Cystic kidney diseases and planar cell polarity signaling.

Renal cystic diseases are a major clinical concern as they are the most common genetic cause of end-stage renal disease. While many of the genes causing cystic disease have been identified in recent years, knowing the molecular nature of the mutations has not clarified the mechanisms underlying cyst formation. Recent research in model organisms has suggested that cyst formation may be because of defective planar cell polarity (PCP) and/or ciliary defects. In this review, we first outline the clinical features of renal cystic diseases and then discuss current research linking our understanding of cystic kidney disease to PCP and cilia.

Direct in situ reverse transcriptase-polymerase chain reaction.

In situ hybridization has been used for localization of specific nucleic acid sequences at the cellular level despite providing relatively low-detection sensitivity. In situ reverse transcriptase-polymerase chain reactions (RT-PCR) enhance sensitivity and thus enable localization of low-abundance mRNA in a cell. However, the available methods are fraught with problems of nonspecific amplifications as a result of mispriming and/or amplification from partially digested residual genomic DNA in tissue. Herein, we demonstrate that nonspecific background amplification can be eliminated by pretreatment of samples with restriction enzymes before DNase I digestion. Primers tagged with a far-red shifted fluorescent dye such as Cy5 in PCR reactions allow identification of target mRNA by fluorescence microscopy. These novel modifications lead to increased specificity and rapid in situ detection of cellular mRNA and thus may be used for pathological diagnosis.

Compromised cytoarchitecture and polarized trafficking in autosomal dominant polycystic kidney disease cells.

Cystogenesis associated with autosomal dominant polycystic kidney disease (ADPKD) is characterized by perturbations in the polarized phenotype and function of cyst-lining epithelial cells. The polycystins, the protein products of the genes mutated in the majority of ADPKD cases, have been described recently, but the pathological mechanism by which causal mutations result in the mislocalization of cell membrane proteins has remained unclear. This report documents the dissociation from the ADPKD cell basolateral membrane of three molecules essential for spatial organization and exocytosis. The adherens junction protein E-cadherin, the subcellular disposition of which governs intercellular and intracellular architecture, was discovered sequestered in an internal ADPKD cell compartment. At the same time, sec6 and sec8, components of a complex critical for basolateral cargo delivery normally arrayed at the apico-lateral apex, were depleted from the ADPKD cell plasma membrane. An analysis of membrane transport revealed that basolateral trafficking of proteins and lipids was impaired as a result of delayed cargo exit from the ADPKD cell Golgi apparatus. Apical transport proceeded normally. Taken together with recent documentation of an association between polycystin-1 and E-cadherin (Huan and van Adelsberg 1999), the data suggest that causal mutations disrupt E-cadherin-dependent cytoarchitecture, adversely affecting protein assemblies crucial for basolateral trafficking.

ADPKD: a human disease altering Golgi function and basolateral exocytosis in renal epithelia.

Epithelial cells explanted from autosomal dominant polycystic kidney disease (ADPKD) tissue exhibit impaired exocytosis, specifically between the Golgi and basolateral membrane (Charron A, Nakamura B, Bacallo R, Wandinger-Ness A. Compromised cytoarchitecture and polarized trafficking in autosomal dominant polycystic kidney disease cells. J Cell Biol 2000; 148: 111-124.). Here the defect is shown to result in the accumulation of the basolateral transport marker vesicular stomatitis virus (VSV) G protein in the Golgi complex. Golgi complex morphology is consequently altered in the disease cells, evident in the noticeable fenestration and dilation of the cisternae. Further detailed microscopic evaluation of normal kidney and ADPKD cells revealed that ineffective basolateral exocytosis correlated with modulations in the localization of select post-Golgi transport effectors. The cytosolic coat proteins p200/myosin II and caveolin exhibited enhanced association with the cytoskeleton or the Golgi of the disease cells, respectively. Most cytoskeletal components with known roles in vesicle translocation or formation were normally arrayed with the exception of Golgi beta-spectrin, which was less prevalent on vesicles. The rab8 GTPase, important for basolateral vesicle targeting, was redistributed from the perinuclear Golgi region to disperse vesicles in ADPKD cells. At the basolateral membrane of ADPKD cells, there was a notable loss of the exocyst components sec6/sec8 and an unidentified syntaxin. It is postulated that dysregulated basolateral transport effector function precipitates the disruption of basolateral exocytosis and dilation of the ADPKD cell Golgi as basolateral cargo accumulates within the cisternae.

Cablin: a novel protein of the capillary basal lamina.

The microvascular wall is remarkably simple, consisting only of the endothelial lining, subjacent basal lamina, and underlying periendothelial cells. This study describes the characterization of a novel microvascular protein. This 80,000-molecular weight protein was predominantly associated with electron-lucent amorphous material in capillary basal laminae and therefore termed cablin (protein of the capillary basal lamina). Consistent with its immunolocalization to the microvasculature, cablin was synthesized and secreted by cultured endothelial cells and vascular smooth muscle cells. Furthermore, cablin expression was induced during neovascularization. The predicted amino acid sequence of cablin revealed a prevalence of polar amino acids. Accounting for the low yet significant homology to several alpha-helical proteins, these residues were best accommodated by secondary structure predictions that aligned the molecule into two large alpha-helical domains. The presence of the integrin-binding RGD tripeptide and a putative elastin-binding sequence suggest that this rodlike molecule is suited to cross-link cells and matrix constituents. In this capacity it could contribute to the mechanical strength or the angiogenic potential of the microvasculature.

Role of the matrix in autosomal dominant polycystic kidney disease.

At present, even though we have accumulated a wealth of knowledge regarding structural, and molecular changes in ADPKD, the primary cause of the disease remains unknown. Obviously the gap in our understanding of the nature of the disease has been narrowed substantially over the past decade. With current techniques and efforts, the ultimate mystery of ADPKD should be resolved during the next decade.

Apiconuclear organization of microtubules does not specify protein delivery from the trans-Golgi network to different membrane domains in polarized epithelial cells.

In nonpolarized epithelial cells, microtubules originate from a broad perinuclear region coincident with the distribution of the Golgi complex and extend outward to the cell periphery (perinuclear [PN] organization). During development of epithelial cell polarity, microtubules reorganize to form long cortical filaments parallel to the lateral membrane, a meshwork of randomly oriented short filaments beneath the apical membrane, and short filaments at the base of the cell; the Golgi becomes localized above the nucleus in the subapical membrane cytoplasm (apiconuclear [AN] organization). The AN-type organization of microtubules is thought to be specialized in polarized epithelial cells to facilitate vesicle trafficking between the trans-Golgi Network (TGN) and the plasma membrane. We describe two clones of MDCK cells, which have different microtubule distributions: clone II/G cells, which gradually reorganize a PN-type distribution of microtubules and the Golgi complex to an AN-type during development of polarity, and clone II/J cells which maintain a PN-type organization. Both cell clones, however, exhibit identical steady-state polarity of apical and basolateral proteins. During development of cell surface polarity, both clones rapidly establish direct targeting pathways for newly synthesized gp80 and gp135/170, and E-cadherin between the TGN and apical and basolateral membrane, respectively; this occurs before development of the AN-type microtubule/Golgi organization in clone II/G cells. Exposure of both clone II/G and II/J cells to low temperature and nocodazole disrupts >99% of microtubules, resulting in: 1) 25-50% decrease in delivery of newly synthesized gp135/170 and E-cadherin to the apical and basolateral membrane, respectively, in both clone II/G and II/J cells, but with little or no missorting to the opposite membrane domain during all stages of polarity development; 2) approximately 40% decrease in delivery of newly synthesized gp80 to the apical membrane with significant missorting to the basolateral membrane in newly established cultures of clone II/G and II/J cells; and 3) variable and nonspecific delivery of newly synthesized gp80 to both membrane domains in fully polarized cultures. These results define several classes of proteins that differ in their dependence on intact microtubules for efficient and specific targeting between the Golgi and plasma membrane domains.

Optical methods in renal research.

Light microscopy is a powerful experimental approach that allows the researcher to study the dynamics of cellular responses and subcellular organization. Recent advances in optics, detectors, computerized image processing, and the development of vital dyes promise to extend the utility of light microscopy. Quantitative approaches measuring intracellular pH, sodium or calcium are well known, but future developments may permit quantitative analysis of immunolabeled specimens. This review will describe the basic features of light microscopes and imaging techniques. Specific types of microscopy such as confocal microscopy, wide field microscopes, and epifluorescence microscopy will be considered. In addition, recent advances in cellular labeling techniques will be described. Post production image processing techniques and computerized deconvolution methods will be discussed, and lastly future developments in optics, improved sensitivity in detectors with enhanced signal-to-noise ratios will be explored. Wherever possible, specific applications that have been applied to renal physiology studies will be cited.

NHE-1 protein in vascular smooth muscle and lymphocytes from the spontaneously hypertensive rat.

The present study examined the abundance of NHE-1 protein in cultured vascular smooth muscle cells (VSMCs), freshly isolated thymocytes, and fresh aortic tissue from spontaneously hypertensive rats (SHRs) and age-matched Wistar-Kyoto (WKY) rats. Two sets of affinity-purified antibodies (Ab[765-778] and Ab[698-711]) against different epitopes of the NHE-1 isoform of the Na+-H+ antiporter were used. Each set of antibodies recognized a major protein band at 105 to 110 kD that was more abundant in protein lysates prepared from cultured VSMCs from the SHR than those from WKY rats (Ab[765-778] 0.047 +/- 0.011 vs 0.010 +/- 0.002 O.D. units/10 microg protein, P<.001 for SHR and WKY, respectively; and Ab(698-711) 0.173 +/- 0.026 vs 0.087 +/- 0.028 O.D. units/10 microg protein, P<.05, for SHR and WKY, respectively). The increase in NHE-1 protein abundance in cultured VSMCs from the SHR was associated with a greater Vmax of the Na+-H+ antiporter as compared to those from WKY rats (17.93 +/- 2.07 vs 8.16 +/- 1.05 mmol H+/min, P<.001, respectively). In contrast to cultured VSMCs, there was no difference in the relative abundance of NHE-1 protein in fresh aortic tissue (0.075 +/- 0.018 vs 0.083 +/- 0.017 O.D. units/10 microg protein, from SHR and WKY, respectively) or in freshly isolated thymocytes (0.158 +/- 0.046 vs 0.226 +/- 0.054 O.D. units/10 microg protein, from SHR and WKY, respectively). We conclude that the increase in the Vmax of the Na+-H+ antiporter in cultured VSMCs from the SHR, compared to those from WKY rats, is due, at least in part, to increased levels of NHE-1 protein.

Recent advances in the understanding of polycystic kidney disease.

Polycystic kidney disease is characterized by localized autonomous cellular proliferation, compartmentalized fluid accumulation within the cysts, and intraparenchymal fibrosis of the kidney. The clinical features include renal failure, liver cysts, and vascular and cardiac valve abnormalities. Recent developments have extended our understanding of cyst formation, fluid secretion, and the genetics of polycystic kidney disease. Two causal genes for polycystic kidney disease, PKD1 and PKD2, that are responsible for greater than 95% of cases of autosomal dominant polycystic kidney disease, have been identified and sequenced. The mechanisms of cystogenesis are being uncovered and the phenotypic features of cystic epithelial cells are being discovered. This review describes recent advances made in the molecular biology of the genetic causes of polycystic kidney disease. The mechanistic details of cystogenesis are discussed and contrasted with the paradigms that guide current experimental approaches.

Actin and villin compartmentation during ATP depletion and recovery in renal cultured cells.

ATP-depletion in renal cultured cells has been used as a model for studying various cytoskeletal and functional alterations induced by renal ischemia. This communication explores the reversibility of these effects utilizing a novel method [1] that depleted ATP (ATP-D) to 2% of control within 30 minutes and caused complete recovery (REC) of ATP in one hour. Under confocal microscopy, ATP-D (30 min) caused thinning of F-actin from the microvilli, cortical region, and basal stress fibers, with the concurrent appearance of intracellular F-actin patches. These changes were more pronounced after 60 minutes of ATP-D. One hour of REC following 30 minutes of ATP-D produced complete recovery of F-actin in each region of the cell. However, after 60 minutes of ATP-D, a heterogeneous F-actin recovery pattern was observed: almost complete recovery of the apical ring and microvilli, thinned cortical actin with occasional breaks along the basolateral membrane, and a dramatic reduction in basal stress fiber density. The time course of cortical actin and actin ring disruption and recovery coincided with a drop recovery in the transepithelial resistance and the cytoskeletal dissociation and reassociation of the Na,K-ATPase. Additionally, the microvilli retracted into the cells during ATP-D, a process that was reversed during REC. Triton extraction and confocal microscopy demonstrated that villin remained closely associated with microvillar actin during both ATP-D and REC. These distinctive regional differences in the responses of F-actin to ATP depletion and repletion in cultured renal epithelial cells may help to clarify some of the differential tubular responses to ischemia and reperfusion in the kidney.

The role of the cytoskeleton in renal development.

The cytoskeleton is comprised of three separate filament networks: microtubules, microfilaments, and intermediate filaments. Collectively, these networks help establish and maintain the structural features of renal epithelial cells. During renal development, the cytoskeleton of the metanephric mesenchyme is extensively reorganized in order to create the cytoarchitectural elements that distinguish tubuloepithelial cells. This reorganization is coordinated with the formation of cell-cell contacts and cell-extracellular matrix interactions that are necessary to complete the developmental program. The actin cytoskeleton, microtubule network, and intermediate filament network all contribute to the development of polarity in the renal epithelial cells. The microtubule network determines the apical-basal axis of the cell. The actin cytoskeleton integrates topographic contacts between the cells and extracellular matrix. The tight junction and microvilli are subcellular structures that are associated with or comprised of actin filaments. Intermediate filament composition changes during the embryonic transition from metanephric mesenchyme to tubular epithelial cells. This review will describe the cell biology of the cytoskeletal elements in epithelial cells and the changes in cytoskeleton that accompany the formation of differentiated epithelial cells.

Impaired tubulogenesis of cyst-derived cells from autosomal dominant polycystic kidneys.

Under appropriate growth factor or hormonal influence, renal epithelial cells cultured in collagen gels form branching tubular elements, reminiscent of metanephric tubulogenesis. This study evaluates the phenotypic characteristics of normal human renal epithelial cells (NK) and epithelial cells from cysts of autosomal dominant polycystic kidneys (ADPKD) grown in collagen gels under the influence of the growth factors (GFs) epidermal (EGF), transforming (TGF-alpha), hepatocyte (HGF) and fibroblast (FGF). All GFs induced cell proliferation with the formation of cell aggregates in both group of cells, however, NK cells exhibited proliferation at a much higher rate compared to ADPKD. All GFs induced formation of branching tubular elements with cell-polarity characteristics in NK cells. Such organized tubular elements were essentially absent in ADPKD cell cultures. Both NK and ADPKD cells expressed cell adhesion and matrix macromolecules. Expression of heparan sulfate-proteoglycan was diminished but enhanced for fibronectin in ADPKD cells. Receptor expression for EGF and FGF was similar. These findings indicate an impairment in tubulogenesis of ADPKD cells, perhaps related to the aberrant morphogenetic cell aggregation. Alternatively, this differentiation arrest may relate to abnormal biosynthesis of secretory matrix glycoproteins rather than those expressed on the plasmalemma.

The pathogenesis of polycystic kidney disease.

Polycystic kidney disease (PKD) is a genetic or acquired disorder characterized by progressive distention of multiple tubular segments and manifested by fluid accumulation, growth of non-neoplastic epithelial cells and remodeling of the extracellular matrix resulting ultimately in some degree of renal functional impairment, with the potential for regression following removal of the inductive agent(s). It is due to an aberration of one or more factors regulating tubular morphogenesis. Human PKD can pursue a rapid course with renal failure occurring perinatally (infantile PKD) or an indolent course without renal failure developing during the life of the individual (adult PKD). Human acquired PKD develops in atrophic and scarred end-stage kidneys with non-cystic forms of renal disease. Cell proliferation, fluid secretion, impaired cell-cell and cell-matrix interaction, defective function of the Golgi apparatus, cell undifferentiation, and an abnormal matrix have been implicated in the pathogenesis of PKD based on clinical and experimental studies. Under normal conditions, the dynamic turnover of tubular epithelia and matrices are tightly regulated to maintain tubular morphology. The basic defect in PKD is tubular dysmorphogenesis. Our finding indicates that the principal phenotypic features of autosomal dominant PKD (ADPKD) are altered structure and function of the Golgi complex, altered structure and composition of the matrix and cell undifferentiation, all of which are probably interrelated. If the gene product of the ADPKD 1 gene results in a defective matrix, the abnormal Golgi function and cell differentiation may be due to faulty matrix-cell communication.

ATP depletion: a novel method to study junctional properties in epithelial tissues. I. Rearrangement of the actin cytoskeleton.

The effect of cellular injury caused by depletion of intracellular ATP stores was studied in the Madin-Darby canine kidney (MDCK) and JTC cell lines. In prior studies, it was shown that ATP depletion uncouples the gate and fence functions of the tight junction. This paper extends these observations by studying the changes in the actin cytoskeleton and tight junction using electron microscopy and confocal fluorescence microscopy in combination with computer-aided three-dimensional reconstruction. Marked regional differences in the sensitivity to the effects of ATP depletion were observed in the actin cytoskeleton. Actin depolymerization appears to first affect the cortical actin network running along the apical basal axis of the cell. The next actin network that is disrupted is the stress fibers found at the basal surface of the cell. Finally, the actin ring at the level of the zonulae occludens and adherens is compromised. The breakup of the actin ring correlates with ultrastructural changes in tight junction strands and the loss of the tight junction's role as a molecular fence. During the process of actin network dissolution, polymerized actin aggregates form in the cytoplasm. The changes in the junctional complexes and the potential to reverse the ATP depletion suggest that this may be a useful method to study junctional complex formation and its relationship to the actin cytoskeletal network.

ATP depletion: a novel method to study junctional properties in epithelial tissues. II. Internalization of Na+,K(+)-ATPase and E-cadherin.

MDCK and JTC cells were subjected to ATP depletion by treating the cells with 10 microM antimycin A and 10 mM 2-deoxyglucose. As visualized by confocal fluorescence microscopy, E-cadherin and Na+,K(+)-ATPase were rapidly internalized following depletion of the intracellular ATP stores. The time course of internalization was similar to the depolymerization of the cortical actin network and dissolution of the actin ring (see companion paper, this volume, pp. 3301-3313). Cell surface biotinylation was used to assay the amount of surface-accessible E-cadherin and Na+,K(+)-ATPase during ATP depletion. At 30 minutes of ATP depletion, 74% and 69% of E-cadherin and Na+,K(+)-ATPase were internalized, respectively, in MDCK cells. By 60 minutes of ATP depletion, internalization increased to 95% and 89%, respectively. The redistribution of both plasma membrane proteins was not microtubule dependent. Similar results were observed in JTC cells. Total biotinylated protein decreased by 67% and 82%, after 30 minutes and 60 minutes of ATP depletion, respectively. The E-cadherin internalization strongly suggests that disruption of adherens junctions occurred following ATP depletion. These results, along with the previously described loss of tight junction integrity, suggest that ATP depletion may be a useful method to study the assembly and disassembly of junctional complexes in epithelial cells.

Method for recovering ATP content and mitochondrial function after chemical anoxia in renal cell cultures.

Cultured renal cells provide a highly reproducible and malleable model to study cellular responses to metabolic perturbations. Nevertheless, there is currently no good method to achieve metabolic inhibition and complete recovery in cultured cells. This study describes a specific method for reversibly inhibiting both glycolytic and oxidative metabolism. Glycolysis was inhibited by removing all glycolytic substrates, and mitochondrial respiration was inhibited with rotenone, a site I inhibitor of the electron transport chain. Within 30 min, ATP values were decreased by 98%. Glycolysis was restored through the reintroduction of glucose. Oxidative metabolism was restored by the addition of heptanoate, a short odd-chain fatty acid, which supplies reducing equivalents to site II of the electron transport chain. Employing Madin-Darby canine kidney and LLC-PK1 cell lines, this protocol caused the immediate and complete recovery of mitochondrial respiration and, by 60 min, the complete recovery of cellular ATP levels. Application of this protocol should allow the investigation of the cellular effects and alterations that occur within cells recovering from sublethal energy depletion.

Cell polarity in human renal cystic disease.

BACKGROUND: In polycystic kidney disease (PKD), altered cellular polarity with mislocation of Na/K-ATPase, and net fluid secretion may have a role in cyst development and progression. EXPERIMENTAL DESIGN: Cell polarity was assessed in surgically excised human normal, autosomal dominant PKD, and acquired PKD occurring in end stage renal disease on long-term dialysis kidneys quick frozen (< 5 minutes) or fixed to minimize ischemic changes. RESULTS: Findings were similar in autosomal dominant PKD and acquired PKD kidneys. By ultrastructure, in cysts, cells were polarized, however, their basement membranes were greatly thickened and reticulated. By immunohistology, in cell-lining cysts, Na/K-ATPase, fodrin, and ankyrin were localized primarily to basolateral cell membranes and uvomorulin was localized to lateral cell membranes. In about 25% of the cells, however, Na/K-ATPase was localized to the apical as well as the basolateral membranes. Both in autosomal dominant PKD and normal kidney cell monolayers in vitro, cationic ferritin was normally absorbed by apical endocytosis, and transferred to apical vacuoles and phagolysosomes. CONCLUSIONS: These findings indicate intact structural and functional polarity in cell-lining cysts; however, in about 25% of the cells, Na/K-ATPase, fodrin, and ankyrin are localized to apical and lateral cell membranes, probably due to cell dedifferentiation. The notable changes in the basement membranes of cysts suggest a key role for the extracellular matrix in the pathogenesis of PKD.