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(The FASEB Journal. 1999;13:523-532.)
© 1999 FASEB

Research Communications

Altered gene expression and functions of mitochondria in human nephrotic syndrome

HARRY HOLTHÖFERa , 1 , MATTHIAS KRETZLERb , ANNI HALTIAa , MARJA-LIISA SOLIN, JAN-WILLEM TAANMANd , HERMANN SCHÄGGERe , WILHELM KRIZf , DONTSCHO KERJASCHKIc and DETLEF SCHLÖNDORFFb


a The Haartman Institute, Division of Bacteriology and Immunology, University of Helsinki, Finland;

b Medical Policlinic, University of Munich, Munich, Germany;

c Department of Pathology, University of Vienna, Vienna, Austria;

d Department of Clinical Neurosciences, University of London, London, U.K.;

e University of Frankfurt, Frankfurt, Germany; and

f Department of Anatomy, University of Heidelberg, Heidelberg, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The molecular basis of glomerular permselectivity remains largely unknown. The congenital nephrotic syndrome of the Finnish type (CNF) characterized by massive proteinuria already present but without extrarenal symptoms is a unique human disease model of pure proteinuria. In search of genes and pathophysiologic mechanisms associated with proteinuria, we used differential display-PCR to identify differences in gene expression between glomeruli from CNF and control kidneys. A distinctly underexpressed PCR product of the CNF kidneys showed over 98% identity with a mitochondrially encoded cytochrome c oxidase (COX I). Using a full-length COX I cDNA probe, we verified down-regulation of COX I mRNA to 1/4 of normal kidney values on Northern blots. In addition, transcripts of other mitochondrially encoded respiratory chain complexes showed a similar down-regulation whereas the respective nuclearly encoded complexes were expressed at comparable levels. Additional studies using histochemical, immunohistochemical, in situ hybridization, RT-PCR, and biochemical and electron microscopic methods all showed a mitochondrial involvement in the diseased kidneys but not in extrarenal blood vessels. As a secondary sign of mitochondrial dysfunction, excess lipid peroxidation products were found in glomerular structures in CNF samples. Our data suggest that mitochondrial dysfunction occurs in the kidneys of patients with CNF, with subsequent lipid peroxidation at the glomerular basement membrane. Our additional studies have revealed similar down-regulation of mitochondrial functions in experimental models of proteinuria. Thus, mitochondrial dysfunction may be a crucial pathophysiologic factor in this symptom.—Holthöfer, H., Kretzler, M., Haltia, A., Solin, M.-L., Taanman, J.-W., Schägger, H., Kritz, W., Kerjaschki, D., Schlöndorff, D. Altered gene expression and functions of mitochondria in human nephrotic syndrome.


Key Words: proteinuria • congenital nephrotic syndrome • glomerular disease • differential display • cytochrome c oxidase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PROTEINURIA REMAINS the clinical hallmark of damage to the kidney glomerular filtration barrier. Despite progress in establishing the immunologic, inflammatory, toxic, metabolic, and genetic factors involved in diseases with proteinuria, the molecular mechanisms maintaining the glomerular filtration barrier are poorly understood (1 , 2 ). This, together with lack of specific markers for the underlying cause, has contributed to the slow progress in establishing etiology-directed treatments for proteinuria.

Congenital nephrotic syndromes (CNS)2 are rare pediatric kidney diseases manifesting at or soon after birth with massive proteinuria. One of the best-characterized CNS is the congenital nephrotic syndrome of the Finnish type (CNF) with treatment-resistant proteinuria and characteristic podocyte changes in the glomeruli (3 , 4 ). Prenatal diagnosis of CNF can be made on the basis of extremely high alpha-fetoprotein in maternal serum or in amniotic fluid by the second trimester of pregnancy. The energy requirements and maturation of kidney specific mitochondrial oxidative phosphorylation complexes appear by the second trimester, whereas in other fetal tissues these are observed considerably later 5-7) . CNF used to be fatal during the first months of life, but current treatment of early bilateral nephrectomy, followed by dialysis and renal transplantation, is curative and provides a unique source of tissue material for search of the basic mechanisms involved. Kestilä et al. (8) recently established the defective gene NPHS1 in CNF patients. This gene, with as yet unknown functions, encodes a transmembrane protein with similarity to cell adhesion molecules.

To search for new molecular clues for glomerular dysfunction and pathophysiology of proteinuria, we used the differential display reverse transcription polymerase chain reaction (DDRT-PCR) method (9) , which permits the detection of genes, known and unknown, that are differentially expressed in control and experimental tissues. For this purpose, we took advantage of kidneys removed from CNF patients. One DDRT-PCR product was distinctly decreased in kidneys from CNF patients and could be identified as the mRNA for subunit I of cytochrome c oxidase (COX I). As terminal component of the mitochondrial respiratory chain, cytochrome c oxidase plays a crucial role in aerobic energy production.

Here we present data with molecular, biochemical, histochemical, immunohistochemical, functional, and morphologic evidence showing consistent mitochondrial respiratory chain abnormalities in kidneys of CNF patients. Furthermore, we found enhanced lipid peroxidation (LPO) in glomeruli from CNF. Defects in mitochondrial respiratory chain functions can lead to excessive reactive oxygen species and LPO (10) . Moreover, LPO has been implicated in the pathophysiology of glomerular proteinuria (11 , 12 ). We therefore postulate that altered mitochondrial function may lead to enhanced lipid peroxidation and proteinuria in the kidney.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Normal and nephrotic human kidneys
Renal tissues of CNF (N=6) were obtained at nephrectomies performed according to an established treatment protocol (13) . Diagnosis of CNF was based on the typical clinical picture at birth (placental weight >40% of the weight of the newborn, edema, massive proteinuria), exclusion of other types of congenital nephroses, and later by the typical pathology at nephrectomy (4) . All procedures were approved by the ethical committee of the University of Helsinki.

The kidneys at nephrectomy were perfused with Ringer's buffer solution and glomeruli were rapidly isolated as described earlier (14) , resulting in over 98% purity of the glomerular preparations. These were immediately processed for RNA isolation (15) . Samples of cortical tissue were also processed for histochemistry, immunohistochemistry measurement of respiratory chain complex activities, and electron microscopy as described previously (15) .

Kidney biopsies from patients with membranous glomerulonephritis (N=3) or adult minimal change disease (N=3; age of all patients >18 years) were obtained from routine diagnostic biopsies. The biopsies were snap-frozen and stored in liquid nitrogen until used.

For normal controls, cadaver kidneys (N=2; age of donors 12 and 18 years) unsuitable for transplantation due to vascular anatomic reasons (Department of Surgery, University of Helsinki) or the normal poles of kidneys removed because of Wilms' tumor (N=2; age 3 and 5 years) were used. Other human tissues used included samples from extrarenal arteries from CNF and control kidneys.

RNA isolation and DDRT-PCR
Total glomerular RNA was isolated from the CNF and control kidneys as described (15 , 16 ). To process the samples for DDRT-PCR analysis (all in duplicate), a method described earlier was used (9 , 17 ), following the manufacturers' instructions (GenHunter, Boston, Mass.). The PCR product analysis was performed as described earlier (17) .

Cloning and sequencing
Differentially displayed bands in the CNF glomerular samples were cut from the dried acrylamide gels, extracted, and finally reamplified using the same primers and conditions as for the initial DDRT-PCR. The PCR products were then gel-purified and cloned using the TA cloning system (In Vitrogen, San Diego, Calif.) (18) . For each PCR product, three to six colonies from a single transformation were prepared (17) . Six clones were then sequenced and screened for homology with database sequences using the BLAST search algorithm via Internet at the National Center for Biotechnology, Washington, D.C.

Northern blotting
Total RNA from five CNF and four normal human cortex samples were prepared (15 , 17 ) and used for reconfirmation of the candidate DDRT-PCR clones in Northern blotting (17) . To control the total RNA content and lack of degradation in the preparations, blots were rehybridized with a human ß-actin probe. For autoradiography, the filters were exposed on Fuji Bas IIIS Imaging Plates and the expression was quantified using a PhosphorImager and accompanying MacBAS software (Fuji Photo Film Co, Japan).

To reveal transcript levels of different COX subunits, we used cloned mtDNA and cDNA probes in Northern analysis. Plasmid pCOX2 contained the complete gene for COX II and 714 bp of the gene for COX I. Plasmid pCOX4 contained a 682 bp COX IV cDNA fragment (courtesy of Dr. M. I. Lomax) and plasmid pCOX6 contained a 450 bp COX VIb cDNA fragment.

Semiquantitative RT-PCR
Because Northern blotting and histological experiments were not sensitive enough for glomerular detection of mitochondrial complexes, we determined the transcript levels of some respiratory chain complex subunits by semiquantitative RT-PCR. RNA samples from isolated glomerular fractions were used as a starting material in this analysis (17) . Sequence-specific oligonucleotide primers were designed for mitochondrially encoded COX I and cytochrome b as well as for COX VIIa, which is encoded in the nucleus. The semiquantiation is based on the serial dilutions of control plasmid DNA and sample cDNAs in the linear range of amplification and on the amount of housekeeping (ß-actin) amplification product (17) .

Histochemistry, immunohistochemistry, and visualization of lipid peroxidation products
Histochemical methods for respiratory chain complex II (succinate dehydrogenase, SDH) and IV (cytochrome c oxidase, COX) were used (19) . For this purpose, fresh cryostat sections were incubated with their specific substrate solutions, producing colored insoluble granular deposits at sites of enzymatic activity.

For immunohistochemistry the tissue sections were fixed for 3 min in acetone at -20°C and incubated with a monoclonal antibody to COX I (clone 1D6-E1-A8) or to COX IV (10G8-D12-C12) 20-22) . Lipid peroxidation products were determined immunohistochemically using antibodies specific for malonyldialdehyde, as described previously (23; kindly provided by Dr. T Montine). For visualization, fluorescein isothiocyanate (FITC) -conjugated second antibodies were used. An Olympus Ox50 microscope with a filter system for FITC fluorescence was used for microscopy.

Quantification of mitochondrial respiratory chain complexes
The steady-state level activities of the mitochondrial respiratory complexes I to V were analyzed by solubilizing 10 mg kidney tissue of CNF patients (N=3) and controls (N=3), and separated in the native state by blue native polyacrylamide gel electrophoresis (BN-PAGE). Lanes from BN-PAGE were then processed by sodium dodecyl sulfate (SDS) -PAGE in a second dimension and quantified by densitometry, as described previously (24 , 25 ). The staining intensities of each individual complex were normalized to the intensity of complex III in each gel.

Electron microscopy
Cortical samples from four CNF patient kidneys and four controls were fixed and processed as previously reported (12 , 26 ).

Statistics
Mitochondrial quantitations are expressed as mean ±SD. Statistics were performed with unpaired two-tailed t tests. Data were obtained by direct counting from the electron micrographs at x4500 magnification. Four to six such sections were analyzed from each sample.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
DDRT-PCR
Using RNA from glomeruli of CNF kidneys and 50 primer pair combinations for the DDRT-PCR analysis, a total of 3800–3900 PCR products could be reproducibly displayed. Twelve PCR products showing distinct changes in expression levels were chosen for additional studies including reconfirmation of expression differences, sequencing, and screening of genetic databases for homologies. One of the products decreased in CNF glomeruli (clone B36) showed 98% identity over 363 bp to the mRNA of mitochondrially encoded subunit I of cytochrome c oxidase (COX I; see Fig. 1 ). Using the cloned PCR product as a probe, an mRNA of the expected size of could be detected (see Fig. 2 ). The intensity of the signal of this mRNA was 70% lower in CNF than in controls (Fig. 2 , Table 1 ).



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  		Figure 1. Sequence comparison of differential display PCR product B36 and cytochrome c oxidase subunit I (COX I) showing 98% similarity to each other. N represents unknown nucleotides.



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  		Figure 2. Northern blot analysis of the differential display product B36, COX I, COX II (mitochondrially encoded), COX IV, and COX VIb (nuclearly encoded) mRNAs. The samples consist of 30 g total cortical RNA extracted from normal (lanes 1–4) or CNF (lanes 5–9) kidneys. Hybridization with ß-actin probe serves as control for even loading.


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  		Table 1. Relative densities of Northern blot hybridization signals detected by densitometric scanning using phosphoimager software (see Fig. 2 )a

Northern blotting and in situ hybridization
Because of the high degree of identity of B36 PCR product with COX I, another mitochondrially encoded COX subunit (COX II) as well as two nuclear encoded subunits (COX IV and VIb) of the cytochrome c oxidase complex were analyzed. In all Northern blotting experiments, RNA isolated from the whole cortex was used. Similar to DDRT-PCR product B36, both COX I and COX II showed consistent underexpression in CNF tissue (Fig. 2) . In contrast, the levels of the nuclearly encoded subunits IV and VIb did not differ from the levels of controls (see Table 1 ). To localize the mRNA expression, we performed in situ hybridization with band B36, COX I, COX II, COX IV, and COX VIb. A decrease of B36, COX I, and COX II based on the amount of grains/cell was observed especially in the proximal and distal tubuli in CNF cortical kidney as compared to the controls (data not shown). Again, in situ hybridization of the nuclearly encoded COX IV and VIb failed to show any difference in signal intensity between CNF and control kidney samples.

Semiquantitative RT-PCR for respiratory chain subunit
Since reactivity in immunohistochemistry and in situ hybridization in the glomeruli was too faint, possibly due to the overwhelming number of mitochondria in tubules, we analyzed the transcript levels of COX subunits directly in isolated glomeruli by semiquantitative RT-PCR (Fig. 3 ). Again, a 2.5x down-regulation of the mitochondrial (mt) encoded COX I subunits (but not the nuclearly encoded subunits) was observed in CNF glomeruli (Table 2 ). To extend the analysis to other complexes of the respiratory chain, we also performed RT-PCR for glomerular cytochrome b (respiratory chain complex III encoded by the mitochondrial genome). This showed an underexpression comparable to that of COX I in CNF glomeruli (Fig. 3 , Table 2 ).



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  		Figure 3. Semiquantitative RT-PCR of mitochondrially encoded (COX I and cytochrome b) and nuclearly encoded (COX VIIa) transcripts in isolated glomeruli of CNF and normal kidneys, using RT-PCR (for details, see Materials and Methods). cDNAs were serially diluted to the range of linear amplification (10-1–10-4); ß-actin expression was used to obtain comparable cDNA concentrations in all samples. The upper bands in COX I quantification represent PCR artifacts.


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  		Table 2. Transcript abundance in isolated glomeruli as determined by RT-PCR (see Fig. 3 )a

Histochemistry and immunohistochemistry
To study the involvement of mitochondria in CNF at the functional and protein levels, we first used histochemical analysis for the respiratory complex II (SDH; Fig. 4a, b ) and complex IV (COX; Fig. 4c, d ). Similar to in situ hybridization, the histochemical reaction product in the glomeruli was weak in both control and CNF kidneys, precluding reliable comparison. The tubular reactivity of both SDH and COX were remarkably decreased in CNF compared with control kidney cortices (see Figs. 4 a–d). Immunohistochemistry with antibodies recognizing the mt encoded COX I showed an intense, finely granular pattern in the proximal and distal tubular cells, whereas glomerular reactivity was diffuse and faint. A consistent decrease in staining was seen in tubuli of CNF kidneys as compared to controls (Fig. 5a, b ). Similar immunohistochemical analysis for the nuclear encoded COX IV showed intense reactivity in tubular cells, but no difference was apparent between CNF and control tissues (Fig. 5c, d ).



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  		Figure 4. Histochemistry of succinate dehydrogenase (SDH), the respiratory complex II, encoded entirely by nuclear DNA, in normal kidney cortex (a) and CNF (b); cytochrome oxidase respiratory complex IV, encoded by both mitochondrial and nuclear genomes, in normal (c) and CNF kidney cortex (d). Only faint remnant reactivity reflecting respective functional activities can be seen, especially in CNF tubules, compared to the normal kidney. Glomeruli (G) appear faintly reactive in both CNF and control samples. x110 (a, b), 340 (c, d).



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  		Figure 5. Immunohistochemistry of cytochrome c oxidase subunit I (COX I, encoded by mtDNA) in cortical kidney of normal (a) and CNF kidneys (b) and COX I subunit IV (encoded by nuclear DNA) in normal (c) and CNF (d) kidneys. A strong decrease of tubular reactivity of COX subunit I in CNF is seen (b), whereas COX subunit IV appears at comparable levels in CNF and control kidneys. G, glomerulus. x260.

Lipid peroxidation
As a secondary indication of mitochondrial dysfunction, we analyzed local lipid peroxidation products within glomeruli. The glomerular malonyl dialdehyde reactivity appeared finely granular, especially within podocytes (Fig. 6A, C ) and along the glomerular basement membrane, whereas negligible reactivity was seen in the control samples (Fig. 6B ).



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  		Figure 6. Immunostaining for lipid peroxidation products (malonyl dialdehyde adducts, see Materials and Methods) in CNF kidney glomeruli (A, C) and control kidney glomeruli (B). Strong staining of podocytes (arrowheads) and epithelia of Bowmans' capsules (BC) as well as diffuse granular labeling of the glomerular basement membranes in panels a, c. By contrast, barely anything is stained in normal glomeruli. x500.

Steady-state levels of the respiratory chain complexes
Steady-state levels of the mitochondrial respiratory chain complexes were determined with the blue native gel electrophoresis technique (Fig. 7 ). In the CNF patient tissues, the normal ratios of the complexes excluded a specific deficiency of complexes II–V. However, CNF tissue showed a consistent decrease of all respiratory chain complexes from 10% (complex I) to 30% (complexes II–V) of controls (Fig. 7 , Table 3 ).



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  		Figure 7. Respiratory chain complexes from kidney cortex analyzed by 2-dimensional blue native protein gel electrophoresis (blue native PAGE; horizontal dimension) and SDS-PAGE (vertical dimension; for details, see Materials and Methods). The Coomassie blue stain intensities of selected protein bands representative of the absolute amount of individual complexes were determined (see Table 3 ) from these analyses.


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  		Table 3. Analysis of selected respiratory chain complex proteins from blue native PAGE (see Fig. 7 )a

Electron microscopy
To find morphological evidence of mitochondrial involvement and to examine whether the changes observed could be due to a decreased number of mitochondria, we studied the EM morphology of CNF and control kidney cortex. In the CNF samples, characteristic concentric intramitochondrial bodies consisting mostly of double membranes could be seen within otherwise normal appearing mitochondria (Fig. 8 ). In the CNF samples studied (N=4), the morphologic changes observed were present in 13.0–44.4% of all mitochondria (mean 26.9%, SD 3.0, standard error 1.7; P <0.001 to controls). In controls, 0–2% of such abnormal mitochondria (mean 0.7%, SD 0.0, standard error 0.0) was found. No statistically significant differences were found in the amount of mitochondria in CNF as compared to controls (51.7 ±6.6 in CNF, 61.5 ±2.1 in controls, P<0.1).



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  		Figure 8. Electron micrograph from CNF kidney cortex. Note the abnormal concentric bodies (arrows) with double membranes within mitochondria, together with normal-appearing cristae. x5400.

Analysis of renal vasculature from CNF patients and of biopsies of other human proteinuric diseases
To analyze mitochondrial involvement in other CNF tissues, histochemistry and immunohistochemistry for nuclearly and mitochondrially encoded COX subunits were performed in renal vessels. Although consistent reactivity was seen, no differences in the staining intensities could be detected between CNF samples and controls (data not shown). Furthermore, kidney biopsies from patients with heavy proteinuria due to minimal change glomerulonephritis and membranous glomerulonephritis were analyzed with histochemical and immunohistochemical methods for COX. No difference could be seen in the level of glomerular or tubular reactivity for COX in these two groups of proteinuric diseases (not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Here we identified the mitochondrially encoded gene COX I as distinctly down-regulated in a human model disease of pure proteinuria. Semiquantitative RT-PCR analysis of glomerular RNA and histochemical, immunohistochemical, biochemical, and electron microscopic analysis verified the mitochondrial involvement further. Prominent increases in local lipid peroxidation products were also seen along the glomerular basement membrane and in podocytes of CNF, but not in the control kidneys. Together these data suggest that mitochondrial dysfunction possibly via enhanced production of reactive oxygen species and lipid peroxidation may be a previously unrecognized effector mechanism of proteinuria and may offer new clues to its treatment.

Kestilä et al. (8) recently reported of a gene defect associated with CNF. This gene, called nephrin, encodes a transmembrane protein with similarity to immunoglobulin-like cell adhesion molecules. Although the functions of nephrin remain to be established, it does not appear to encode known mitochondrial components. However, it is interesting that a mitochondrial enzyme proline oxidase shows remarkable similarity to nephrin as determined by comparing the respective amino acid sequences. Moreover, the immediate nephrin locus in chromosome 19q13.1 contains at least two nuclearly encoded subunits of mitochondrial respiratory chain enzymes (COX VIb and COX VIIa) as well as the major protective enzyme against lipid peroxidation, the phospholipid hydroperoxide glutathione peroxidase. Whether nephrin is involved in the regulation of these mitochondrial components remains to be studied. Männikkö et al. (27) have reported an apparent mitochondrial involvement in 2 of 30 CNF patients; another 2 patients suffer from impaired hearing, a finding often observed in mitochondrial diseases (10) . Additional studies have shown involvement of mitochondria also in other human and experimental glomerular diseases, especially in the puromycin nephrosis of the rat (H. Holthöfer et al., unpublished results). Thus, our finding of altered mitochondrial functions appears to be an important pathophysiologic feature in glomerular permeability changes in general.

Cytochrome c oxidase is a 13 subunit complex of the respiratory chain crucial for cellular ATP production (6) . Three of the COX subunits are encoded by the mitochondrial genome, whereas the remaining subunits are encoded by the nucleus and are needed for the fully functional complex (28 , 29 ). Some nuclear encoded subunits are expressed in a strictly developmental stage-, tissue-, and even cell type-specific manner obviously reflecting individual cellular energy needs (30 , 31 ). It is noteworthy that the kidney shows strong COX activity in histochemical staining earlier than most other tissues at the second trimester of pregnancy (32) , coinciding with the onset of glomerular filtration. These data suggest that the glomerular filtration process may be crucially dependent on tissue- and cell type-specific mitochondrial energy production.

In addition to demonstrating identity and down-regulation of mitochondrially encoded COX I subunit in CNF kidneys, we subsequently demonstrated involvement of other mitochondrially encoded respiratory chain components, while the respective nuclearly encoded respiratory chain components remained at control levels. This suggests a defective nuclear–mitochondrial interaction rather than a primary mitochondrial defect 33-35) . The maintenance of an optimal energy balance requires finely tuned participation of tens of nuclear encoded, tissue-, cell type-, and developmental stage-specific factors, which must be correctly targeted, recognized, processed, and assembled into the mitochondria in stoichiometric amounts (36) to become fully functional.

In any tissue, both normally and abnormally functioning mitochondria (heteroplasmic) may be found simultaneously in individual cells (10) . Furthermore, tissues with a high mitotic index are able to select against heteroplasmic cells, whereas in tissues with little mitotic activity the ratio of mutant to normal mitochondria increases (37) . The threshold value (above which a mitochondrial defect leads to clinical symptoms) appears to reflect the level of dependency of energy metabolism (10 , 37 , 38 ). In our study the number of mitochondria in CNF and controls did not differ, yet steady-state levels of all the respiratory chain complexes in CNF were decreased to a level of 10–30% of controls. In the central nervous system, neuron type-specific symptoms have been observed at a similar remnant mitochondrial activity in symptomatic disease (29 , 30 ). This was explained by the fact that terminally differentiated neurons cannot compensate functional over nonfunctional mitochondria due to lack of cell (and interrelated mitochondrial) proliferation (10 , 34 , 38 ). This could also apply to glomerular podocytes, which in general do not proliferate (39) . Mandel et al. (40) showed that a decline in cellular ATP levels leads to increased epithelial permeability in MDCK cells. Moreover, Gabai and Kabakov reported (41) that such a decrease of ATP content specifically disrupts the architecture of actin and intermediate filaments, which are considered especially important for the maintenance of the complex structure of podocytes (26) . Together, these findings could explain the common morphologic changes, including flattening and retraction of podocyte foot processes, seen in proteinuria and indicate that podocytes may be ill-prepared for challenges in ATP level maintenance.

Recently, a deficiency of mitochondrial complex I has been shown to result in excessive production of superoxide radicals (42) , which in turn causes excessive formation of hydroxyl radicals and aldehydic lipid peroxidation (43) . These are intriguing findings, as glomerular lipid peroxidation has been implicated as an important factor in the generation of proteinuria (44) . Mitochondrial diseases often show changes in the kidney as a part of multiorgan involvement (10 , 45 ). Massive proteinuria and proximal tubular damage have occasionally been reported in newborns as the presenting symptoms (45 , 46 ). There is convincing evidence of excess reactive oxygen species and lipid peroxidation products in glomeruli in various experimental models, including the puromycin nephrosis of the rat 47-49) . We also found (H. Holthöfer et al, unpublished results) that in the glomerular damage induced by puromycin, mitochondrial dysfunction develops before the onset of proteinuria, suggesting a causal relationship.

Based on these considerations and on our findings of decreased renal mitochondrial function and enhanced glomerular LPO in CNF kidneys, we propose the following hypothesis: In CNF, a change in nephrin gene leads to a kidney-specific defect in the mitochondrial respiratory chain functions. This leads to excessive reactive oxygen species generation and accumulation of local LPO adducts. This, in turn, causes glomerular proteinuria. This hypothesis can now be tested in additional studies.


   ACKNOWLEDGMENTS
 
This study was supported by The University of Helsinki, The Paulo Foundation, Päivikki and Sakari Sohlberg Foundation, The Academy of Finland, and Deutsche Academische Austauschdienst. The expert comments and suggestions of Dr. Mårten Wikström and Dr. Aaro Miettinen are gratefully acknowledged.


   FOOTNOTES
 
1 Correspondence: The Haartman Institute, Department of Bacteriology and Immunology, PB 21 (Haartmaninkatu 3), FIN-00014 University of Helsinki, Finland. E-mail: Harry.Holthofer{at}Helsinki.fi

2 Abbreviations: BN, blue native; CNS, congenital nephrotic syndromes; CNF, congenital nephrotic syndrome of the Finnish type; COX, cytochrome c oxidase; DDRT-PCR, differential display reverse transcription polymerase chain reaction; FITC, fluorescein isothiocyanate; LPO, lipid peroxidation; mt, mitochondrial; PAGE, polyacrylamide gel electrophoresis; SDH, succinate dehydrogenase; SDS, sodium dodecyl sulfate.

Received for publication June 15, 1998. Revision received October 23, 1998.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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