activation. Gα12 activation or Pkd1 knockout increases ectodomain shedding of E‐cadherin and N‐cadherin. In addition, the early form of N‐cadherin is increased and the late form is decreased after Gα12 activation. In renal cystic fluid collected from Pkd1 knockout mice and ADPKD patients, shed fragments from both E‐cadherin and N‐cadherin are increased but no change in K‐cadherin, which indicates a specific change of E‐cadherin and N‐cadherin by Gα12 activation. Immunostaining of kidney tissue showed that N‐cadherin is localized in nucleus after Gα12 activation, consistent with WNT/β‐catenin signaling pathway. In conclusion, our data shows that Gα12 regulates shedding of E‐cadherin and N‐cadherin and affects the cell‐cell adhesion and cell polarity of kidney epithelial cells in the development of kidney cysts in ADPKD.

Targeting of Receptor for Advanced Glycation End Products Suppresses Cyst Growth in Polycystic Kidney Disease

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited renal disorder. Although a myriad of research groups have attempted to identify a new therapeutic target for ADPKD, no drug has worked well in clinical trials. Our research group has focused on the receptor for advanced glycation end products (RAGE) gene as a novel target for ADPKD. This gene is involved in inflammation and cell proliferation. We have already confirmed that blocking RAGE function attenuates cyst growth in vitro. Based on this previous investigation, our group examined the effect of RAGE on cyst enlargement in vivo. PC2R mice, a severe ADPKD mouse model that we generated, were utilized. An adenovirus containing anti-RAGE shRNA was injected intravenously into this model. We observed that RAGE gene knockdown resulted in loss of kidney weight and volume. Additionally, the cystic area that originated from different nephron segments decreased in size because of down-regulation of the RAGE gene. Blood urea nitrogen and creatinine values tended to be lower after inhibiting RAGE. Based on these results, we confirmed that the RAGE gene could be an effective target for ADPKD treatment.

Sirtuin 1 inhibition delays cyst formation in autosomal-dominant polycystic kidney disease

Autosomal-dominant polycystic kidney disease (ADPKD) is caused by mutations in either PKD1 or PKD2 and is characterized by the development of multiple bilateral renal cysts that replace normal kidney tissue. Here, we used Pkd1 mutant mouse models to demonstrate that the nicotinamide adenine dinucleotide-dependent (NAD-dependent) protein deacetylase sirtuin 1 (SIRT1) is involved in the pathophysiology of ADPKD. SIRT1 was upregulated through c-MYC in embryonic and postnatal Pkd1-mutant mouse renal epithelial cells and tissues and could be induced by TNF-α, which is present in cyst fluid during cyst development. Double conditional knockouts of Pkd1 and Sirt1 demonstrated delayed renal cyst formation in postnatal mouse kidneys compared with mice with single conditional knockout of Pkd1. Furthermore, treatment with a pan-sirtuin inhibitor (nicotinamide) or a SIRT1-specific inhibitor (EX-527) delayed cyst growth in Pkd1 knockout mouse embryonic kidneys, Pkd1 conditional knockout postnatal kidneys, and Pkd1 hypomorphic kidneys. Increased SIRT1 expression in Pkd1 mutant renal epithelial cells regulated cystic epithelial cell proliferation through deacetylation and phosphorylation of Rb and regulated cystic epithelial cell death through deacetylation of p53. This newly identified role of SIRT1 signaling in cystic renal epithelial cells provides the opportunity to develop unique therapeutic strategies for ADPKD.

HDAC6 regulates epidermal growth factor receptor (EGFR) endocytic trafficking and degradation in renal epithelial cells.

We present for the first time that histone deacetylase 6 (HDAC6) regulates EGFR degradation and trafficking along microtubules in Pkd1 mutant renal epithelial cells. HDAC6, the microtubule-associated α-tubulin deacetylase, demonstrates increased expression and activity in Pkd1 mutant mouse embryonic kidney cells. Targeting HDAC6 with a general HDAC inhibitor, trichostatin (TSA), or a specific HDAC6 inhibitor, tubacin, increased the acetylation of α-tubulin and downregulated the expression of EGFR in Pkd1 mutant renal epithelial cells. HDAC6 was co-localized with EGF induced endocytic EGFR and endosomes, respectively. Inhibition of the activity of HDAC6 accelerated the trafficking of EGFR from early endosomes to late endosomes along the microtubules. Without EGF stimulation EGFR was randomly distributed while after stimulation with EGF for 30 min, EGFR was accumulated around α-tubulin labeled microtubule bundles. These data suggested that the Pkd1 mutation induced upregulation of HDAC6 might act to slow the trafficking of EGFR from early endosomes to late endosomes along the microtubules for degradation through deacetylating α-tubulin. In addition, inhibition of HDAC activity decreased the phosphorylation of ERK1/2, the downstream target of EGFR axis, and normalized EGFR localization from apical to basolateral in Pkd1 knockout mouse kidneys. Thus, targeting HDAC6 to downregulate EGFR activity may provide a potential therapeutic approach to treat polycystic kidney disease.

Polycystic Kidney Disease Evaluation by Magnetic Resonance Imaging in Ischemia-Reperfusion Injured PKD1 Knockout Mouse Model

To investigate the efficacy of true fast imaging with steady state precession (true-FISP) compared with T2-weighted fast spin echo (T2W-FSE) in monitoring changes in cyst volume and renal expansion in an ischemia reperfusion (IR) injured PKD1 knockout (IKO) mouse model. The animal study was approved by the local institutional authority. A total of 24 mice (14 PKD1 IKO mice and 10 control mice) were sequentially scanned on a 4.7-T scanner from 4 to 12 weeks after IR injury and the cysts and kidney volumes were measured from true-FISP and T2W-FSE MR images. Signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) were calculated for the images generated from both methods. Cyst SNR and CNR in true-FISP images were significantly higher than in images employing the T2W-FSE sequence (P < 0.05). Both methods demonstrated kidney volume increase as well as cyst growth after IR injury in the mouse model. The kidney and cysts volume at different time points were strongly correlated between true-FISP and T2W-FSE measurements (r(kidney) = 0.99, r(cysts) = 0.97). The true-FISP method offers shorter scan times and higher cyst SNR and cyst to kidney tissue contrast than T2W-FSE. Both sequences have identical accuracy in measuring the cysts and kidney volume changes in mice with PKD.

A Multiplexed Quantitative Strategy for Membrane Proteomics: Opportunities for Mining Therapeutic Targets for Autosomal Dominant Polycystic Kidney Disease

Toward multiplexed, comprehensive, and robust quantitation of the membrane proteome, we report a strategy combining gel-assisted digestion, iTRAQ (isobaric tags for relative and absolute quantitation) labeling, and LC-MS/MS. Quantitation of four independently purified membrane fractions from HeLa cells gave high accuracy (<8% error) and precision (<12% relative S.D.), demonstrating a high degree of consistency and reproducibility of this quantitation platform. Under stringent identification criteria (false discovery rate = 0%), the strategy efficiently quantified membrane proteins; as many as 520 proteins (91%) were membrane proteins, each quantified based on an average of 14.1 peptides per integral membrane protein. In addition to significant improvements in signal intensity for most quantified proteins, most remarkably, topological analysis revealed that the biggest improvement was achieved in detection of transmembrane peptides from integral membrane proteins with up to 19 transmembrane helices. To the best of our knowledge, this level of coverage exceeds that achieved previously using MS and provides superior quantitation accuracy compared with other methods. We applied this approach to the first proteomics delineation of phenotypic expression in a mouse model of autosomal dominant polycystic kidney disease (ADPKD). By characterizing kidney cell plasma membrane from wild-type versus PKD1 knock-out mice, 791 proteins were quantified, and 67 and 37 proteins showed > or =2-fold up-regulation and down-regulation, respectively. Some of these differentially expressed membrane proteins are involved in the mechanisms underlying major abnormalities in ADPKD, including epithelial cell proliferation and apoptosis, cell-cell and cell-matrix interactions, ion and fluid secretion, and membrane protein polarity. Among these proteins, targeting therapeutics to certain transporters/receptors, such as epidermal growth factor receptor, has proven effective in preclinical studies of ADPKD; others are known drug targets in various diseases. Our method demonstrates how comparative membrane proteomics can provide insight into the molecular mechanisms underlying ADPKD and the identification of potential drug targets, which may lead to new therapeutic opportunities to prevent or retard the disease.

Loss of polycystin-1 causes centrosome amplification and genomic instability

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenetic disease predominantly caused by alteration or dysregulation of the PKD1 gene, which encodes polycystin-1 (PC1). The disease is characterized by the progressive expansion of bilateral fluid-filled renal cysts that ultimately lead to renal failure. Individual cysts, even within patients with germline mutations, are genetically heterogeneous, displaying diverse chromosomal abnormalities. To date, the molecular mechanisms responsible for this genetic heterogeneity remain unknown. Using a lentiviral-mediated siRNA expression model of Pkd1 hypomorphism, we show that loss of PC1 function is sufficient to produce centrosome amplification and multipolar spindle formation. These events lead to genomic instability characterized by gross polyploidism and mitotic catastrophe. Following these dramatic early changes, the cell population rapidly converges toward a stable ploidy in which centrosome amplification is significantly decreased, though cytological abnormalities such as micronucleation, chromatin bridges and aneuploidy remain common. In agreement with our in vitro findings, we provide the first in vivo evidence that significant centrosome amplification occurs in kidneys from conditional Pkd1 knockout mice at early and late time during the disease progression as well as in human ADPKD patients. These findings establish a novel function of PC1 in ADPKD pathogenesis and a genetic mechanism that may underlie the intrafamilial variability of ADPKD progression.

Small-Molecule CFTR Inhibitors Slow Cyst Growth in Polycystic Kidney Disease

Cyst expansion in polycystic kidney disease (PKD) involves progressive fluid accumulation, which is believed to require chloride transport by the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Herein is reported that small-molecule CFTR inhibitors of the thiazolidinone and glycine hydrazide classes slow cyst expansion in in vitro and in vivo models of PKD. More than 30 CFTR inhibitor analogs were screened in an MDCK cell model, and near-complete suppression of cyst growth was found by tetrazolo-CFTR(inh)-172, a tetrazolo-derived thiazolidinone, and Ph-GlyH-101, a phenyl-derived glycine hydrazide, without an effect on cell proliferation. These compounds also inhibited cyst number and growth by >80% in an embryonic kidney cyst model involving 4-d organ culture of embryonic day 13.5 mouse kidneys in 8-Br-cAMP-containing medium. Subcutaneous delivery of tetrazolo-CFTR(inh)-172 and Ph-GlyH-101 to neonatal, kidney-specific PKD1 knockout mice produced stable, therapeutic inhibitor concentrations of >3 microM in urine and kidney tissue. Treatment of mice for up to 7 d remarkably slowed kidney enlargement and cyst expansion and preserved renal function. These results implicate CFTR in renal cyst growth and suggest that CFTR inhibitors may hold therapeutic potential to reduce cyst growth in PKD.

Small‐Molecule CFTR Inhibitors Slow CYST Growth in Culture and Mouse Models of Polycystic Kidney Disease

Polycystic kidney disease (PKD) is a major cause of chronic renal insufficiency. Cyst expansion in PKD involves progressive fluid accumulation, which is believed to require chloride transport by the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The Aim of this study is to test if small‐molecule CFTR inhibitors of the thiazolidinone and glycine hydrazide classes slow cyst expansion in in vitro and in vivo models of PKD. Screening of more than 30 CFTR inhibitor analogs in an MDCK cell cyst model indicated near complete suppression of cyst growth by the tetrazolo‐derivatized thiazolidinone, tetrazolo‐CFTRinh‐172, and the phenyl‐derivatized glycine hydrazide, Ph‐GlyH‐101, without effect on cell proliferation. These compounds also inhibited cyst number and growth by &gt; 80 % in an embryonic kidney cyst model involving 4‐day organ culture of E13.5 mouse kidneys in 8‐Br‐cAMP‐containing medium. Subcutaneous delivery of tetrazolo‐CFTRinh‐172 and Ph‐GlyH‐101 to neonatal, kidney‐specific PKD1 knock‐out mice produced stable and ‘therapeutic’ inhibitor concentrations of &gt;3 μM in urine and kidney tissue. Treatment of mice for up to 7 days remarkably slowed kidney enlargement and cyst expansion, and preserved renal function. These results indicate the involvement of CFTR in renal cyst growth in PKD, and provide proof‐of‐concept for application of CFTR inhibitors to reduce cyst growth in PKD.

Lowering of Pkd1 expression is sufficient to cause polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is a major cause of renal failure and is characterized by the formation of many fluid-filled cysts in the kidneys. It is a systemic disorder that is caused by mutations in PKD1 or PKD2. Homozygous inactivation of these genes at the cellular level, by a 'two-hit' mechanism, has been implicated in cyst formation but does not seem to be the sole mechanism for cystogenesis. We have generated a novel mouse model with a hypomorphic Pkd1 allele, Pkd1(nl), harbouring an intronic neomycin-selectable marker. This selection cassette causes aberrant splicing of intron 1, yielding only 13-20% normally spliced Pkd1 transcripts in the majority of homozygous Pkd1(nl) mice. Homozygous Pkd1(nl) mice are viable, showing bilaterally enlarged polycystic kidneys. This is in contrast to homozygous knock-out mice, which are embryonic lethal, and heterozygous knock-out mice that show only a very mild cystic phenotype. In addition, homozygous Pkd1(nl) mice showed dilatations of pancreatic and liver bile ducts, and the mice had cardiovascular abnormalities, pathogenic features similar to the human ADPKD phenotype. Removal of the neomycin selection-cassette restored the phenotype of wild-type mice. These results show that a reduced dosage of Pkd1 is sufficient to initiate cystogenesis and vascular defects and indicate that low Pkd1 gene expression levels can overcome the embryonic lethality seen in Pkd1 knock-out mice. We propose that in patients reduced PKD1 expression of the normal allele below a critical level, due to genetic, environmental or stochastic factors, may lead to cyst formation in the kidneys and other clinical features of ADPKD.

Recurrent fetal loss associated with bilineal inheritance of type 1 autosomal dominant polycystic kidney disease

Background: Autosomal dominant polycystic kidney disease (ADPKD) is a common Mendelian disorder that affects approximately 1 in 500 to 1,000 live births. Mutations in one of two genes, PKD1 and PKD2, account for the disease in most ADPKD families. Despite the relative high frequency of PKD1 mutant alleles, compound heterozygotes or diseased homozygotes have not been described. Methods and Results: We report a family with type 1 ADPKD in which the marriage between affected first-degree cousins resulted in two live-born heterozygous offspring and two fetuses lost in late pregnancy. Genetic analysis with PKD1 and PKD2 flanking markers showed that this family is PKD1 linked (zmax = 1.66 and −2.54 at θ = 0.0 for intragenic markers for PKD1 [ie, KG8] and PKD2 [ie, SPP1], respectively). Conclusion: Given a 25% chance for mutant homozygosity in the offspring of this family, our findings suggest that homozygosity of PKD1 mutations in humans is embryonically lethal, as recently documented in Pkd1 knockout mice. © 2002 by the National Kidney Foundation, Inc.

Current advances in molecular genetics of autosomal-dominant polycystic kidney disease.

Autosomal-dominant polycystic kidney disease results from at least two causal genes, PKD1 and PKD2. The identical clinical phenotype in human patients and targeted Pkd1 and Pkd2 mutant mouse models provides evidence that both gene products act in the same pathogenic pathway. The discovery of direct PKD1 and PKD2 interactions implies that both gene products, polycystin-1 and polycystin-2, play a functional role in the same molecular complex. The spectrum of germ-line mutations in both genes and the somatic mutations identified from individual PKD1 or PKD2 cysts indicate that loss of function of either PKD1 or PKD2 is the mechanism of cystogenesis in autosomal-dominant polycystic kidney disease. A novel mouse model, Pkd2WS25/-, has proved that loss of heterozygosity is the molecular mechanism of autosomal-dominant polycystic kidney disease. Recently, studies on the expression patterns of PKD1 and PKD2 in humans or mice indicate that polycystin 1 and polycystin 2 seem to have their own respective functional roles, even though most of the functions of these polycystins are parallel during human and mouse development. Pkd2-deficient mice have cardiac septum defects, but Pkd1 knockout mice do not have this phenotype. On the other hand, Pkd2 has a very low level of expression in the central nervous system when compared with Pkd1. In addition, the level of expression of Pkd1 is increased during mesenchymal condensation, whereas Pkd2 expression is unchanged. Preliminary data have shown that the PKD1/PKD2 compound trans-heterozygous has a more severe cystic phenotype in the kidney than that of an age-matched heterozygous type 1 or type 2 of autosomal-dominant polycystic kidney disease alone. This finding suggests that PKD1 may be a modifier of disease severity for PKD2, and vice versa. The characteristics of the contiguous PKD1/TSC2 syndrome phenotypes and the data from Krd mice imply that TSC2 and PAX2 may also serve as potential modifiers for the disease severity of autosomal-dominant polycystic kidney disease.

A human PKD1 transgene generates functional polycystin-1 in mice and is associated with a cystic phenotype.

Three founder transgenic mice were generated with a 108 kb human genomic fragment containing the entire autosomal dominant polycystic kidney disease (ADPKD) gene, PKD1, plus the tuberous sclerosis gene, TSC2. Two lines were established (TPK1 and TPK3) each with approximately 30 copies of the transgene. Both lines produced full-length PKD1 mRNA and polycystin-1 protein that was developmentally regulated, similar to the endogenous pattern, with expression during renal embryogenesis and neonatal life, markedly reduced at the conclusion of renal development. Tuberin expression was limited to the brain. Transgenic animals from both lines (and the TPK2 founder animal) often displayed a renal cystic phenotype, typically consisting of multiple microcysts, mainly of glomerular origin. Hepatic cysts and bile duct proliferation, characteristic of ADPKD, were also seen. All animals with two copies of the transgenic chromosome developed cysts and, in total, 48 of the 100 transgenic animals displayed a cystic phenotype. To test the functionality of the transgene, animals were bred with the Pkd1(del34) knockout mouse. Both transgenic lines rescued the embryonically lethal Pkd1(del34/del34) phenotype, demonstrating that human polycystin-1 can complement for loss of the endogenous protein. The rescued animals were viable into adulthood, although more than half developed hepatic cystic disease in later life, similar to the phenotype of older Pkd1(del34/+) animals. The TPK mice have defined a minimal area that appropriately expresses human PKD1. Furthermore, this model indicates that over-expression of normal PKD1 can elicit a disease phenotype, suggesting that the level of polycystin-1 expression may be relevant in the human disease.