Background Polycystic kidney disease is characterized by theenlargement of renal cysts, interstitial fibrosis, and gradualloss of normal renal tissue in association with progressivedeterioration of renal function. The process causing the progressiveloss of renal tissue is unknown, but it could be the resultof a form of programmed cell death known as apoptosis.
Methods We assayed apoptotic DNA fragmentation in normal andpolycystic kidneys biochemically by gel electrophoresis andhistochemically by in situ end-labeling. A DNA-specific dye,Hoechst 33258, was used to detect morphologic apoptosis in renalsamples from patients with normal kidneys, polycystic kidneydisease, and other kidney diseases.
Results Apoptotic DNA fragmentation was detected in polycystickidneys from 5 patients without renal failure and 11 patientswith renal failure but not in kidneys from 12 patients withno renal disease. In situ end-labeling revealed apoptotic cellsin glomeruli, in cyst walls, and in both cystic and noncystictubules of the polycystic kidneys. No tubular apoptosis wasdetected in renal-biopsy specimens from five patients with IgAnephropathy, three patients with nephrosclerosis, two patientswith focal glomerulosclerosis, one patient with diabetic nephropathy,six patients with acute tubular necrosis, or four patients withacute and four patients with chronic renal-transplant rejection.The capacity of polycystic kidney cells to undergo apoptosiswas retained in vitro in the absence of uremia, ischemia, andother confounding pathologic conditions.
Conclusions Apoptotic loss of renal tissue may be associatedwith the progressive deterioration of renal function that occursin patients with polycystic kidney disease.
The polycystic kidney diseases are a group of disorders characterizedby the presence of numerous cysts throughout grossly enlargedkidneys. In humans, they are inherited as autosomal dominantor autosomal recessive disorders. Autosomal dominant polycystickidney disease is a major cause of chronic renal failure, accountingfor 10 percent of all cases requiring long-term dialysis orrenal transplantation. Despite the recent cloning of the generesponsible for 80 to 90 percent of the cases of autosomal dominantpolycystic kidney disease, the primary pathogenic mechanismof polycystic kidney disease remains unknown.
Three types of defects have been implicated in the process wherebyrenal tubules enlarge to become cystic: abnormalities in basement-membranecomponents,1,2 abnormal proliferation of cystic epithelia,3and abnormal development and polarization of cystic epithelia.4,5,6Renal insufficiency is attributed to the compression of normalrenal tissue and interstitial disease.
Each human kidney is made up of approximately 1 million nephrons.In all types of polycystic kidney disease examined to date,the cysts numbered in the hundreds or thousands. Microdissectionstudies indicate that each cyst arises from the focal dilatationof a small segment of a nephron.7,8 The loss of a small percentageof nephrons due to cyst formation should not result in renalinsufficiency, because many functional nephrons should remain.
Apoptosis is a type of physiologic cell deletion that occursduring embryonic development and in the renewal of mature tissues.In this study, we evaluated the possibility that the loss ofnoncystic renal tubules in polycystic kidneys could be causedby apoptosis.
Methods
Polycystic human kidneys were obtained from the surgical pathologydepartment of the UCLA Medical Center, the International Institutefor the Advance of Medicine (Exton, Pa.), the National DiseaseResearch Interchange (Philadelphia), and the Polycystic KidneyResearch Foundation (Kansas City, Mo.). Five of the 16 kidneysfrom patients with autosomal dominant disease (Polycystic KidneySpecimens 1, 3, 5, 8, and 13) were originally removed from brain-deadorgan donors for transplantation but were not used because theywere polycystic. These organs were not subjected to warm ischemia,and the donors had normal serum creatinine concentrations. Polycystickidneys were also collected from 10 patients with end-stagerenal disease who were undergoing renal transplantation. A polycystickidney was obtained from a one-day-old infant with terminalautosomal recessive polycystic kidney disease. Twelve normalhuman kidneys obtained from either the International Institutefor the Advance of Medicine or the National Disease ResearchInterchange had been obtained for transplantation, but suitablerecipients were not found. These kidneys were not subjectedto warm ischemia.
The kidney tissues were trimmed into cubes measuring 1 to 3mm and were processed for the isolation of genomic DNA, cryopreservedin a 1:1 mixture of DMEM and F12 medium containing 10 percentdimethyl sulfoxide, or processed further for in vitro culture.In addition, fine-needle-biopsy specimens of renal tissue inBouin's fixative from five patients with IgA nephropathy, threepatients with nephrosclerosis, two patients with focal glomerulosclerosis,one patient with diabetic nephropathy, six patients with acutetubular necrosis, and four patients with acute and four patientswith chronic renal-transplant rejection were obtained from Dr.Arthur Cohen (Cedars-Sinai Medical Center, Los Angeles). Thesefine-needle-biopsy samples were used only for the histologicdetection of apoptotic cells.
We obtained cpk mice9 from the Jackson Laboratory (Bar Harbor,Me.); they were maintained by mating known heterozygotes. Weobtained pcy mice10 from Dr. H. Takahashi (Fujita Health University,Toyoake, Japan) through Dr. Jared Grantham (University of Kansas,Kansas City); they were maintained by back-crossing heterozygousfemale mice with homozygous polycystic male mice.
Cells from 1-mm3 fragments of normal and polycystic human kidneyswere prepared for in vitro culture as described elsewhere.11Both primary cultured cells and cryopreserved primary cellsrevived and cultured for up to three passages were studied.The cells were plated at a density of 106 cells per 10-mm cultureplate (Corning, Cambridge, Mass.) and incubated at 37 °Cin 5 percent carbon dioxide.
Genomic DNA from tissues and cultured cells was prepared withthe proteinase K-sodium dodecyl sulfate digestion method.12Oligonucleosome-length DNA fragments were detected by electrophoresison 1 percent agarose gels containing 0.5 microg of ethidiumbromide per milliliter.
For in situ detection of apoptotic nuclei, normal and polycystickidney cells and tissues were fixed with 1 percent paraformaldehydeand embedded in paraffin. Tissue sections on ProbeOn Plus slides(Fisher Scientific, Pittsburgh) were processed for in situ end-labelingas described below. Cultured cells were grown in four-compartmentglass chamber slides (Nunc, Naperville, Ill.) and fixed overnightbefore in situ end-labeling.
For in situ end-labeling, the ends of DNA fragments generatedduring apoptosis in the nuclei of apoptotic cells were enzymaticallylabeled in situ with biotin-16-deoxyuridine triphosphate (Boehringer-Mannheim,Indianapolis) or biotin-21-deoxyuridine triphosphate (Clontech,Palo Alto, Calif.) and terminal transferase (Promega, Madison,Wis., or Boehringer-Mannheim) with a modification of the homopolymertailing method.13 The reactions were carried out in the capillaryspace between a pair of ProbeOn Plus slides. First, endogenousperoxidase activity was stopped by incubation with 3 percenthydrogen peroxide in phosphate-buffered saline for 15 minutes.The slides were washed and then incubated at 37 °C for onehour with 200 microl of a solution containing 50 units of terminaltransferase and 10 microM biotin-16-deoxyuridine triphosphate(or biotin-21-deoxyuridine triphosphate) in 100 mM cacodylate(pH 6.8), 1.5 mM cobalt chloride, and 0.1 mM dithiothreitol.The biotin incorporated in the sections was coupled to horseradishperoxidase with Vectorstain Elite ABC reagents (Vector Laboratories,Burlingame, Calif.). The slides were washed and then developedwith a diaminobenzidine substrate kit (Vector Laboratories)for 10 minutes. The slides were then stained with periodic acid-Schiffreagent and counterstained with hematoxylin.
To detect apoptotic nuclei in histologic sections of fine-needle-biopsysamples of kidney tissue from the patients with other renaldiseases, the sections were deparaffinized, rehydrated in phosphate-bufferedsaline, and stained with 10 microg of the DNA-specific bisbenzimidedye Hoechst 33258 (Molecular Probes, Eugene, Oreg.) per milliliterin 100 mM sodium chloride, 10 mM TRIS, and 1 mM EDTA (pH 7.5)for 10 minutes and mounted in VectaShield mounting medium forfluorescence microscopy. Apoptotic nuclei are identified bytheir condensed appearance and intense blue fluorescence ascompared with normal nuclei.14
Results
Progressive loss of renal function is a common feature of polycystickidney disease and other chronic renal diseases. Kidneys frommost patients with nonpolycystic end-stage renal disease containsome remnant nephrons and abnormal glomeruli. However, withingrossly enlarged kidneys of patients with end-stage polycystickidney disease, fibrotic tissue filled the interstitium andfew or no nephrons remained (Figure 1A, Figure 1B, Figure 1C,Figure 1D, Figure 1E, Figure 1F). These observations promptedthe search for evidence of apoptosis as a possible mechanismunderlying the progressive loss of renal tissue in polycystickidney diseases. The range of disease progression representedin the 16 kidneys from patients with polycystic kidney diseaseis shown in Figure 2A, Figure 2B, Figure 2C, Figure 2D, Figure 2E,Figure 2F.
Figure 1. Loss of Renal Tissue in Polycystic Kidney Disease. There are abundant glomeruli and tubules in a normal human kidney (Panel A) and a normal mouse kidney (Panel D). In contrast, there is a lack of glomeruli and renal tubules and there are multiple cysts in kidneys from patients with end-stage autosomal dominant (Panel B) or autosomal recessive (Panel C) polycystic kidney disease, a kidney from a cpk mouse with end-stage polycystic kidney disease (Panel E), and a kidney from a pcy mouse with end-stage polycystic kidney disease (Panel F). Interstitial fibrosis is apparent in the kidneys from patients with end-stage polycystic kidney disease (Panels B and C) and in the kidney from a six-month-old pcy mouse with polycystic kidney disease (Panel F). Bars represent 0.1 mm. Sections were stained with periodic acid-Schiff stain and counterstained with hematoxylin.
Figure 2. Range of Disease Progression in Polycystic Kidney Disease in Humans. The full range of cystic involvement was represented in the 16 kidney specimens from patients with autosomal dominant polycystic kidney disease that were examined in this study (5 of which are shown). Polycystic Kidney Specimens 1, 5, 8, and 13, shown in Panels A, B, C, and D, respectively, were functionally normal. A kidney from a patient with end-stage polycystic kidney disease (Specimen 9) is shown in Panel E. A pair of kidneys from an infant who died of polycystic kidney disease 24 hours after birth is shown in Panel F. Bars represent 1 cm.
In contrast to necrosis, which is the passive result of traumaticcell death, apoptosis is a type of programmed cell death inwhich each cell actively synthesizes new RNA and proteins tomediate its own demise.15 In apoptosis, cell death is precededby the cleavage of chromatin into oligonucleosomes. The DNAin these oligonucleosomes can be detected by agarose-gel electrophoresisas a ladder of DNA fragments with lengths in multiples of about180 base pairs.16 Oligonucleosome-length DNA ladders indicativeof apoptotic DNA fragmentation were detected in DNA preparedfrom all the kidneys we studied from patients with autosomalrecessive and autosomal dominant polycystic kidney disease (Figure 3A),except for one (Specimen 1) in which the disease was ata very early stage. An apoptotic DNA ladder was not found inthe 12 samples of normal human kidneys. Apoptotic DNA fragmentationwas evident in the preuremic kidneys of pcy mice (Figure 3B)and cpk mice (Figure 3C), but not in kidneys of age- and sex-matchednormal littermates. In addition, apoptotic DNA fragmentationwas detected in the lung and liver of cpk mice. Apoptotic DNAfragmentation in the thymus and spleen of 10-day-old mice isnormal.17
Figure 3. Apoptotic Fragmentation of DNA in Kidneys from Patients and Mice with Polycystic Kidney Disease. In Panel A, DNA extracted from five normal human kidneys (NHK 0, 5, 6, 7, and 8) shows no signs of apoptotic fragmentation. DNA extracted from a kidney from a patient with autosomal recessive polycystic kidney disease (ARPKD) and six kidneys from patients with autosomal dominant polycystic kidney disease (PKD 1, 5, 6, 7, 8, and 9) had various degrees of apoptotic DNA fragmentation. The patients from whom Polycystic Kidney Specimens 1, 5, and 8 were obtained had normal renal function, whereas those from whom Polycystic Kidney Specimens 6, 7, and 9 were obtained had end-stage renal failure. Although apoptotic nuclei were detected in all examples of early polycystic kidney disease in humans with use of the in situ homopolymer tailing technique (as shown in Fig. 4), no apoptotic DNA fragmentation was detected in Polycystic Kidney Specimen 1. The level of DNA fragmentation in Polycystic Kidney Specimens 7, 8, and 9 did not correlate with the stage of disease. In Panel B, apoptotic DNA fragmentation is evident in the preuremic polycystic kidneys of three-month-old pcy mice. Apoptotic DNA fragmentation cannot be detected in other organs tested in these mice at three months of age or later. No apoptotic DNA fragmentation was detected in the organs of normal littermates. In Panel C, apoptotic fragmentation of DNA is evident in the thymus and spleen of normal 10-day-old C57/BL6 mice. In 10-day-old preuremic cpk mice, apoptotic DNA fragmentation was detected in the lung, liver, and kidneys, in addition to the thymus and spleen. Sub denotes DNA samples from the submaxillary gland. In Panel D, apoptotic fragmentation of DNA in cells cultured from Polycystic Kidney Specimens 8 and 9 and ARPKD can be seen. No apoptotic fragmentation of DNA was detected in cells cultured from normal human kidneys (NHK 8, 7, 6, 5, and 0); from the established cell lines mdck, bsc-1, llc-pk, bhk, or nrk; or from Polycystic Kidney Specimens 5, 6, and 7. All cultured human cells were cryopreserved, revived, and passaged twice in culture. All lanes were loaded with 50 microg of total genomic DNA from each sample. MW denotes the molecular-weight marker, a DNA ladder of 100 base pairs (BRL, Gaithersburg, Md.).
Apoptotic DNA fragmentation was detected in most cultures ofpolycystic kidney cells from humans but was not found in anyculture of normal human kidney cells (Figure 3D). The absenceof DNA fragmentation in some cultures of polycystic kidney cellsprobably reflects variations in sampling, because DNA fragmentationcould be demonstrated in other cultures from the same polycystickidneys (data not shown). Apoptotic fragmentation of chromosomalDNA was apparent in cultured polycystic kidney cells from cpkand pcy mice but not from normal littermates (data not shown).
The in situ end-labeling technique labels the large number ofDNA ends in oligonucleosomes that are generated within apoptoticnuclei. Figure 4A, Figure 4B, Figure 4C, Figure 4D, Figure 4E,Figure 4F, Figure 4G, and Figure 4H shows apoptotic nuclei inkidney tissue from patients with autosomal dominant and autosomalrecessive polycystic kidney disease and mice with congenitalpolycystic kidney disease. Apoptotic nuclei can be detectedin noncystic tubular epithelial cells, in cells within glomeruli,and in cells lining renal cysts. With the use of this technique,all the polycystic human kidneys but none of the normal humankidneys we examined had evidence of apoptotic nuclei. In addition,apoptosis was readily detected in all proliferating culturesof human and murine polycystic kidney cells.
Figure 4. In Situ Demonstration of Apoptotic Nuclei in Polycystic Kidneys. With the use of in situ end-labeling, apoptotic nuclei are stained brown by the horseradish peroxidase-diaminobenzidine reaction, whereas normal nuclei are stained purple by the hematoxylin counterstain (in all panels except Panel G). Panels A and B show apoptotic nuclei within the tubules and a glomerulus of a kidney from a patient with autosomal dominant polycystic kidney disease (Specimen 1) (x470). Panels C and D show cells with apoptotic nuclei next to cells with normal nuclei in undilated tubules and in cells lining the expanded cyst wall of a kidney from a patient with autosomal recessive polycystic kidney disease (x470). Apoptotic nuclei are apparent in normal tubular cells and cells lining cysts within the polycystic kidneys of a 10-day-old cpk mouse (Panel E; x470) and a 3-month-old pcy mouse (Panel F; x470). In Panel G, after Hoechst-dye staining, apoptotic nuclei within tubules of a kidney from a patient with early polycystic kidney disease (Specimen 1) appear condensed (pyknotic) and bright blue (x470). Panel H shows apoptotic nuclei in cultured kidney cells from a patient with autosomal dominant polycystic kidney disease (x950).
The detection of apoptosis in undilated nephrons in preuremicpolycystic human kidneys and in polycystic cpk and pcy mousekidneys and the contrasting absence of apoptosis in kidneysfrom normal humans and mice strongly suggest that apoptosismay be associated with the progressive loss of renal tissuein polycystic kidney disease. However, the reported detectionof apoptosis in rats with obstructive hydronephrosis18,19 andischemic renal atrophy20 raised the possibility that renal apoptosismay be a common manifestation of uremia and ischemia associatedwith renal disease. Therefore, kidney-biopsy specimens frompatients with various types of nephropathy, acute tubular necrosis,and acute and chronic transplant rejection were analyzed forthe presence of apoptotic nuclei. Because low pH induces randomstrand breaks in DNA and Bouin's fixative contains 5 percentacetic acid, samples in Bouin's fixative produced unacceptablyhigh levels of background nuclear staining in the terminal transferaselabeling assay.
All histologic samples of normal kidneys, polycystic kidneys,and kidneys with other types of diseases were stained with theDNA-specific dye Hoechst 33258 and examined with ultravioletfluorescence microscopy to detect the morphologic features ofapoptotic nuclei. With the use of this technique, apoptoticnuclei were detected in all polycystic kidney samples (Figure 4G).No tubular apoptosis was seen in normal kidneys or in anyof the samples of tissue from patients with noncystic renaldiseases (100 to 200 tubules studied per sample), indicatingthat, if present, tubular apoptosis is not easily detected inthese forms of renal diseases. Apoptotic nuclei were seen intwo glomeruli in one tissue sample from a patient with IgA nephropathyand in a single glomerulus in one tissue sample from a patientwith acute transplant rejection. In the absence of tubular apoptosis,glomerular apoptosis may be associated with recovery ratherthan injury.21,22
Discussion
Programmed cell death occurs during normal embryonic developmentand morphogenesis, in the maturation of the immune system, andin the turnover of renewing tissues,23 and it may have a rolein tumor formation24 and degenerative diseases such as the acquiredimmunodeficiency syndrome and Alzheimer's disease.25,26 Thisstudy shows that in addition to cyst enlargement and interstitialfibrosis, apoptosis is a pathological feature of polycystickidney disease.
Within solid tissues, apoptotic cells are phagocytized withina few hours by neighboring cells or by phagocytes in a processinvolving the vitronectin receptor or the phosphatidylserinereceptor.27,28 Thus, apoptosis can be demonstrated histologicallyonly during the interval before the apoptotic cell is phagocytizedand digested. This limits the detection of apoptosis to samplesin which apoptosis is occurring continually. Except in cpk micewith polycystic kidney disease and in the most severe formsof autosomal recessive polycystic kidney disease, in which thereis rapid loss of renal tissues, renal insufficiency usuallydevelops very gradually in polycystic kidney disease. In themore slowly progressive forms, in which loss of nephrons isslow, apoptosis was nevertheless detected before the onset ofuremia. The failure to detect renal tubular apoptosis in severalnonpolycystic forms of chronic renal disease suggests that eithercell death occurs in polycystic kidney disease more than inother renal diseases with comparable or higher rates of lossof renal function, or, if cell death occurs in these other chronickidney diseases, a nonapoptotic form of programmed cell deathmay be involved.29,30
The finding of extrarenal apoptosis in the cpk mice is in agreementwith the well-known fact that polycystic kidney disease is nota kidney-specific disease.31 The ability of the lung and liverto maintain cell-number homeostasis by cellular proliferationmay explain the absence of abnormalities in these organs. Apoptoticdegeneration of vessel walls could lead to the aneurysms thatare frequently associated with polycystic kidney disease. Thefinding of apoptosis in both primary cultures and serially passagedpolycystic kidney cells in vitro, in the absence of uremia,ischemia, obstruction, compression, or other undefined pathologicconditions associated with the in vivo disease state, suggeststhat apoptotic cell death may be an innate abnormality of cellsin polycystic kidney disease.
The mature mammalian kidney is a quiescent organ with littleor no mitotic activity, and little or no apoptosis was foundin adult human kidneys. Although the mature kidney is capableof cellular proliferation in special circumstances, such asafter acute tubular necrosis,32 renal cells, like differentiatedneurons, seldom divide. Usually, the kidney compensates forloss in mass and function or responds to increased functionaldemands by hypertrophy and not by hyperplasia.33 The evolutionof this hypertrophic response is dictated by the complex cellularand functional architecture of the individual nephrons. Developmentally,each nephron results from the amalgamation of the inducing uretericbud, which differentiates into collecting tubules, and of theresponding metanephric mesenchyme, which differentiates intothe remainder of the nephrons.34 Large-scale apoptosis occursduring metanephric development,14,35 which may serve to matchthe numbers of collecting ducts developed from the uretericbud to the number of tubules developing from the metanephricmesenchyme. As a result, after all the differentiated nephronsare formed, the mature kidney can no longer generate new nephrons.In polycystic kidneys, uncompensated apoptosis and the inabilityto regenerate new nephrons would result in the progressive lossof renal tissue.
The progressive apoptotic death of rod cells in retinitis pigmentosacould result from mutations in several different genes,36 andso too the various mutations in polycystic kidney disease coulddirectly or indirectly trigger the aberrant activation of oneof the apoptotic pathways. For example, basement-membrane componentsregulate the proliferation, differentiation, and apoptosis ofepithelial cells.37 Since polycystic kidneys have abnormal basementmembranes,1,2 the apoptotic loss of intact nephrons, the increasedproliferative potential of cystic epithelia, and the aberrantepithelial-cell polarization and differentiation in polycystickidneys could be a result of defective basement-membrane components.
In mice with a homozygous deletion of the bcl-2 gene, polycystickidney disease develops and is fatal38,39; bcl-2 is a memberof a family of genes that regulate apoptosis. Polycystic kidneyshave also been reported in transgenic mice expressing c-myc40or SV40-LT.41 The fact that all these divergent models of polycystickidney disease can be linked to the aberrant expression of regulatorsof apoptosis provides circumstantial support for the hypothesisthat apoptosis may be central to the pathogenesis of polycystickidney diseases.
Supported by grants from the National Institutes of Health (DK40700and DK45663).
Source Information
From the Division of Nephrology, Department of Medicine, University of California at Los Angeles, Los Angeles, CA 90024-1689, where reprint requests should be addressed to Dr. Woo.
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