Role of SIRT1 in HIV-associated kidney disease.

Human immunodeficiency virus (HIV) infection of kidney cells can lead to HIV-associated nephropathy (HIVAN) and aggravate the progression of other chronic kidney diseases. Thus, a better understanding of the mechanisms of HIV-induced kidney cell injury is needed for effective therapy against HIV-induced kidney disease progression. We have previously shown that the acetylation and activation of key inflammatory regulators, NF-κB p65 and STAT3, were increased in HIVAN kidneys. Here, we demonstrate the key role of sirtuin 1 (SIRT1) deacetylase in the regulation of NF-κB and STAT3 activity in HIVAN. We found that SIRT1 expression was reduced in the glomeruli of human and mouse HIVAN kidneys and that HIV-1 gene expression was associated with reduced SIRT1 expression and increased acetylation of NF-κB p65 and STAT3 in cultured podocytes. Interestingly, SIRT1 overexpression, in turn, reduced the expression of negative regulatory factor in podocytes stably expressing HIV-1 proviral genes, which was associated with inactivation of NF-κB p65 and a reduction in HIV-1 long terminal repeat promoter activity. In vivo, the administration of the small-molecule SIRT1 agonist BF175 or inducible overexpression of SIRT1 specifically in podocytes markedly attenuated albuminuria, kidney lesions, and expression of inflammatory markers in Tg26 mice. Finally, we showed that the reduction in SIRT1 expression by HIV-1 is in part mediated through miR-34a expression. Together, our data provide a new mechanism of SIRT1 regulation and its downstream effects in HIV-1-infected kidney cells and indicate that SIRT1/miR-34a are potential drug targets to treat HIV-related kidney disease.

Extracellular vesicles from human bone marrow mesenchymal stem cells repair organ damage caused by cadmium poisoning in a medaka model.

Treatment modalities for kidney disease caused by long-term exposure to heavy metals, such as cadmium (Cd), are limited. Often, chronic, long-term environmental exposure to heavy metal is not recognized in the early stages; therefore, chelation therapy is not an effective option. Extracellular vesicles (EVs) derived from stem cells have been demonstrated to reduce disease pathology in both acute and chronic kidney disease models. To test the ability of EVs derived from human bone marrow mesenchymal stem cells (hBM-MSCs) to treat Cd damage, we generated a Cd-exposed medaka model. This model develops heavy metal-induced cell damage in various organs and tissues, and shows decreased overall survival. Intravenous injection of highly purified EVs from hBM-MSCs repaired the damage to apical and basolateral membranes and mitochondria of kidney proximal tubules, glomerular podocytes, bone deformation, and improved survival. Our system also serves as a model with which to study age- and sex-dependent cell injuries of organs caused by various agents and diseases. The beneficial effects of EVs on the tissue repair process, as shown in our novel Cd-exposed medaka model, may open new broad avenues for interventional strategies.

Epigenetic regulation of RCAN1 expression in kidney disease and its role in podocyte injury.

Mounting evidence suggests that epigenetic modification is important in kidney disease pathogenesis. To determine whether epigenetic regulation is involved in HIV-induced kidney injury, we performed genome-wide methylation profiling and transcriptomic profiling of human primary podocytes infected with HIV-1. Comparison of DNA methylation and RNA sequencing profiles identified several genes that were hypomethylated with corresponding upregulated RNA expression in HIV-infected podocytes. Notably, we found only one hypermethylated gene with corresponding downregulated RNA expression, namely regulator of calcineurin 1 (RCAN1). Further, we found that RCAN1 RNA expression was suppressed in glomeruli in human diabetic nephropathy, IgA nephropathy, and lupus nephritis, and in mouse models of HIV-associated nephropathy and diabetic nephropathy. We confirmed that HIV infection or high glucose conditions suppressed RCAN1 expression in cultured podocytes. This suppression was alleviated upon pretreatment with DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine, suggesting that RCAN1 expression is epigenetically suppressed in the context of HIV infection and diabetic conditions. Mechanistically, increased expression of RCAN1 decreased HIV- or high glucose-induced nuclear factor of activated T cells (NFAT) transcriptional activity. Increased RCAN1 expression also stabilized actin cytoskeleton organization, consistent with the inhibition of the calcineurin pathway. In vivo, knockout of RCAN1 aggravated albuminuria and podocyte injury in mice with Adriamycin-induced nephropathy. Our findings suggest that epigenetic suppression of RCAN1 aggravates podocyte injury in the setting of HIV infection and diabetic nephropathy.

Proteomics analysis of the non-muscle myosin heavy chain IIa-enriched actin-myosin complex reveals multiple functions within the podocyte.

MYH9 encodes non-muscle myosin heavy chain IIA (NMMHCIIA), the predominant force-generating ATPase in non-muscle cells. Several lines of evidence implicate a role for MYH9 in podocytopathies. However, NMMHCIIA's function in podocytes remains unknown. To better understand this function, we performed immuno-precipitation followed by mass-spectrometry proteomics to identify proteins interacting with the NMMHCIIA-enriched actin-myosin complexes. Computational analyses revealed that these proteins belong to functional networks including regulators of cytoskeletal organization, metabolism and networks regulated by the HIV-1 gene nef. We further characterized the subcellular localization of NMMHCIIA within podocytes in vivo, and found it to be present within the podocyte major foot processes. Finally, we tested the effect of loss of MYH9 expression in podocytes in vitro, and found that it was necessary for cytoskeletal organization. Our results provide the first survey of NMMHCIIA-enriched actin-myosin-interacting proteins within the podocyte, demonstrating the important role of NMMHCIIA in organizing the elaborate cytoskeleton structure of podocytes. Our characterization of NMMHCIIA's functions goes beyond the podocyte, providing important insights into its general molecular role.

Inhibition of Notch pathway attenuates the progression of human immunodeficiency virus-associated nephropathy.

The Notch pathway is an evolutionarily conserved signaling cascade that is critical in kidney development and has also been shown to play a pathogenetic role in a variety of kidney diseases. We have previously shown that the Notch signaling pathway is activated in human immunodeficiency virus-associated nephropathy (HIVAN) as well as in a rat model of the disease. In this study, we examined Notch signaling in the well established Tg26 mouse model of HIVAN. Notch signaling components were distinctly upregulated in the kidneys of these mice as well as in immortalized podocytes derived from these mice. Notch1 and Notch4 were upregulated in the Tg26 glomeruli, and Notch4 was also expressed in tubules. Notch ligands Jagged1, Jagged2, Delta-like1, and Delta-like 4 were all upregulated in the tubules of Tg26 mice, but glomeruli showed minimal expression of Notch ligands. To examine a potential pathogenetic role for Notch in HIVAN, Tg26 mice were treated with GSIXX, a gamma secretase inhibitor that blocks Notch signaling. Strikingly, GSIXX treatment resulted in significant improvement in both histological kidney injury scores and renal function. GSIXX-treated Tg26 mice also showed diminished podocyte proliferation and dedifferentiation, cellular hallmarks of the disease. Moreover, GSIXX blocked podocyte proliferation in vitro induced by HIV proteins Nef and Tat. These studies suggest that Notch signaling can promote HIVAN progression and that Notch inhibition may be a viable treatment strategy for HIVAN.

Three-dimensional imaging of fetal mouse kidneys.

Three-dimensional imaging is a valuable tool for analyzing kidney growth and development. This technique provides information about spatial relationships between the branching ureteric bud, nephrons, and other structures within the kidney. Availability of user-friendly volume-rendering software now puts this technique within the capability of most laboratories with access to a confocal microscope. This paper describes how to prepare samples and acquire images and three-dimensional volume-rendered images.

Glomerular MYH9 expression is reduced by HIV-1.

BACKGROUND: The continuing disease burden of HIV-associated nephropathy (HIVAN) warrants better elucidation of its pathogenic mechanisms. Given that loss of MYH9 function causes a Mendelian renal disease, we hypothesized that renal expression of MYH9 is down-regulated by HIV-1 in HIVAN pathogenesis. METHOD AND RESULTS: Using immunofluorescence, we determined that glomerular expression of MYH9 was reduced in the kidneys of HIV-1 transgenic mice. We further determined that Myh9 expression was reduced in HIV-1 transgenic podocytes, statistically significantly at the protein level, and that MYH9 expression was significantly reduced at protein and message level in human podocytes transduced with HIV-1. In analyzing expression in human tissue, we confirmed that MYH9 is abundantly expressed in glomeruli, and podocytes specifically. Finally, we found that MYH9 expression was significantly reduced in human glomeruli in the setting of HIVAN. CONCLUSION: We conclude that the podocyte host response to HIV-1 includes down-regulation of MYH9 expression, and hypothesize that this down-regulation might play a role in the pathogenesis of HIVAN.

PRKX critically regulates endothelial cell proliferation, migration, and vascular-like structure formation.

Angiogenesis is a fundamental step in several important physiological events and pathological conditions including embryonic development, wound repair, tumor growth and metastasis. PRKX was identified as a novel type-I cAMP-dependent protein kinase gene expressed in multiple developing tissues. PRKX has also been shown to be phylogenetically and functionally distinct from PKA. This study presents the first evidence that PRKX stimulates endothelial cell proliferation, migration, and vascular-like structure formation, which are the three essential processes for angiogenesis. In contrast, classic PKA demonstrated an inhibitory effect on endothelia vascular-like structure formation. Our findings suggest that PRKX is an important protein kinase engaged in the regulation of angiogenesis and could play critical roles in various physiological and pathological conditions involving angiogenesis. PRKX binds to Pin-1, Magi-1 and Bag-3, which regulate cell proliferation, apoptosis, differentiation and tumorigenesis. The interaction of PRKX with Pin-1, Magi-1 and Bag-3 could contribute to the stimulating role of PRKX in angiogenesis.

Receptor protein tyrosine phosphatases are novel components of a polycystin complex.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutation of PKD1 and PKD2 that encode polycystin-1 and polycystin-2. Polycystin-1 is tyrosine phosphorylated and modulates multiple signaling pathways including AP-1, and the identity of the phosphatases regulating polycystin-1 are previously uncharacterized. Here we identify members of the LAR protein tyrosine phosphatase (RPTP) superfamily as members of the polycystin-1complex mediated through extra- and intracellular interactions. The first extracellular PKD1 domain of polycystin-1 interacts with the first Ig domain of RPTPσ, while the polycystin-1 C-terminus of polycystin-1 interacts with the regulatory D2 phosphatase domain of RPTPγ. Additional homo- and heterotypic interactions between RPTPs recruit RPTPδ. The multimeric polycystin protein complex is found localised in cilia. RPTPσ and RPTPδ are also part of a polycystin-1/E-cadherin complex known to be important for early events in adherens junction stabilisation. The interaction between polycystin-1 and RPTPγ is disrupted in ADPKD cells, while RPTPσ and RPTPδ remain closely associated with E-cadherin, largely in an intracellular location. The polycystin-1 C-terminus is an in vitro substrate of RPTPγ, which dephosphorylates the c-Src phosphorylated Y4237 residue and activates AP1-mediated transcription. The data identify RPTPs as novel interacting partners of the polycystins both in cilia and at adhesion complexes and demonstrate RPTPγ phosphatase activity is central to the molecular mechanisms governing polycystin-dependent signaling. This article is part of a Special Issue entitled: Polycystic Kidney Disease.

Actin depolymerizing factors cofilin1 and destrin are required for ureteric bud branching morphogenesis.

The actin depolymerizing factors (ADFs) play important roles in several cellular processes that require cytoskeletal rearrangements, such as cell migration, but little is known about the in vivo functions of ADFs in developmental events like branching morphogenesis. While the molecular control of ureteric bud (UB) branching during kidney development has been extensively studied, the detailed cellular events underlying this process remain poorly understood. To gain insight into the role of actin cytoskeletal dynamics during renal branching morphogenesis, we studied the functional requirements for the closely related ADFs cofilin1 (Cfl1) and destrin (Dstn) during mouse development. Either deletion of Cfl1 in UB epithelium or an inactivating mutation in Dstn has no effect on renal morphogenesis, but simultaneous lack of both genes arrests branching morphogenesis at an early stage, revealing considerable functional overlap between cofilin1 and destrin. Lack of Cfl1 and Dstn in the UB causes accumulation of filamentous actin, disruption of normal epithelial organization, and defects in cell migration. Animals with less severe combinations of mutant Cfl1 and Dstn alleles, which retain one wild-type Cfl1 or Dstn allele, display abnormalities including ureter duplication, renal hypoplasia, and abnormal kidney shape. The results indicate that ADF activity, provided by either cofilin1 or destrin, is essential in UB epithelial cells for normal growth and branching.

HIV type-1 transgene expression in mice alters adipose tissue and adipokine levels: towards a rodent model of HIV type-1 lipodystrophy.

BACKGROUND: Lipodystrophy in HIV type-1 (HIV-1)-infected patients is the consequence of effects originating from antiretroviral treatment and HIV-1 infection. We have studied adipose tissues and circulating parameters in mice bearing the HIV-1 transgene as a model to provide insight into the role of HIV-1-infection-related events in fat alterations. METHODS: Heterozygous transgenic mice expressing a 7.7 kb HIV-1 construct (Tg26+/-) were used. Cytokine and adipokine levels were quantified using multiplex procedures. Gene expression and mitochondrial DNA abundance in visceral and subcutaneous white adipose tissues and in brown fat were determined using quantitative real-time PCR. RESULTS: The amount of visceral, but not subcutaneous, adipose depot was lower in Tg26+/- mice. Serum proinflammatory cytokine levels were increased in Tg26+/- mice, whereas adiponectin and leptin levels were reduced. Gene expression of monocyte chemoattractant protein-1 was induced in visceral and subcutaneous fat, whereas tumour necrosis factor-α and interleukin-6 were induced in visceral and subcutaneous white adipose tissues, respectively. Adiponectin and leptin gene expression was repressed in all white fat depots, in concert with reduced expression of peroxisome proliferator-activated receptor γ, a master controller of adipogenesis. In brown fat, a coordinate induction in the expression of thermogenesis marker genes was observed. CONCLUSIONS: HIV-1 transgene expression in mice causes changes in adipose tissue reminiscent of those in patients with HIV-1 lipodystrophy, particularly early pretreatment changes. These data support a role for HIV-1-infection-related events in eliciting adipose tissue dysfunction. The Tg26+/- mouse appears as a promising model to assess the effects of HIV-1 infection on adipose tissue and for determining the effects of antiretroviral drugs on an HIV-1-infected background.

Kidney development in the absence of Gdnf and Spry1 requires Fgf10.

GDNF signaling through the Ret receptor tyrosine kinase (RTK) is required for ureteric bud (UB) branching morphogenesis during kidney development in mice and humans. Furthermore, many other mutant genes that cause renal agenesis exert their effects via the GDNF/RET pathway. Therefore, RET signaling is believed to play a central role in renal organogenesis. Here, we re-examine the extent to which the functions of Gdnf and Ret are unique, by seeking conditions in which a kidney can develop in their absence. We find that in the absence of the negative regulator Spry1, Gdnf, and Ret are no longer required for extensive kidney development. Gdnf-/-;Spry1-/- or Ret-/-;Spry1-/- double mutants develop large kidneys with normal ureters, highly branched collecting ducts, extensive nephrogenesis, and normal histoarchitecture. However, despite extensive branching, the UB displays alterations in branch spacing, angle, and frequency. UB branching in the absence of Gdnf and Spry1 requires Fgf10 (which normally plays a minor role), as removal of even one copy of Fgf10 in Gdnf-/-;Spry1-/- mutants causes a complete failure of ureter and kidney development. In contrast to Gdnf or Ret mutations, renal agenesis caused by concomitant lack of the transcription factors ETV4 and ETV5 is not rescued by removing Spry1, consistent with their role downstream of both RET and FGFRs. This shows that, for many aspects of renal development, the balance between positive signaling by RTKs and negative regulation of this signaling by SPRY1 is more critical than the specific role of GDNF. Other signals, including FGF10, can perform many of the functions of GDNF, when SPRY1 is absent. But GDNF/RET signaling has an apparently unique function in determining normal branching pattern. In contrast to GDNF or FGF10, Etv4 and Etv5 represent a critical node in the RTK signaling network that cannot by bypassed by reducing the negative regulation of upstream signals.

Reduction of Stat3 activity attenuates HIV-induced kidney injury.

HIV-1 Nef induces podocyte proliferation and dedifferentiation by activating the Stat3 and MAPK1,2 pathways. Activation of Stat3 also occurs in human kidneys affected by HIV-associated nephropathy (HIVAN), but its contribution to the development of HIVAN is unknown. Here, we generated HIV-1 transgenic mice (Tg26) with either 75% Stat3 activity (Tg26-SA/+) or 25% Stat3 activity (Tg26-SA/-). The kidneys of Tg26-SA/+ mice, but not Tg26-SA/- mice, showed increased Stat3 phosphorylation. The Tg26-SA/+ phenotype was not different from Tg26 mice, but Tg26-SA/- mice developed significantly less proteinuria, glomerulosclerosis, and tubulointerstitial injury. Tg26-SA/+ mice exhibited reduced expression of podocyte differentiation markers and increased expression of VEGF and proliferation markers as compared to Tg26-SA/- mice. Primary podocytes isolated from Tg26-SA/+ mice showed increased Stat3 phosphorylation and reduced expression of podocyte differentiation markers. The tubulointerstitial compartment and isolated tubules of Tg26-SA/+ mice also had increased Stat3 phosphorylation and expression of Stat3 target genes. We confirmed that the expression of the HIV-1 transgene and reduction of Stat3 activity did not affect T and B cell development. In conclusion, Stat3 plays a critical role in the pathogenesis of HIVAN.

Protein kinase-X interacts with Pin-1 and Polycystin-1 during mouse kidney development.

The regulation of epithelial branching morphogenesis by bone morphogenetic protein-7 depends, in part, on functionally defined cyclic adenosine monophosphate (cAMP)-dependent protein kinases. We previously identified protein kinase-X (PRKX), a cAMP-dependent kinase, as a regulator of epithelial morphogenesis during kidney development and found that it binds to and phosphorylates Polycystin-1. Overexpression of PRKX stimulates renal epithelial cell migration, tubulogenesis, ureteric bud branching, and glomerular induction in embryonic mouse kidney explants in organ cultures. Here we determined the physiological functions of endogenous PRKX. Knockdown by siRNA of PRKX gene expression in a human fetal collecting tubule (HFCT) cell line exceeded 70% and resulted in decreased cell migration and increased adhesion of the cells to a collagen I matrix. In embryonic mouse kidney explants, the same degree of knockdown decreased ureteric bud branching and glomerular induction. Because PRKX BAG-3 PIN-1 and MAGI-1 are all expressed in ureteric bud derivatives, we tested for interactions among them and found that PRKX binds to all three proteins through its WW domain as determined by TransSignal domain arrays, and it coimmunoprecipitated with Pin-1 in HFCT cell lysates. These studies suggest that Polycystin-1 and Pin-1 may mediate the function of PRKX in kidney development.

A transgenic mouse that reveals cell shape and arrangement during ureteric bud branching.

Understanding the cellular events that underlie epithelial morphogenesis is a key problem in developmental biology. Here, we describe a new transgenic mouse line that makes it possible to visualize individual cells specifically in the Wolffian duct and ureteric bud, the epithelial structures that give rise to the collecting system of the kidney. myr-Venus, a membrane-associated form of the fluorescent protein Venus, was expressed in the ureteric bud lineage under the control of the Hoxb7 promoter. In Hoxb7/myr-Venus mice, the outlines of all Wolffian duct and ureteric bud epithelial cells are strongly labeled at all stages of urogenital development, allowing the shapes and arrangements of individual cells to be readily observed by confocal microscopy of freshly excised or cultured kidneys. This strain should be extremely useful for studies of cell behavior during ureteric bud branching morphogenesis in wild type and mutant mouse lines.

Protein kinase X (PRKX) can rescue the effects of polycystic kidney disease-1 gene (PKD1) deficiency.

Autosomal dominant polycystic kidney disease (ADPKD) is a common, genetically determined developmental disorder of the kidney that is characterized by cystic expansion of renal tubules and is caused by truncating mutations and haplo-insufficiency of the PKD1 gene. Several defects in cAMP-mediated proliferation and ion secretion have been detected in ADPKD cyst-lining epithelia. Unlike the ubiquitous PKA, the cAMP-dependent CREB-kinase, Protein Kinase X (PRKX) is developmentally regulated, tissue restricted and induces renal epithelial cell migration, and tubulogenesis in vitro as well as branching morphogenesis of ureteric bud in developing kidneys. The possibility of functional interactions between PKD1-encoded polycystin-1 and PRKX was suggested by the renal co-distribution of PRKX and polycystin-1 and the binding and phosphorylation of the C-terminal of polycystin-1 by PRKX at S4166 in vitro. Early consequences of PKD1 mutation include increased tubule epithelial cell-matrix adhesion, decreased migration, reduced ureteric bud branching and aberrant renal tubule dilation. To determine whether PRKX might counteract the adverse effects of PKD1 mutation, human ADPKD epithelial cell lines were transfected with constitutively active PRKX and shown to rescue characteristic adhesion and migration defects. In addition, the co-injection of constitutively active PRKX with inhibitory pMyr-EGFP-PKD1 into the ureteric buds of mouse embryonic kidneys in organ culture resulted in restoration of normal branching morphogenesis without cystic tubular dilations. These results suggest that PRKX can restore normal function to PKD1-deficient kidneys and have implications for the development of preventative therapy for ADPKD.

Mouse embryonic stem cell-derived embryoid bodies generate progenitors that integrate long term into renal proximal tubules in vivo.

The metanephric kidney is a mesodermal organ that develops as a result of reciprocal interactions between the ureteric bud and the blastema. The generation of embryonic stem (ES) cell-derived progenitors offers potential for regenerative therapies but is often limited by development of tumor formation. Because brachyury (T) denotes mesoderm specification, a mouse ES cell line with green fluorescence protein (GFP) knocked into the functional T locus as well as lacZ in the ROSA26 locus (LacZ/T/GFP) was used in cell selection and lineage tracing. In the absence of leukemia inhibitory factor, mouse ES cells give rise to embryoid bodies that can differentiate into mesoderm. Culture conditions were optimized (4 d, 10 ng/ml Activin-A) to generate maximal numbers of renal progenitor populations identified by expression of the specific combination of renal markers cadherin-11, WT-1, Pax-2, and Wnt-4. LacZ/T/GFP+ cells were further enriched by FACS selection. Five days after injection of LacZ/T/GFP+ cells into embryonic kidney explants in organ culture, beta-galactosidase immunohistochemistry showed incorporation into blastemal cells of the nephrogenic zone. After a single injection into developing live newborn mouse kidneys, co-localization studies showed that the LacZ/T/GFP+ cells were stably integrated into proximal tubules with normal morphology and normal polarization of alkaline phosphatase and aquaporin-1 for 7 mo, without teratoma formation. It is concluded that defined differentiation of ES cells into embryoid bodies with Activin-A and selection for T expression provides a means to isolate and purify renal proximal tubular progenitor cells with the potential for safe use in regenerative therapies.

Branching morphogenesis of the ureteric epithelium during kidney development is coordinated by the opposing functions of GDNF and Sprouty1.

Branching of ureteric bud-derived epithelial tubes is a key morphogenetic process that shapes development of the kidney. Glial cell line-derived neurotrophic factor (GDNF) initiates ureteric bud formation and promotes subsequent branching morphogenesis. Exactly how GDNF coordinates branching morphogenesis is unclear. Here we show that the absence of the receptor tyrosine kinase antagonist Sprouty1 (Spry1) results in irregular branching morphogenesis characterized by both increased number and size of ureteric bud tips. Deletion of Spry1 specifically in the epithelium is associated with increased epithelial Wnt11 expression as well as increased mesenchymal Gdnf expression. We propose that Spry1 regulates a Gdnf/Ret/Wnt11-positive feedback loop that coordinates mesenchymal-epithelial dialogue during branching morphogenesis. Genetic experiments indicate that the positive (GDNF) and inhibitory (Sprouty1) signals have to be finely balanced throughout renal development to prevent hypoplasia or cystic hyperplasia. Epithelial cysts develop in Spry1-deficient kidneys that share several molecular characteristics with those observed in human disease, suggesting that Spry1 null mice may be useful animal models for cystic hyperplasia.

Inhibition of HER-2(neu/ErbB2) restores normal function and structure to polycystic kidney disease (PKD) epithelia.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a very common lethal monogenetic disease with significant morbidities and a high likelihood of progression to renal failure for which there is no proven disease-specific therapy currently available for clinical use. Human ADPKD cystic epithelia have proliferative abnormalities mediated by EGFR over-expression and mispolarization leading autocrine response to EGF family ligands. We now show that apical localization of EGFR complexes in normal fetal and ADPKD epithelia is associated with heterodimerization of EGFR(HER-1) with HER-2(neu/ErbB2), while basal membrane localization in normal adult renal epithelia is associated with EGFR(HER-1) homodimers. Since ADPKD epithelial cells have reduced migratory function, this was used as a bioassay to evaluate the ability of compounds to rescue the aberrant human ADPKD phenotype. General tyrosine kinase inhibition by herbimycin and specific inhibition of HER-2(neu/ErbB2) by AG825 or pretreatment with ErbB2 siRNA reversed the migration defect of ADPKD epithelia. Selective inhibition of EGFR(HER-1) showed partial rescue. Increased ADPKD cell migration after inhibition of p38MAP kinase but not of PI3-kinase implicated p38MAPK downstream of HER-2(neu/ErbB2) stimulation. Daily administration of AG825 to PKD1 null heterozygous mice significantly inhibited the development of renal cysts. These studies implicate HER2(neu/ErbB2) as an effector of apical EGFR complex mispolarization and that its inhibition should be considered a candidate for clinical therapy of ADPKD.

Image processing software for 3D light microscopy.

Advances in microscopy now enable researchers to easily acquire multi-channel three-dimensional (3D) images and 3D time series (4D). However, processing, analyzing, and displaying this data can often be difficult and time- consuming. We discuss some of the software tools and techniques that are available to accomplish these tasks.