*Division of Nephrology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Japan; Department of Nephrology, Hitachi General Hospital, Hitachi, Japan; and Division of Nephrology, Tokyo Medical University, Kasumigaura Hospital, Ami, Japan.
Correspondence to Dr. Kunihiro Yamagata, Division of Nephrology, Institute of Clinical Medicine, University of Tsukuba, 1-1-1, Ten-oudai, Tsukuba, 305-8575, Japan. Phone: +81-298-53-3202; Fax: +81-298-53-3202; E-mail: k-yamaga{at}md.tsukuba.ac.jp
ABSTRACT. Glomerular epithelial cells are primary pathogenicsites in focal segmental glomerulosclerosis (FGS) lesions. Glomerularepithelial cells are regarded as terminally differentiated cellsthat do not proliferate. These characteristics are also notedfor neurons and muscular cells, which are major sites of mitochondrialDNA (mtDNA) mutation accumulation. Screening for mtDNA mutationswas performed with renal biopsy specimens from patients withprimary FGS and patients with IgA nephropathy (as subjects withsecondary FGS and as control subjects). mtDNA extracted fromkidney biopsy specimens was amplified with appropriate primerpairs for study of the mtDNA point mutations 3243AG, 3271TC,8344AG, and 8993TG/C, as well as the common deletion (a 4977-bpdeletion spanning mtDNA nucleotide pairs 8469 to 13447). Insitu amplification of both total mtDNA and the common deletionwas also performed. Two patients with FGS demonstrated the 3243AGpoint mutation; 12 patients with FGS and seven patients withIgA nephropathy accompanied by glomerulosclerotic lesions exhibitedthe common deletion in their kidney tissue. No patient demonstratedthe mtDNA mutations 3271TC, 8344AG, or 8993TG/C. The degreeof heteroplasmy for the 3243AG point mutation was >85%; however,the heteroplasmy for the common deletion was <1%. As determinedwith in situ PCR, normal mtDNA was mainly distributed in thetubular epithelium and mtDNA with the common deletion was mainlydistributed among glomerular epithelial cells. In conclusion,it is suggested that mtDNA mutations are distributed in glomerularepithelial cells among some patients with primary FGS or secondaryFGS with IgA nephropathy. These mutations may be related toglomerular epithelial cell damage.
Focal segmental glomerulosclerosis (FGS) lesions represent thefinal common pathway for nephron degeneration in many formsof chronic progressive renal failure (1). The initial pathologicchanges in FGS are thought to occur in glomerular epithelialcells (1,2). Renal epithelial cell damage attributable to mitochondrialdysfunction in congenital nephrotic syndrome was recently reported(3,4). Furthermore, glomerular involvement of an AG transitionat mitochondrial DNA (mtDNA) position 3243 in the gene for tRNALeuyielded minor glomerular abnormalities (5) or FGS (6–9).
mtDNA is a 16,569-bp closed circular duplex and is the onlyextranuclear DNA in the human species. mtDNA encodes 13 respiratorychain polypeptides, two rRNA, and 22 tRNA (10,11). mtDNA isprone to oxidative damage (12), because it lacks histone-likecoverage and is very close to the inner mitochondrial membrane,which is the major intracellular source of reactive oxygen species(13–15). Furthermore, accumulation of age-dependent mtDNAmutations has been reported (15,16); in particular, a 4977-bpdeletion spanning mtDNA nucleotide pairs 8469 to 13447 (thecommon deletion) has frequently been reported (17–19).Age-dependent accumulation of an AG transition at mtDNA position3243 has also been reported (20–22). Blood cells oftenexhibit low levels of mutated mtDNA, with mutations such asthe common deletion and the 3243AG transition (23–25).In general, glomerular epithelial cells are regarded as terminallydifferentiated cells that do not proliferate (26–28).These characteristics are also noted for neurons and muscularcells, which are regarded as major sites for the accumulationof mtDNA mutations. In this study, to clarify the relationshipbetween FGS lesions and mtDNA mutations, we performed mtDNAmutation screening of kidney biopsy specimens and studied thein situ accumulation of mtDNA mutations in glomerular epithelialcells.
Kidney Samples and DNA Isolation
Renal tissues from patients with primary FGS or IgA nephropathywere obtained in kidney biopsies performed at the Universityof Tsukuba or one of its affiliated hospitals. The patientswere assigned to one of three groups, i.e., (1) primary FGS(16 patients), (2) familial FGS (seven patients with maternallyinherited renal diseases and/or neurologic diseases), or (3)IgA nephropathy (16 patients). The characteristics of the patientsare presented in Table 1. All patients were accepted with writteninformed consent. The indications for renal biopsy were as follows:for 11 patients with primary FGS and one patient with IgA nephropathy,nephrotic syndrome; for the other patients, close examinationof chance proteinuria and/or hematuria. The kidney biopsy specimenswere processed for light microscopy, immunofluorescence assays,and electron microscopy by using routine methods. Total DNAwas extracted from paraffin-embedded samples by using DEXPAT(Takara Biomedicals Inc., Tokyo, Japan), following the recommendationsof the manufacturer.
Qualitative PCR
Regions of the mitochondrial genome between positions 3130 and3301, positions 8257 and 8386, and positions 8837 and 9017 wereamplified by PCR with a GeneAmp PCR 9600 system (Perkin-Elmer,Norwalk, CT). PCR amplifications were performed in 50-µlvolumes with 200 µM levels of each dNTP, 0.5 µMlevels of each primer, 1 U of Ex Taq polymerase (Takara Biomedicals),PCR reaction buffer (Takara Biomedicals), and 200 ng of eachDNA template. The primer sets used in this study are indicatedin Table 2. The amplification conditions included 35 cyclesof denaturation at 94°C for 30 s, annealing at 55 to 57°Cfor 30 s, and extension at 72°C for 1 min, with an initial3-min denaturation step at 94°C. The amplified fragmentswere digested with restriction endonucleases (ApaI, AflII, BglI,or HpaII; Takara Biomedicals). The digested DNA fragments wereseparated on 8% polyacrylamide gels, and the gels were stainedwith SYBR Green I nucleic acid stain (Takara Biomedicals). Fordetection of the common deletion (a 4977-bp deletion betweenpositions 8482 and 13459), the common deletion primer set (Table 2)was used. The amplification conditions included 30 cyclesof denaturation at 94°C for 30 s and annealing at 55°Cfor 30 s, using Ampri Wax PCR Gem 50 (Perkin-Elmer) for hot-startPCR, according to the instructions provided by the manufacturer.
Quantitative PCR
The frequency of the AG mutation at nucleotide position 3243of mtDNA was determined by colony-directed PCR and restrictionfragment length polymorphism analysis of subcloned PCR productsfrom >40 randomly selected colonies in each specimen. Subcloningof the PCR products was performed by using an Original TA cloningkit (Invitrogen, San Diego, CA), according to the instructionsprovided by the manufacturer, as follows. Fresh PCR productswere ligated into pCR2.1 vector, transformed into One Shot cells,and incubated overnight at 37°C. More than 40 growth colonieswere selected, and colony-directed PCR and restriction fragmentlength polymorphism analyses were performed.
The frequency of the common deletion was determined in triplicateby using a Perkin-Elmer 7700 TaqMan PCR system. The forwardprimer for the mitochondrial common deletion was 5'-CCCCCATACTCCTTACACTA-3'(positions 8406 to 8425), and the reverse primer was 5'-TGCGGTTTCGATGATGTGGT-3'(positions 13533 to 13514). The probe was FAM-CCTACCTCCCTCACCATTGGCAGCCTAG-TAMRA(positions 8467 to 8482 and13459 to 13471). For total mtDNA,the primers were 5'-GACGAGCTACCTAAGAACAG-3' (forward; positions1913 to 1932), 5'-GAGGGTTCTGTGGGCAAATT-3' (reverse; positions2070 to 2051), and FAM-CGACAAACCTACCGAGCCTGG-TAMRA (probe; positions1989 to 2009). The amplification conditions included 50 cyclesof denaturation at 95°C for 15 s and annealing at 53°Cfor 60 s, with an initial 10-min denaturation step at 95°C.The deletion levels, expressed as percentages of both the commondeletion and the total mtDNA level, were calculated from linear-rangeamplification plots of the sequence detector v1.6 (Perkin-Elmer).Extrapolation of the plots to the zero-amplification cycle yieldedthe relative amounts of mtDNA before the PCR amplification.
In Situ PCR In situ PCR was performed as described previously (29), withadjustments as follows. Paraffin-embedded, 5-µm, kidneybiopsy sections were used. The slides were deparaffinized andrehydrated by using standard protocols. The slides were thentransferred into phosphate-buffered saline (PBS) (pH 7.4) andbriefly equilibrated. The tissue sections were permeabilizedby a 10-min treatment with proteinase K (10 µg/ml) in100 mM Tris-HCl (pH 7.5), 5 mM ethylenediaminetetraacetate,in a humidified chamber, and were boiled in citrate buffer (pH6.0) with microwave exposure. The slides were transferred intocold PBS for at least 5 min and were refixed with 4% paraformaldehydein PBS. The slides were removed from the PBS and carefully spot-driedaround the tissue sections with Kimwipes. PCR cocktail (50-µlreaction volume with final concentrations of 0.5 µM forwardand reverse primers and 0.2 µM TaqMan probe, in 25 µlof TaqMan Universal PCR Master Mix; Perkin-Elmer) was appliedto the slide, and a Takara slide seal (Takara Biomedicals) forin situ PCR under hot-start conditions was applied, to sealthe reaction mixture over the tissue. The PCR profile consistedof an initial 2-min denaturation step at 50°C and 10 minat 95°C, typically followed by 28 to 40 cycles of denaturationat 94°C for 15 s and annealing at 53°C for 1 min, ina thermal cycler (PTC-100-16MS; MJ Research, Watertown,glomerulosclMA).In situ amplification of both total mtDNA and the common deletionregion was observed with confocal laser microscopy (TCS SP2;Leica, Wetzlar, Germany). In control experiments, PCR cocktailsomitting both forward and reverse primers were used for eachin situ PCR amplification.
Statistical Analyses
Data were expressed as median values and ranges, because ofthe asymmetrical distribution of the data. The Mann-WhitneyU test was used for comparisons. A P value of <0.05 was consideredstatistically significant.
Screening for mtDNA Point Mutations
Of the patients with FGS, two patients with familial FGS demonstratedmtDNA 3243AG point mutations (Figure 1). Both patients exhibitedsensory hearing loss. One patient had a family history of hearingloss (his mother), and his mother demonstrated the same pointmutation in her peripheral lymphocyte DNA sample. The otherpatient had a family history of end-stage renal disease (hermother, who died 8 yr earlier) and hearing loss (her motherand younger sister). Figure 2 presents the light-microscopicfindings for a renal biopsy specimen from a patient with the3243AG point mutation. Typical segmental sclerotic changes andhyaline lesions were observed near the vascular pole. No vascularlesions were observed in the glomeruli. Cystic tubular dilations,tubular degeneration, and interstitial fibrosis were obviousin a serial renal biopsy specimen obtained from this patient3 yr later. Figure 3 presents the electron-microscopic findingsfor a patient with the 3243AG point mutation. Abnormally developedmitochondria were observed in the glomerular epithelium. Quantitativeevaluation of wild-type and mutant mtDNA demonstrated that theproportions of mutant mtDNA were 88% for one patient and 85%for the other patient in kidney biopsy samples and 80 and 56%,respectively, in peripheral lymphocyte DNA samples. No patientdemonstrated mtDNA 3271TC, 8344AG, or 8993TG/C point mutationsin the analyzed kidney biopsy samples (Table 3).
Figure 1. Mitochondrial DNA (mtDNA) fragments from patients with the 3243AG point mutation and a normal control subject, amplified by PCR and digested with ApaI. M, 100-bp ladder; lanes 1 and 2, patients; lanes 3 and 4, normal control subject. The 184- and 112-bp products were observed for the patients; only a 294-bp product was observed for the other patients.
Figure 2. Light-microscopic findings for a patient with the 3243AG mutation. Focal and segmental hyalinosis and sclerosis at the vascular pole were observed. Periodic acid-Schiff stain. Magnification, x400.
Figure 3. Electron-microscopic findings for a patient with the 3243AG mutation. Large and small mitochondria with abnormally developed cristae were observed in the cytoplasm of the glomerular epithelium.glomerulosclMagnification, x10,000.
Table 3. Mitochondrial DNA mutations in kidney biopsy specimens
Screening for the mtDNA Common Deletion
The mtDNA common deletion was observed for nine patients withprimary FGS, three patients with familial FGS, and seven patientswith IgA nephropathy (Table 3). Figure 4 presents the amplificationplots for total mtDNA and the common deletion, with real-timePCR. The degree of heteroplasmy for the common deletion was<1% for all patients (Table 3). Age-dependent accumulationof the common deletion was not observed in this study, and thecommon deletion was not related to renal function or proteinuria.The common deletion was not related to glomerular alterationsin primary or familial FGS; however, the total numbers of segmentalsclerotic glomeruli and glomeruli with adhesion of the capillarytuft to Bowman’s capsule were significantly increasedin IgA nephropathy with the common deletion (Table 4). Figure 5presents the electron-microscopic findings for a patient withprimary FGS and the common deletion. Accumulated dysmorphicmitochondria, including abnormally developed mitochondria, wereobserved in the glomerular epithelium. However, the other patientsdid not demonstrate epithelial mitochondrial abnormalities inextensive electron-microscopic examinations.
Figure 4. PCR amplification of the common deletion (inset) and TaqMan PCR amplification plots of total mtDNA and the common deletion. A 160-bp product was observed. M, 100-bp ladder; lane 1, patient 1; lane 2, patient 2; lane 3, patient 15; lane 4, normal control sample. Total mtDNA amplification was observed after 17 cycles, and common deletion amplification was observed after 35 cycles.
Figure 5. Electron-microscopic findings for a patient with primary focal segmental glomerulosclerosis. (A) Extensive foot process fusion was observed. (B) A higher-magnification view of the abnormal cytoplasm of a glomerular epithelial cell revealed a markedly increased number of mitochondria, of varied size and shape. Some of the mitochondria exhibited abnormally developed cristae.glomerulosclMagnification, x2000 in A; x10,000 in B.
In Situ Detection of the Common Deletion Figure 6 presents the in situ PCR results for total mtDNA. Mostof the mtDNA signals were observed in the cytoplasm of the renaltubular epithelium. Figure 7 presents the in situ PCR resultsfor the common deletion. The common deletion was observed mainlyin the glomerular epithelium. In both experiments, neither mtDNAnor common deletion signals were detected when assays were performedwith a PCR mixture omitting both forward and reverse primers(data not shown).
Figure 7. In situ PCR for the common deletion. The mtDNA common deletion was observed mainly in the glomerular epithelium. Occasional signals were observed in the tubular epithelium and interstitium. Magnification, x200.
FGS is morphologically characterized by segmental areas of sclerosisin some glomeruli. FGS lesions represent a heterogeneous categorythat includes several types of glomerular lesions and severalclinical syndromes or manifestations. FGS associated with mtDNAmutations was recently reported (6–9). In general, glomerularepithelial cells are regarded as terminally differentiated cellsthat do not proliferate (26–28). These characteristicsare also noted for neurons and muscular cells, which are regardedas major sites for the accumulation of mtDNA mutations. In ourscreening of mtDNA mutations in renal biopsy specimens, twopatients with a mtDNA 3243AG point mutation and 19 patientswith a common deletion were identified.
A mtDNA 3243AG point mutation was originally noted for patientswith mitochondrial myopathy, encephalopathy, lactic acidosis,and stroke-like episodes (10). However, several studies havesuggested that this mutation is associated with diabetes mellitusand/or deafness without neurologic involvement (7–9).Our patients with the mtDNA 3243AG point mutation exhibitedmaternally inherited sensory hearing loss or renal disease.Several forms of familial FGS have been reported, includingthose involving mutations in glomerular epithelial cytoskeletoncomponents such as podocin (30) and -actinin 4 (31) or structuralcomponents of the slit diaphragm such as nephrin (32). The mtDNA3243AG point mutation is regarded as another form of familialFGS. This mutation has been observed in approximately 0.6 to1.5% of patients with type 2 diabetes mellitus (25,33) and inapproximately 16.3/100,000 individuals in the general adultpopulation (34). Guillausseau et al. (35) reported that 28%of patients with type 2 diabetes mellitus and the mtDNA 3243AGpoint mutation exhibited kidney disease, and renal histologicanalyses for three patients who underwent renal biopsies demonstratedFGS. Therefore, the mtDNA 3243AG point mutation may be the mostfrequent etiologic mutation in familial FGS.
The pathogenesis of FGS lesions with the mtDNA 3243AG pointmutation exhibits some discrepancies. Mochizuki et al. (7) andDoleris et al. (8) reported that vascular smooth muscle cellinjury attributable to mitochondrial damage led to arteriolarhyaline lesions, which abolished the autoregulatory mechanismfor glomerular pressure; subsequent renal hemodynamic alterationsmight occur, resulting in FGS lesions. In contrast, the studyby Hotta et al. (9) and our study demonstrated that abnormalmitochondria accumulated in glomerular epithelial cells, whichled to glomerular epithelial dysfunction, resulting in FGS lesions.More than 85% of mtDNA in analyzed kidney samples exhibitedthis point mutation. Although the distribution of mtDNA is abundantin the renal tubular epithelium, early renal manifestationsof the mtDNA 3243AG point mutation involve glomeruloscleroticchanges. In general, most mitochondrial diseases exhibit a delayedonset and a progressive course. The phenotypic expressions ofthese diseases are affected by both the predisposing mutationand an age-related factor, which causes a decline in mitochondrialfunction (36). Although the characteristics of glomerular epithelialcells may cause an accumulation of mutated mtDNA during mtDNAreplication, aging and continuous oxidative stress also damagenot only the glomeruli but also tubular tissues.
In screening for the common deletion, this mutation was detectedfor nine patients with primary FGS, three patients with familialFGS, and seven patients with IgA nephropathy; the level of themutation was extremely low. The common deletion was thus observednot only in primary FGS but also in other glomerular diseases,including IgA nephropathy. Indeed, we also detected the commondeletion in renal biopsy samples from patients with diabeticglomerulosclerosis (37). However, Simonetti et al. (18) reportedthat the common deletion was not observed in mtDNA from a kidneysample from an aged normal subject, and Liu et al. (20) reportedthat the common deletion was not observed in mtDNA from normalkidney samples from <37-yr-old subjects and the maximal mutationrate among normal aged subjects was 0.001% in a kidney samplefrom a 76-yr-old subject. Although the level of this mutationmight be extremely low, most of the patients in this study withthe common deletion were thought to exhibit abnormal conditions.Furthermore, Hayashi et al. (38) reported that >60% accumulationof the common deletion mutation was needed for progressive inhibitionof mitochondrial translation and reduction of cytochrome c oxidaseactivity. Therefore, it may be difficult for a pathologic changewith this level of deletion mutation to manifest itself. Bhatet al. (19) reported that, among patients with unilateral peripheralarterial disease, muscles from hemodynamically affected limbsexhibited greater proportions of the common deletion mutation,because of oxidative stress. The common deletion detected inthis study might be the consequence of oxidative stress duringthe glomerular disease process. However, in situ distributionof the common deletion in glomerular epithelial cells was detectedamong our subjects. It is speculated that a few glomerular epithelialcells might exhibit accumulation of the common deletion in >60%of mtDNA in the cytoplasm, resulting in functional damage tothe cells. Furthermore, a mtDNA deletion mutant mouse modelhas been produced (39). These mutant mice, which accumulated>80% deletion mutations in their kidney mtDNA, experiencedsevere renal diseases and died within 6 mo because of renalfailure. The kidney is also the most likely target and accumulationorgan for the mtDNA deletion mutant in this mouse model (39).Furthermore, patients with IgA nephropathy and the common deletionexhibited significantly more segmental lesions, including adhesions,than did patients without the common deletion. There were nosignificant relationships among other parameters, and otherpatient groups demonstrated no relationships with segmentallesions. Adhesion of the capillary tuft to Bowman’s capsuleis regarded as an initial change of FGS lesions (1). Most ofthe renal cells that accumulate the common deletion might continueto degenerate during the disease process. Once FGS lesions areestablished, glomerular epithelial cells that accumulate thecommon deletion might be lost. In the brains of patients withAlzheimer’s disease, mtDNA common deletion levels wereobserved to be very low, in contrast to the presence of highlevels of 8'-hydroxy-2'-deoxyguanosine, a marker of oxidativeDNA damage (40). Continuous oxidative stress to renal cellsduring the FGS disease process might hinder the mtDNA replicationapparatus (40). Solin et al. (4) observed a 30% decrease inmtDNA contents, compared with normal control samples, in congenitalnephrotic syndrome of the Finnish type. This might have resultedin the lack of a relationship between the accumulation of apparentlylower levels of the mtDNA common deletion in our patients withFGS and aging. We used renal biopsy samples, which yielded verylimited amounts of tissue for mtDNA analyses; the proportionsof renal cortex and medulla, and thus the number of glomeruli,might be different for each sample. This could be another explanationfor our difficulty in evaluating the level of mtDNA heteroplasmyin this study.
Mitochondria play a major role in apoptosis (41). Mitochondria-mediatedapoptosis has been observed in rat hypertensive nephrosclerosis(42). Further studies are needed to clarify the role of mitochondriain hereditary renal diseases, as well as in aging and progressiverenal diseases, and especially to determine whether distributionof the common deletion in the glomerular epithelium is the consequenceof stress to the cells or the cause of glomerular epithelialdamage attributable to mitochondrial dysfunction and glomerulosclerosis.
Acknowledgments
We thank Dr. Kazuto Nakata and Dr. Jun-ichi Hayashi (Instituteof Biological Science, University of Tsukuba) for valuable discussion,Dr. Michio Nagata (Department of Pathology, University of Tsukuba)and Dr. Tatsuro Shimokama (Department of Pathology, HitachiGeneral Hospital) for pathologic interpretation, Devin Oglesbee(Institute of Molecular Biology, University of Oregon) for manuscriptpreparation, as well as valuable discussion and suggestions,and Rie Kikko for excellent technical assistance. This studywas supported in part by a Grant-in-Aid for Scientific Researchfrom the Ministry of Education, Science, Sports, and Cultureof Japan (Grant 13671097, to Dr. Yamagata) and by a grant fromthe Disease Control Division, Health Service Bureau, Ministryof Health, Labor, and Welfare of Japan.
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Received for publication August 23, 2001.
Accepted for publication April 11, 2002.
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M. S. Janes, B. J. Hanson, D. M. Hill, G. M. Buller, J. Y. Agnew, S. W. Sherwood, W. G. Cox, K. Yamagata, and R. A. Capaldi Rapid Analysis of Mitochondrial DNA Depletion by Fluorescence In Situ Hybridization and Immunocytochemistry: Potential Strategies for HIV Therapeutic Monitoring
J. Histochem. Cytochem.,
August 1, 2004;
52(8):
1011 - 1018.
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Abstract
Introduction
G, 3271T
-actinin 4 (31) or structural components of the slit diaphragm such as nephrin (32). The mtDNA 3243A
