Polycystic kidney disease: In danger of being X-rated?
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Animals↗
Biomedical subjects
J P Calvet
Publications and source records attributed to J P Calvet.
Polycystin-1 transforms the cAMP growth-responsive phenotype of M-1 cells.
BACKGROUND: Polycystic kidney disease (PKD) is characterized by the abnormal proliferation of tubular epithelial cells. It was recently shown that the growth of PKD cyst-lining cells is stimulated by cyclic adenosine monophosphate (cAMP), whereas the growth of normal human kidney cortex cells is inhibited. METHODS: We have examined the effects of overexpressing the C-terminal cytosolic tail of mouse polycystin-1, as a membrane-targeted fusion protein, on cAMP-responsive cell proliferation in stably transfected M-1 cortical collecting duct cells. Two cell lines that express high levels of the polycystin-1 fusion protein and two control cell lines that do not express the fusion protein were tested. RESULTS: Growth of parental M-1 cells and the control cell lines was inhibited by 8-Br-cAMP and by a variety of cAMP agonists. In contrast, growth of the polycystin-1-expressing clones was stimulated by cAMP. Consistent with this, the protein kinase A (PKA) inhibitor H-89 caused either a positive or a negative growth effect depending on the primary response to cAMP. PD98059 blocked the cAMP stimulation of cell proliferation, indicating that the pathway is MEK1 dependent. CONCLUSIONS: Expression of the polycystin-1 C-terminal tail disrupts normal cellular signaling and transforms the stably transfected M-1 cells to an abnormal PKD cell proliferation phenotype.
8-Bromo Cyclic Adenosine Monophosphate↗
The genetics and physiology of polycystic kidney disease.
Autosomal dominant polycystic kidney disease (ADPKD) is a major, inherited disorder that is characterized by the growth of large, fluid-filled cysts from the tubules and collecting ducts of affected kidneys, and by a number of extrarenal manifestations including liver and pancreatic cysts, hypertension, heart valve defects, and cerebral and aortic aneurysms. Mutations in either of 2 different genes (PKD1 or PKD2) give rise to ADPKD. Most mutations identified in affected families appear to inactivate the PKD genes, and accumulating evidence suggests that a 2-hit mechanism, in which the normal PKD1 or PKD2 allele is also mutated, may be required for cyst growth. The protein products of the PKD genes (polycystin-1 and polycystin-2) are thought to function together as part of a multiprotein membrane-spanning complex involved in cell-cell or cell-matrix interactions. Polycystin-1 and polycystin-2 can initiate signal transduction, leading to the activation of a number of downstream effectors, including heterotrimeric G-proteins, protein kinase C, mitogen-activated protein kinases, beta-catenin, and the AP-1 transcription factor. In addition, polycystin-2 may function in mediating calcium flux. The pathogenesis of cyst formation is currently thought to involve increased cell proliferation, fluid accumulation, and basement membrane remodeling. It now appears that cyclic adenosine monophosphate (cAMP) metabolism is a central component of cyst formation, stimulating apical chloride secretion and driving the accumulation of cyst fluid. Recent evidence has shown that ADPKD cells also have an altered responsiveness to cyclic AMP. In contrast to normal kidney cells whose cell proliferation is inhibited by cyclic AMP, ADPKD cells are stimulated to proliferate. Thus, it is likely that an alteration in polycystin function transforms the normal cellular phenotype to one that responds to elevated cyclic AMP by an increased rate of cell proliferation and that the enlarging cyst expands by an increased rate of cyclic AMP-driven fluid secretion. Cyclic AMP and growth factors, including epidermal growth factor, have complementary effects to accelerate the enlargement of ADPKD cysts, and thereby to contribute to the progression of the disease. This knowledge should facilitate the discovery of inhibitors of signal transduction cascades that can be used in the treatment of ADPKD.
Cyclic AMP↗
Identification of the major site of in vitro PKA phosphorylation in the polycystin-1 C-terminal cytosolic domain.
Sequence analysis of the C-terminal cytosolic domain of human and mouse polycystin-1 has identified three RxS consensus protein kinase A (PKA) phosphorylation motifs. GST-fusion proteins containing the full-length and truncated C-terminal cytosolic domain of murine polycystin-1 were phosphorylated in vitro by the purified catalytic subunit of PKA. This identified a sequence of 25 amino acids, immediately downstream of a previously identified heterotrimeric G-protein activation sequence, as the major site of PKA phosphorylation. Phosphorylation of wild-type and alanine substituted synthetic peptides containing this motif demonstrated that alanine substitution of serine 4159 largely eliminated phosphorylation. Mutation of this residue in the fusion protein reduced phosphorylation by about 70%, whereas mutation of the other two conserved phosphorylation motifs had little effect. We conclude that serine 4159 is the major site of PKA phosphorylation in the C-terminal cytosolic domain of murine polycystin-1.
Amino Acid Sequence↗
Matrix metalloproteinase-2 in a murine model of infantile-type polycystic kidney disease.
It was previously found that elevated levels of matrix metalloproteinase (MMP)-2 (gelatinase A) and -9 (gelatinase B) were synthesized and secreted into the medium by cultured kidney tubules derived from cystic C57BL/6J-cpk mice. To determine whether increased synthesis and secretion occur in vivo in this mouse model of polycystic kidney disease, kidney protein extracts, mRNA, and tissue sections were compared for expression and activity of MMP-2 and -9. Although both MMP were detected in tissue extracts, the differences in expression levels and activity in normal and cystic kidneys were far greater for MMP-2. High levels of MMP-2 seemed to result from increased expression by the cystic kidneys predominantly in the second and third postnatal weeks (a time when the kidneys are undergoing rapid cystic enlargement). Much of the increased MMP was present in the inactive zymogen form, although active enzyme was readily detected by sodium dodecyl sulfate-polyacrylamide gel zymography and in situ zymography. MMP-2 was abnormally localized to the interstitium and to foci between cysts, suggesting that MMP-2 may regulate collagen accumulation at those sites, thus allowing cyst enlargement and limiting the severity of interstitial fibrosis.
Animals↗
The polycystic kidney disease-1 protein, polycystin-1, binds and activates heterotrimeric G-proteins in vitro.
Analysis of the C-terminal cytosolic domain of human and mouse polycystin-1 has identified a number of conserved protein motifs, including a 20-amino-acid heterotrimeric G-protein activation sequence. A peptide specific for this sequence was synthesized and shown to activate purified bovine brain heterotrimeric Gi/Go in vitro. To test whether the C-terminal cytosolic domain of polycystin-1 stably binds G-proteins, GST-fusion constructs were used in pull-down and co-immunoprecipitation assays with purified bovine brain Gi/Go and rat brain lysates. This identified a 74-amino-acid minimal binding domain that includes the G-protein activation sequence. This region of polycystin-1, including the G-protein activation peptide and flanking amino acid sequences, is highly conserved in mouse, human, and puffer fish, lending further support to the functional importance of the minimal binding domain. These results suggest that polycystin-1 may function as a heterotrimeric G-protein coupled receptor.
Amino Acid Sequence↗
Kid-1 expression is high in differentiated renal proximal tubule cells and suppressed in cyst epithelia.
The cDNA coding for the transcriptional repressor protein Kid-1 was cloned in a screen for zinc finger proteins, which are regulated during renal development and after renal ischemia. Kid-1 mRNA levels increase in the course of postnatal renal development and decrease after acute renal injury caused by ischemia or administration of folic acid. We have raised a monoclonal anti-Kid-1 antibody and demonstrate that the Kid-1 protein is strongly expressed in the proximal tubule of the adult rat kidney. During nephron development, the Kid-1 protein appears after the S-shaped body stage concomitantly with the brush-border enzyme alkaline phosphatase. In two animal models of polycystic kidney disease, the expression of Kid-1 is downregulated. The loss of expression of Kid-1 in cyst wall cells correlates with the loss of alkaline phosphatase histochemical staining. Kid-1 mRNA levels are also reduced in rodent renal cell carcinomas, another condition characterized by epithelial cell dedifferentiation and increased proliferation. We propose that Kid-1 plays an important role during the differentiation of the proximal tubule.
Aging↗
[Anatomy and physiology of the perineal anus. 1957].
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Anal Canal↗
A mouse kidney- and liver-expressed cDNA having homology with a prokaryotic parathion hydrolase (phosphotriesterase)-encoding gene: abnormal expression in injured and polycystic kidneys.
To investigate abnormalities in gene expression associated with cyst formation in polycystic kidney disease, differential cDNA library screening was carried out using RNA from normal and cystic kidneys of the C57BL/6J-cpk mouse. Among a number of genes found to be abnormally expressed was one (cDNA clone 56-1) that was significantly underexpressed in cystic kidneys. Hybridization analyses revealed that the 56-1 mRNA is expressed primarily in kidney and liver, and that the kidney expression begins postnatally and continues in the adult. Expression of this mRNA was found to be significantly decreased upon acute renal injury induced by a single intraperitoneal injection of folic acid, and to return to normal levels upon recovery of kidney function. Analysis of the cDNA sequence predicted a protein of 349 amino acids (aa), which was confirmed by in vitro translation of a sense-strand transcript, producing a protein of approx. 39 kDa. The aa sequence shows similarity to Flavobacterium sp. and Pseudomonas diminuta parathion hydrolase (phosphotriesterase or PTE), an enzyme that hydrolyzes toxic organophosphates and other phosphotriesters, and to the predicted product of an Escherichia coli open reading frame of unknown function (phosphotriesterase homology protein or PHP). Use of optimal alignment programs demonstrated a significant overall homology between the bacterial and mouse sequences, with greater than 50% aa sequence similarity. This cDNA represents the first eukaryotic sequence showing similarity to these prokaryotic genes. Based on this apparent homology, it has been named mpr56-1 (for mouse phosphotriesterase-related 56-1).
Amino Acid Sequence↗
Growth characteristics of cells cultured from two murine models of polycystic kidney disease.
Polycystic kidney disease (PKD) is characterized by multiple renal cysts that are lined by epithelium and filled with fluid. PKD may result from one of a number of factors, either inherited or environmental. In this study, we have compared two mouse models in which PKD results from a genetic cause. In the C57BL/6J-cpk model, the mutated gene is unknown. In the other model, an SV40 large T antigen transgene causes renal cysts. We examined cultured cells from the kidneys of these mouse models, comparing growth characteristics. Although several features of PKD lead one to expect that the epithelial cells lining the cysts would have an increased rate of proliferation in culture, we found that they did not. The implications of these findings are discussed.
Animals↗
Renal expression of a transforming growth factor-alpha transgene accelerates the progression of inherited, slowly progressive polycystic kidney disease in the mouse.
Polycystic kidney disease (PKD) is a prevalent inherited disease in human beings. The pathogenesis of PKD is as yet unclear. The epidermal growth factor family of proteins has been implicated in PKD based largely on in vitro data. To determine whether these growth factors contribute to the progression of inherited PKD in vivo, we crossed mice with a transgene for human transforming growth factor-alpha (TGF-alpha, a member of the epidermal growth factor (EGF) family) and mice with the pcy gene (which causes a slowly progressive form of PKD very similar to human autosomal dominant PKD). Renal expression of the TGF-alpha transgene in cystic mice (homozygous for the pcy gene) accelerated the development of PKD as shown by an increased kidney weight as a percent of body weight and an increased volume density of renal cysts at 8.5 weeks of age. However, renal expression of the TGF-alpha transgene did not appear to precociously initiate cyst development (at 6.5 weeks), nor did it cause an increase in the final degree of renal enlargement (at 29 weeks). Thus TGF-alpha accelerated the enlargement of cysts once initiated. At 8.5 weeks of age, renal expression of the TGF-alpha mRNA correlated positively with the amount of renal enlargement. At all time points studied, cystic kidneys exhibited increased expression of c-myc mRNA as compared with phenotypic normal kidneys, consistent with PKD being a hyperplastic disease of renal tubules. However, the renal expression of c-myc in 8.5 week cystic kidneys, with or without the transgene, did not correlate with the degree of renal enlargement. The results of this study suggest that EGF-like proteins may accelerate the progression of inherited renal cystic disease. However, the final degree of cystic change is dictated by the primary disease process rather than by the continued presence of growth factor.
Aging↗
The cystic fibrosis transmembrane conductance regulator mediates transepithelial fluid secretion by human autosomal dominant polycystic kidney disease epithelium in vitro.
Transepithelial fluid secretion promotes the progressive enlargement of cysts in autosomal dominant polycystic kidney disease (ADPKD). Recent indirect evidence indicated that active chloride transport may drive net fluid secretion in cultures of epithelia derived from ADPKD cysts. We now report that forskolin, which stimulates adenylate cyclase, increased the efflux rate constant for 36Cl in monolayers of ADPKD cells in vitro from 0.23 +/- 0.02 min-1 to 0.44 +/- 0.05 min-1 (N = 4) and that diphenylamine 2-carboxylate (DPC), which blocks chloride channels, eliminated the forskolin-stimulated chloride efflux from these cells. To establish whether the cAMP-regulated chloride transporter, cystic fibrosis transmembrane conductance regulator (CFTR), may potentially be involved in the chloride transport and fluid secretion of ADPKD epithelia, we examined CFTR mRNA and protein in these cultures. Northern blot hybridization using a human (h) CFTR cDNA probe demonstrated the presence of an approximately 6.5 kb transcript in total RNA from polarized cultures of ADPKD, normal human kidney cortex (HKC), and T84 cells. Utilizing several antibodies to hCFTR, immunocytochemistry and confocal fluorescence microscopy localized an immunoreactive protein primarily in the apical region of forskolin-stimulated ADPKD cells grown on permeable supports. This immunoreactivity could be eliminated by preincubation of antibody with immunizing peptide. To determine the effect of CFTR abundance on the magnitude of net fluid secretion, polarized ADPKD cultures were treated with deoxyoligonucleotides that were either complementary (antisense), homologous (sense), or partially complementary (misantisense) to a sequence near the translation initiation site in hCFTR mRNA. Treatment with 5.0 microM antisense oligonucleotide resulted in a 73% reduction in forskolin-stimulated fluid secretion and a comparable reduction in the abundance of CFTR as detected by immunocytochemistry. By contrast, treatment with 5.0 microM sense oligonucleotide reduced fluid secretion by only 34% and had less of an effect on CFTR abundance, while the effects of 5.0 microM misantisense oligonucleotide on both fluid secretion and CFTR abundance were insignificant. On the basis of these results we suggest that CFTR is a major mediator of forskolin-stimulated chloride and fluid secretion by epithelial cells of human polycystic kidneys in vitro.
Cells, Cultured↗
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.
Animals↗
Analysis of differential gene expression in the kidney by differential cDNA screening, subtractive cloning, and mRNA differential display.
It is becoming increasingly evident that significant changes in gene expression occur during the course of disease progression in both genetic and nongenetic kidney diseases. Knowledge of the differentially expressed genes may yield information about the abnormal biochemical events that occur in the initiation and pathogenesis of these diseases. The purpose of this review is to provide an overview of some of the current approaches for identifying and analyzing differentially expressed genes. The power of these techniques lies in their their ability to detect differences in the levels of specific mRNAs in the diseased compared to the nondiseased kidney without prior knowledge of their identity. The three basic techniques considered are differential cDNA library screening, subtracted cDNA libraries, and PCR-based differential display. Emphasis is placed on cDNA library construction and differential screening. Also reviewed are the analysis of differentially expressed cDNAs by Southern and Northern blot hybridization, S1-protection, RT-PCR, DNA sequencing, and DNA sequence analysis.
Base Sequence↗
Mouse plasma glutathione peroxidase. cDNA sequence analysis and renal proximal tubular expression and secretion.
A mouse kidney cDNA isolated by differential screening was found to be highly homologous to rat, human, and bovine plasma glutathione peroxidase (GPx) sequences. Analysis of the full-length coding region sequence demonstrated an in-frame selenocysteine-encoding opal codon and putative signal sequence, suggesting that the sequence represents the mouse homolog of plasma GPx. The level of expression of plasma GPx in various mouse tissues and during development was investigated by Northern blot analysis. Plasma GPx mRNA was observed to be very abundant in kidney compared with placenta, epididymis, intestine, lung, heart, testis, ovary, salivary gland, spleen, thymus, stomach, brain, and fetal kidney and could not be detected in pancreas or in liver except from pregnant mice. In addition, plasma GPx mRNA levels were shown to increase during postnatal development of the kidney. In situ hybridization localized plasma GPx mRNA to proximal tubules, while primary cell culture demonstrated that plasma GPx is synthesized and secreted by proximal tubular epithelial cells. The relative abundance of plasma GPx mRNA in mouse kidney suggests that proximal tubules may be the primary source of the enzyme detectable in plasma and further suggests that plasma GPx has an important function in protecting the kidney from oxidative damage.
Amino Acid Sequence↗
Injury and development in polycystic kidney disease.
The large diversity of genetic and nongenetic causes of polycystic kidney disease begs the question of whether there is a common denominator in the process of cyst formation. Accumulating evidence now indicates that cysts are made up of immature epithelium, which suggest that cystic transformation may be caused by an arrested developmental process that locks cells in a unique differentiated state. Although there may be any number of possible causes of this cystic change, it is proposed that one unifying mechanism may be subcritical injury-induced cellular dedifferentiation.
Animals↗
Primary and secondary genetic responses after folic acid-induced acute renal injury in the mouse.
Folic acid-induced acute renal injury results in dramatic changes in gene expression. Among the genes affected by folic acid treatment are the primary response genes, c-fos and c-myc, which are thought to function to initiate cell cycle events. In this report, changes in the expression of three other genes in response to folic acid injury have been investigated: ornithine decarboxylase, epidermal growth factor (EGF), and sulfated glycoprotein-2 (SGP-2). Renal injury was found to cause a rapid decrease in EGF mRNA, which remained absent for several days after the initial injury, gradually returning to normal levels over an approximately 3-wk regeneration and recovery period. Ornithine decarboxylase mRNA showed a similar decrease. In contrast, folic acid caused a rapid increase in SGP-2 mRNA, which peaked several days after treatment, decreasing to normal levels over the 3-wk period. The mRNAs for the primary response genes were superinduced in the injured kidneys in the presence of the protein synthesis inhibitor cycloheximide. In contrast, the changes in EGF and SGP-2 mRNA levels were blocked by cycloheximide, indicating that these responses required new protein synthesis during the first few hours after folic acid injury. The opposite but parallel responses in the expression of the EGF and SGP-2 genes suggest that their regulation is coupled to the initial injury-induced dedifferentiation and subsequent return to the fully differentiated state.
Animals↗
Renal cysts in transgenic mice expressing transforming growth factor-alpha.
Transforming growth factor-alpha (TGF-alpha) is a member of the epidermal growth factor (EGF) family of proteins and, like EGF, elicits its cellular function by binding to the EGF receptor. EGF stimulation may have a role in several normal and pathologic processes in the kidney, and EGF has been implicated in the development of renal cysts in vitro and in human autosomal dominant polycystic kidney disease. We sought to determine whether renal expression of an EGF-like protein (TGF-alpha) could lead to the formation of renal cysts in vivo. We examined morphologic alterations to the normal kidney caused by renal expression of a TGF-alpha transgene linked to a mouse metallothionein promoter stably integrated into the genome of the CD1 mouse. TGF-alpha transgene expression was induced with exogenous zinc treatment starting at 4 weeks of age, and mice were killed at 8 weeks of age. The transgene was expressed at higher levels in female transgenic mice than in male transgenic mice. The augmented expression of the TGF-alpha transgene in females was associated with increased renal size and the development of renal epithelial cysts. Both male and female mice exhibited increases in glomerular size and mesangial volume density. These results provide evidence that stimulation by an endogenous EGF-like protein can lead to renal enlargement, glomerular mesangial expansion, and renal cyst formation.
Animals↗