Polycystic kidney disease: in search of a cause.
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Animals↗
Biomedical subjects
F A Carone
Publications and source records attributed to F A Carone.
Biology of polycystic kidney disease.
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Animals↗
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.
Adult↗
Tubular cell and matrix changes in renal cystic disease.
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Animals↗
Decreased synthesis and delayed processing of sulfated glycoproteins by cells from human polycystic kidneys.
BACKGROUND: In polycystic kidney disease, there is progressive distention of tubules and remodeling of an altered extracellular matrix. Recent findings indicate a defect(s) in the synthesis, post-translational modification, cellular transport, and extracellular matrix incorporation of proteoglycans in PKD that may have a fundamental role in its pathogenesis. EXPERIMENTAL DESIGN: Pulse-chase techniques and quantitative autoradiography were used to investigate in vitro the de novo synthesis and the kinetics of intracellular transport and extracellular matrix assembly of sulfated glycoproteins by epithelial cells from normal human kidneys (NK) and from cysts of autosomal dominant polycystic kidneys (ADPKD). NK and ADPKD cell monolayers were pulsed with [35S]sulfate for 150 min and chased for four intervals between 15 and 120 min. RESULTS: In ADPKD versus NK cells there was a significant reduction in the de novo synthesis of sulfated glycoproteins and, during the chase, a marked prolongation of the disappearance time for cellular sulfated glycoproteins. Transport kinetics revealed a substantial delay in processing and release of these macromolecules by the Golgi apparatus in ADPKD cells. During the chase, kinetics for the cellular transport of sulfated glycoproteins by secretory vesicles and their assembly into extracellular matrix were comparable in ADPKD and NK cells. However, the percentage of total sulfated glycoproteins incorporated into the matrix of ADPKD versus NK cell monolayers was notably diminished. CONCLUSIONS: The findings indicate that in ADPKD versus NK cell monolayers, synthesis of sulfated glycoproteins is impaired, processing of sulfated glycoproteins by the Golgi apparatus is prolonged, and assembly of these macromolecules into the extracellular matrix is reduced. These alterations may have a fundamental role in the pathogenesis of autosomal dominant polycystic kidney disease.
Autoradiography↗
Altered synthesis of proteoglycans by cyst-derived cells from autosomal-dominant polycystic kidneys.
Normal human renal epithelial cells (NK) and cells from cysts of autosomal-dominant polycystic kidneys (ADPKD) were radiolabeled with [35S]sulfate. A two- to three-fold decrease in the radioactivity incorporated into the proteoglycan (PG) fraction, as ascertained by tissue autoradiography and biochemical techniques, was observed in the ADPKD group. In subconfluent NK cells, PGs eluted as two peaks with different proportions of chondroitin sulfate (CS) and heparan sulfate (HS) in the cellular and media fractions. In the confluent stage, only a single major peak in the media and matrix fractions was seen and had variable proportions of CS and HS. In subconfluent ADPKD monolayers, cellular PGs eluted as two peaks, with the major peak of higher molecular weight compared with NK cells. In confluent stage, there was a single PG peak of a relatively higher molecular weight, with a variable increase in the proportions of CS vs. HS and lower charge-density characteristics. These findings indicate that size and species of PGs vary during subconfluent and confluent stages of culture and elucidate a defect in the biosynthesis of PGs in human ADPKD cells.
Autoradiography↗
Mannose-induced dysmorphogenesis of metanephric kidney. Role of proteoglycans and adenosine triphosphate.
Because various fetal anomalies are seen in diabetic offspring, we examined the effects of sugars on proteoglycans (PGs): extracellular matrix (ECM) macromolecules modulating morphogenesis. 13-d-old mouse metanephric kidney explants were exposed to mannose for 7 d and labeled with [35S]sulfate, [35S]-methionine, or [3H]thymidine. Mannose exposure caused reduction in kidney size and disorganization of ureteric bud branches with inhibition of glomerulogenesis. Tissue autoradiographic and immunofluorescence studies indicated decreased expression of sulfated PGs in ECMs. Helix pomatia lectin binding to D-GalNAc residues of glomerular epithelial cells was also reduced. Biochemical studies revealed decreased synthesis of sulfated PGs. PGs were of lower molecular weight with reduced charge density and increased chondroitin/heparan sulfate ratio. Immunoprecipitation of [35S]methionine-labeled proteins confirmed the reduction of PG core peptides. Intracellular ATP levels were reduced. The addition of 0.1 mM ATP to culture media restored kidney size, the population of glomeruli, and the synthesis and characteristics of PGs to almost normal, with no detectable effect on the replication of cells as determined by [3H]thymidine incorporation. The effect of ATP could be partially blocked by the P2y-purinoreceptor, i.e., reactive blue-2. Data suggest that mannose causes energy depletion by cellular ATP consumption and thus selectively alters the synthesis of heavily glycosylated proteins with rapid turnover, such as PGs, resulting in renal dysmorphogenesis.
Adenosine Triphosphate↗
Altered synthesis and intracellular transport of proteoglycans by cyst-derived cells from human polycystic kidneys.
Employing in vitro pulse-chase techniques, we investigated the de novo synthesis and the kinetics of intracellular transport and extracellular matrix incorporation of proteoglycans (PG) by normal human renal epithelial cell and by epithelial cells isolated from cysts of autosomal dominant kidneys (ADPKD). Cell monolayers were pulsed either with (3H)leucine for 15 min and chased for seven intervals between 15 and 270 min or with (35S)sulfate for 150 min and chased for a single interval of 120 min. Total proteins and PG were isolated from cell, media, and matrix fractions and characterized by Sepharose CL-6B and DEAE-Sephacel chromatographies. ADPKD and NK cells synthesized comparable amounts of total proteins; however, the de novo synthesis of PG by ADPKD cells was significantly reduced. ADPKD versus NK cells exhibited a substantial delay in the cellular transport and extracellular release of de novo synthesized PG, indicating an impairment at the level of the Golgi complex and/or secretory vacuoles. PG synthesized by ADPKD versus NK cells had decreased charge density characteristics, probably due to a posttranslational defect in the sulfation of the PG glycosaminoglycan chains. ADPKD versus NK cells synthesized PG of higher molecular weight and had an increased proportion of chondroitin sulfate PG versus heparan sulfate PG. Collectively, these findings suggest a defect in the synthesis and intracellular transport of sulfated PG in human ADPKD cells.
Biological Transport, Active↗
Sequential tubular cell and basement membrane changes in polycystic kidney disease.
Tubular basement membrane (BM) changes (dysmorphogenesis), cell proliferation, and fluid accumulation related to the altered location of Na,K-ATPase are purported essential key events in the development and progression of renal cysts. These changes were assessed daily in Phenol II (2-amino-4-hydroxyphenyl-5-phenyl thiazole)-treated rats, which rapidly develop marked and progressive cystic change of all collecting tubules (CT). At Day 1, 12% of CT were cystic and their BM were thickened severalfold. At Day 4, 30% of CT were cystic and their BM remained thickened. BM of cystic tubules showed decreased staining for heparan sulfate proteoglycan and increased staining for fibronectin. Proliferation, as determined by (3H)thymidine, incorporation, was not significant until Day 2 and involved cystic and noncystic tubular cells as well as interstitial cells. As cystic changes progressed, cell proliferation decreased. By immunohistochemistry, the altered location of Na,K-ATPase in epithelial cells lining cysts was primarily detected after Day 2 and consisted of focal loss from basal and/or lateral cell membranes and localization in the cell cytoplasm. Only rarely was Na,K-ATPase localized to the apical cell membrane. After the removal of Phenol II, cystic tubular cells, BM, and Na,K-ATPase returned to normal. Thus, in this model of polycystic kidney disease, initial cyst formation occurred in tandem with BM structural change whereas cell proliferation and altered location of Na,K-ATPase occurred after the appearance of cysts.
Animals↗
Comparison of proteoglycans synthesized by porcine normal and polycystic renal tubular epithelial cells in vitro.
Newly synthesized porcine tubular epithelial cell proteoglycans were labeled in vitro with Na2[35S]SO4. At the beginning of the labeling period (24 h) [35S] sulfate incorporated into macromolecules was measured following PD-10 chromatography. There was a significant reduction in the amount of 35S-labeled macromolecules isolated from polycystic cells compared to that from normal cells. The distribution of recovered radiolabeled material among the medium, cell surface, and intracellular fractions was similar for both normal and polycystic cells. Analysis of the proteoglycans in polycystic cells demonstrated that 86 and 73% of 35S-labeled macromolecules were of the heparan sulfate type in polycystic and normal cells, respectively. The remainder was chondroitin sulfate. Proteoglycans were characterized using DEAE-Sephacel ion-exchange chromatography, chondroitinase ABC, heparitinase, and nitrous acid digestion followed by Sepharose CL-4B gel permeation chromatography. The majority of radiolabeled material in the medium, cell surface, and intracellular fractions eluted between 0.35 and 0.39 M NaCl. However, a second peak (peak II) that eluted at 0.25 M NaCl was found in the medium from polycystic cells. This peak accounted for 27% of the total macromolecules secreted into the medium. Proteoglycans in the major peak were susceptible to nitrous acid and chondroitinase ABC digestion. A similar proportion of peak II was degraded by chondroitinase ABC. However, the remainder was only slightly susceptible to treatment with nitrous acid or heparitase. In normal cells a small amount of material eluted at a similar low charge; the proteoglycans were the same as those found in the major peak and appeared as a shoulder on this peak.
Animals↗
Cell fractionation and electron microscope studies of kidney folate-binding protein.
The subcellular distribution of folate-binding protein (FBP) and [3H]folate in the proximal tubule was examined using cell fractionation and different electron microscope (EM) techniques. Cell fractionation of rabbit proximal tubules revealed that FBP distributed into two modes: 50% of FBP distributed with alanylaminopeptidase activity (brush border), and the remaining FBP distributed with organelles of lower density that did not show a large digitonin-induced shift to greater density. Infusion of [3H]folate into the kidney followed by isolation and fractionation of the proximal tubules revealed a time-dependent shift of [3H]folate from the heavy (brush border) mode to the lighter organelle mode. By EM immunocytochemistry, rat kidney FBP locates in the brush border, endocytic invaginations, endocytic vacuoles, and dense apical tubules of proximal tubule cells. EM autoradiography of rat kidney 10 min after intravenous infusion of [3H]folate revealed that the label was significantly concentrated only in the brush border, endocytic vesicles, and lysosomes. These data support a mechanism of receptor-mediated endocytosis for the process of FBP-mediated folate transport in the kidney.
Animals↗
Inhibition of catalase and epoxide hydrolase by the renal cystogen 2-amino-4,5-diphenylthiazole and its metabolites.
Subchronic feeding of 2-amino-4,5-diphenylthiazole (DPT) to rats results in the development of renal cysts and has been used as a model system to study polycystic kidney disease. Because previous studies revealed changes in renal enzymes following DPT administration, a possible direct effect of DPT and its phenolic metabolites on catalase and a related enzyme, epoxide hydrolase, was examined. Experiments with three in vitro systems (suspensions of rabbit renal tubules, rat kidney homogenates, and commercially obtained bovine liver catalase) revealed direct inhibition of catalase activity by the diphenolic metabolite (diOH- DPT: 2-amino-4,5di(4'-hydroxyphenyl)-thiazole), the known renal cystogen nordihydroquaiaretic acid (NDGA) 2-amino-4(4'-hydroxyphenyl),5-phenyl-thiazole (4OH-DPT), and the known catalase inhibitor 3-amino-1,2,4-triazole; DPT did not inhibit catalase activity. Following oral administration to rats of the DPT congeners, 4OH-DPT caused the greatest decrease in both renal catalase and cytosolic epoxide hydrolase activities and the shortest time to onset of cystic lesions. In vitro, mouse liver cytosolic epoxide hydrolase activity was substantially inhibited by 4OH-DPT and dioH-DPT, and NDGA, but not by 2-amino-4-phenyl,5-(4'-hydroxyphenyl)-thiazole (5OH-DPT) or DPT itself. Microsomal epoxide hydrolase (mEH) activity was inhibited by 4OH-DPT, unaffected by DPT or dioH-DPT, and stimulated 2-fold by 5OH-DPT. Finally, mEH activity was substantially higher in samples of normal human kidney than in samples of kidney derived from a patient with autosomal recessive polycystic kidney disease; no differences were observed in cEH activity in these samples. Although the role of altered catalase and epoxide hydrolase activities in cystogenesis is unknown, DPT-induced cyst formation is associated with loss of these enzyme activities in kidney tissue. To our knowledge, this is the first report of an in vivo diminution of cytosolic epoxide hydrolase activity by xenobiotics.
8,11,14-Eicosatrienoic Acid↗
The pathogenesis of polycystic kidney disease.
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Animals↗
Cyst-derived cells do not exhibit accelerated growth or features of transformed cells in vitro.
Progressive renal enlargement is a prominent feature in autosomal dominant polycystic kidney disease (ADPKD), suggesting that the disease is due to hyperplasia and/or preneoplastic transformation of renal epithelial cells. In this study in vitro methods were developed to grow and propagate large numbers of cyst-derived epithelial cells from ADPKD kidneys and cortical epithelial cells from normal human kidneys (NK). In order to study their biologic features during early cell passages, cells were grown on Vitrogen (bovine dermal collagen)-FCS (fetal calf serum) coated dishes and fed a basic medium (DME:F12) supplemented with 10% FCS or a defined medium (Sens) containing insulin, transferrin, selenium, hydrocortisone, tri-iodothyronine and epidermal growth factor (EGF). Both ADPKD and NK cells grew as monolayers, were positive for keratin by immunohistochemistry and flow cytometry and had ultrastructural features of renal epithelial cells. Confluent NK and ADPKD monolayers formed domes. In contrast to NK cells, the growth and propagation of ADPKD cells were not supported by defined medium alone but required serum supplementation and ADPKD cells did not respond to growth factors (insulin, transferrin, EGF) that promoted the growth of NK cells. In serum supplemented media, the growth rate, cell doubling time and end cell number of ADPKD and NK cells were the same. Moreover, ADPKD cells did not exhibit any in vitro features of transformed cells: they were not immortal, they were sensitive to contact inhibition, they were anchorage dependent and they were not tumorigenic in nude mice. These findings do not support an increased rate of cell growth or cell transformation as causative factors in ADPKD.
Adult↗
Tubular basement membrane change occurs pari passu with the development of cyst formation.
Our previous studies have shown that 2-amino-4,5-diphenyl thiazole hydrochloride (DPT) administered orally to rats induces a urine concentrating defect (within 1 to 2 days) and progressive, but reversible, cystic change of all collecting tubules (prominent between 4 and 8 weeks). Cystic change was characterized by tubular cell and basement membrane changes consisting of alterations in cellular biosynthetic/secretory organelles, followed by thickening of the basement membrane with marked reduction (approximately 50%) of the de novo synthesis of sulfated proteoglycans, suggesting that altered synthesis of tubular basement membrane plays a role in the development of cystic disease. In this study, following the administration of [14C]-DPT in vivo, a major urinary metabolite (greater than 70%) was isolated by HPLC and characterized by gas chromatographic-mass spectral and NMR analyses as 2-amino-4-hydroxyphenyl-5 phenyl thiazole, designated phenol II. Phenol II was synthesized and administered orally to rats for four days to compare its biological effects with DPT. Phenol II induced a significantly greater impairment of concentrating ability and tubular cystic transformation than DPT. At day 5, in phenol II treated animals, basement membranes lining cysts were thickened several-fold and exhibited extensive loss and disorder of ruthenium red binding sites, indicative of loss of sulfated proteoglycans (heparin sulfate proteoglycan). The basement membrane changes occurred in tandem with the development of cystic transformation and strongly suggests that the basement membrane has a key role in the pathogenesis of PKD. The findings support the hypothesis that PKD may be due to a defect in the synthesis/degradation of one or more basement membrane components (sulfated proteoglycans) resulting in faulty tubular morphogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Contraluminal uptake of serine in the proximal nephron.
Rabbit proximal nephron segments were microperfused in vitro to determine whether active contraluminal uptake of serine occurs in the renal proximal tubule during bath-to-lumen transport (influx) of the L- and D-isomers in the convoluted (pars convoluta) and straight (pars recta) segments. It is known that several amino acids are actively reabsorbed in the proximal nephron by a mechanism involving co-transport with sodium at the luminal membrane. There is some evidence that certain amino acids may also be accumulated across the contraluminal membrane by an energy-dependent mechanism, indicating that net reabsorption is the result of two oppositely directed active transport processes. During in vitro microperfusion of rabbit proximal nephron segments in this study, inward movement of L- and D-serine occurred in a bath-to-cell direction against a concentration gradient in the range 305-2735:1, indicating active uptake at the contraluminal membrane. The concentration gradients were maintained during influx of both isomers of serine in the proximal tubule. L-Serine accumulation by tubular cells was similar in the pars convoluta and recta, and significantly greater than that of D-serine, which was the same in both regions of the proximal tubule. The data support the conclusion that renal handling of serine involves active contraluminal uptake of the L- and D-isomers in both regions of the proximal tubule, and suggest that contraluminal events play an important role in renal handling of amino acids.
Animals↗
Mechanisms affecting the development of renal cystic disease induced by diphenylthiazole.
To provide information on the possible influence that hypertension or its treatment might have on the development of cysts in autosomal dominant polycystic kidney disease, we studied the effects of the sodium content of the diet, DOCA-salt hypertension, renovascular hypertension, and the administration of enalapril or furosemide on the development of 2-amino-4-5-diphenylthiazole (DPT)-induced renal cystic disease. DOCA-salt hypertension caused vascular and glomerular lesions and proteinuria, but it did not enhance the development of cysts. Cytogenesis was enhanced in experimental conditions where the renin-angiotensin system is known to be activated. On the other hand, interventions known to suppress the renin-angiotensin system lessened the development of cysts. We hypothesize that this effect might be mediated by intrarenal angiotensin II and its capacity to promote cell growth and to control the postglomerular vascular resistances and the compliance of the renal interstitium.
Animals↗
Renal functional changes in experimental cystic disease are tubular in origin.
Chronic (30 weeks) structural and functional changes were correlated in diphenylthiazole (DPT)-induced polycystic kidney disease (PKD) in rats. DPT induced two different types of progressive tubular changes: cystic transformation and hyperplastic/atrophic tubular changes. Cystic changes diffusely involved collecting tubules in the outer medulla and cortex, and they were progressive over 30 weeks. Hyperplastic/atrophic changes occurred as clusters of tubules in the cortex and involved between 25% and 50% of tubular profiles after 12 and 30 weeks of drug treatment. Thus, the two types of tubular change were independent of each other and represent different cellular responses to the drug. DPT treatment induced no detectable light- and electron-microscopic or histochemical alterations in glomeruli or renal blood vessels. Renal functional changes consisted of: (1) early (4 weeks) and persistent impairment of concentrating ability; (2) a progressive drop in creatinine clearance and elevation in BUN; and (3) the late onset (30 weeks) of moderate proteinuria. These findings suggest that cystic as well as hyperplastic-atrophic tubular changes contribute to the loss of tubular and renal function in DPT-induced PKD. Both types of tubular lesions may have a role in the development of impaired renal function in other forms of experimental and clinical PKD.
Animals↗