1 Department of Bacteriology and Immunology, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland
2 Department of Medicine, Division of Nephrology, Helsinki University Central Hospital and Folkhälsan Research Centre, Biomedicum Helsinki, Helsinki, Finland
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ABSTRACT |
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Diabetes is a rapidly increasing health care problem all over the world, showing a fivefold increase in the prevalence figures during the past 15 years. In parallel, one of the main diabetic complications, nephropathy, is also increasing. Diabetic nephropathy is a devastating chronic event that is characterized by persistent proteinuria, elevated arterial blood pressure, and decline in renal function. Overt nephropathy is diagnosed when the albumin excretion rate (AER) persistently exceeds 300 mg in a 24-h urine collection (1).
The decline in renal function and glomerular filtration rate arises from damage to the glomerular filtration barrier. This is accompanied by deterioration of tubular reabsorption, increase of circulating creatinine, and morphologically by accumulation of extracellular matrix and thickening of the basement membrane within the glomeruli (2,3). Ultimately, diabetic nephropathy leads to excessive scarring and a nonfunctional end-stage kidney requiring dialysis and transplantation therapies. Microalbuminuria, which is the earliest detectable marker of nephropathy, precedes overt nephropathy and is defined as a persistent AER ranging from 30 to 300 mg/24 h. It is also a strong predictor of subsequent nephropathy in type 1 diabetes (4,5). At the time of microalbuminuria, the glomeruli already demonstrate advanced glomerulopathy (6).
The filtration barrier of a glomerulus comprises capillary endothelium; glomerular basement membrane; and specialized visceral epithelial cells, the podocytes, which are located on the outer surface of the barrier (7). Podocytes are important for the maintenance of the dynamic functional filtration barrier (8), and recent data suggest that the podocyte number may be reduced in the glomeruli of both type 1 and type 2 diabetic patients (9,10). Furthermore, longitudinal data provide evidence for an association of podocyte loss and increasing AER in type 1 diabetes (11). It is presumable that there are earlier signs of perturbed podocyte metabolism before podocyte loss. Nephrin, a transmembrane protein of the immunoglobulin superfamily, seems to be a key molecular component of the filtration slit diaphragm between neighboring podocytes (1214). Its major mutations lead to the massively proteinuric disease congenital nephrotic syndrome of the Finnish type (12,15). Pancreatic ß-cells also express nephrin (16). Various experimental models of diabetes and hypertension show alterations in nephrin mRNA or protein levels in the glomeruli (17,18). In line with this, nephrin protein expression seems to be altered in various human proteinuric kidney diseases (1921).
Given the rapidly increasing burden of diabetes and its complications, it is important to establish earlier predictive markers than microalbuminuria to allow early detection and early aggressive treatment. At the moment, no such markers are available. This study assessed the presence of immunoreactive nephrin in the urine of type 1 diabetic patients with or without nephropathy, reflecting damage of the filtration barrier.
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RESEARCH DESIGN AND METHODS |
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Sample preparation.
AER was measured using commercial radioimmunoassay (Pharmacia, Uppsala, Sweden; 2 mg/l detection limit and interassay coefficient of variance 5%) and total urinary protein concentration with colorimetric RC CD Protein Assay (Bio-Rad Laboratories, Hercules, CA) using microfuge tube assay protocol according to the manufacturers instructions. Sample volumes corresponding to 30 µg of total protein were precipitated with 10% (wt/vol) trichloroacetic acid in PBS on ice for 30 min and then centrifuged for 10 min at 13,100g at 4°C, and the pellets were washed twice with ice-cold acetone. The samples were air-dried and dissolved in Laemmli buffer (62.5 mmol/l Tris-HCl [pH 6.8], 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, and 0.05% bromophenol blue) followed by heating at 95°C for 5 min. Because the relative proportion of albumin from total urinary proteins was so much higher in the Micro and Macro groups, there was a possibility that the negative patients were false negatives. Therefore, we also analyzed a corrected urine sample from each negative patient of the Micro and Macro groups. A larger urine volume corresponding to 30 µg of other proteins than albumin (other proteins = amount of total protein - amount of albumin) was precipitated as above. Indeed, 9 of the 11 nephrinuric samples in the Macro group were found by analyzing a corrected sample, whereas no new positives were found in the Micro group after correction. Five samples from the Micro group had run out and could not be analyzed again as the corrected sample.
SDS-PAGE and Western blotting.
The proteins were run through reducing 10% polyacrylamide gels in the Protean Mini-gel electrophoresis system (Bio-Rad Laboratories) and then transferred to nitrocellulose filters (Amersham Biosciences, Buckinghamshire, U.K.). After blocking for 2 h at room temperature with 3% nonfat dried milk (Valio, Helsinki, Finland) in PBS, the filters were incubated with antibodies Aff338 (1:5) and Aff380 (1:5) in PBS containing 1% nonfat dried milk and 0.02% sodium azide for 1 h at room temperature. The filters were then washed several times in PBS containing 0.2% Tween 20, further incubated with peroxidase-conjugated affinity-purified goat anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA; 1:40,000) for 1 h at room temperature, and washed as above. The bound antibodies were detected with Super Signal enhanced chemiluminescence substrate (Pierce, Rockford, IL). The Aff338 and Aff380 rabbit polyclonal antibodies were originally raised against the major splicing variant designated as nephrin using a recombinant fusion protein
-435 as the antigen as described earlier (16). It carries parts of the intra- and extracellular regions of nephrin (amino acids 10311055 and 10961215), thus lacking the exon 24. Presence of the characteristic protein bands visible with both antibodies in Western blots was regarded as positive for nephrin.
Immunofluorescence.
Normal kidney tissue was obtained from cadaver kidneys taken for transplantation but not grafted because of vascular anatomic abnormalities (Department of Surgery, University of Helsinki, Helsinki, Finland) in accordance with the principles of the Declaration of Helsinki. Five-micrometerthick human cortex cryostat sections were air-dried and then fixed in ice-cold acetone for 5 min before washes in PBS. The sections were then incubated with Aff338 (1:1), Aff380 (1:1), rabbit anti-human glucagon antibody (Zymed, San Francisco, CA; 1:50) or no primary antibody in 1% normal goat serum in PBS overnight at 6°C. After washes in PBS, the sections were incubated with tetramethylrhodamine-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch; 1:200) for 30 min at room temperature. After washes in PBS, the slides were covered with Immu-mount mounting medium (Shandon, Pittsburgh, PA). Microscopy was performed with an Olympus BX50 microscope (Olympus Optical, Tokyo, Japan) equipped with a cooled digital camera (Hamamatsu Photonics, Hamamatsu City, Japan). Openlab 2.2.3 (Improvision, Coventry, U.K.) and Adobe Photoshop (Adobe Systems, San Jose, CA) software was used for image documentation.
Statistical analysis.
Data are expressed as mean ± SE unless otherwise stated. Differences between group means were tested with ANOVA, Mann-Whitney, Kruskall-Wallis, or Pearson 2 tests when appropriate. The correlation between AER and urinary albumintototal protein ratio was tested with linear regression analysis after loge transformation of AER (lnAER). All analyses were performed using the BMDP statistical package (BMDP Statistical Software, Los Angeles, CA). P < 0.05 was considered significant.
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RESULTS |
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DISCUSSION |
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We used affinity-purified antibodies in our analyses, revealing the typical 185-kDa protein band of nephrin in Western blot of isolated glomeruli and glomerular immunofluorescence staining typical for nephrin. Unlike our earlier results in experimental diabetic nephropathy (22), we did not find the full-length nephrin in the urine of the study patients; thus, the nephrin released from the podocytes is most likely split into typical fragments during the passage through the nephrons, especially in the tubular system, where degradation and reabsorption of proteins leaking from the glomerulus takes place (23,24). No protease inhibitors were added to the samples, so some nephrin degradation may also have occurred while the urine was collected and stored. Most of the nephrinuric urine demonstrated the characteristic banding pattern, but a few nephrinuric patients lacked some of the specific bands, which could mean that protease activities or tubular reabsorption may differ between individuals.
Our earlier results have shown that in diabetes induced experimentally by streptozotocin, nephrin was found in the urine earlier than albumin in the course of kidney damage (22). In human glomeruli, an alternatively spliced nephrin mRNA that lacks the transmembrane area exists, allowing production of a soluble form of the protein nephrin (25). The observed fragments of nephrin in the present study may represent this form. Alternatively, the fragments could derive from the full-length nephrin from detaching podocytes. Nakamura et al. (26) found podocytes in the urine of micro- and macroalbuminuric type 2 diabetic patients, but patients with chronic renal failure failed to show urinary podocytes, suggesting that urinary podocytes may represent the active phase of diabetic nephropathy. In contrast, they did not find podocytes in the urine of normoalbuminuric patients (26). If the situation is the same in type 1 diabetes, then nephrinuria at the normoalbuminuric stage could represent secretion of nephrin
rather than detached podocytes. In support of this, our preliminary data show that fragments of other podocyte-specific proteins, including podocalyxin and podocin, are found only in a few nephrinuric urine samples of our study (unpublished data). It may be possible that before being shed, podocytes undergo a period of perturbed metabolism, leading to active secretion of its distinct molecular components.
Whether various human kidney diseases show changes in nephrin expression remains controversial (19,2729). The expression is shown to be altered in effaced foot processes toward a more granular cytoplasmic localization, whereas in diseased glomeruli, the preserved foot processes show normal expression (20,30). Nephrin may be lost in urine and the granular staining could represent newly synthesized nephrin (30), or activation of podocyte foot process cytoskeleton could alter the surface distribution of nephrin (21). It is possible that primary reduction in nephrin may destabilize and disrupt the filtration slit structure and lead to podocyte loss, proteinuria, and nephrinuria.
Agents that modulate the renin-angiotensin system (RAS), such as ACE inhibitors or angiotensin-receptor antagonists, reduce proteinuria (31,32). In experimental models of diabetes, glomerular nephrin mRNA expression was reduced and the ACE inhibitors and angiotensin II antagonists were able to normalize the reduction in expression (17,33,34). In addition, ACE inhibitors and angiotensin II antagonists were able to normalize the ultrastructural changes in an experimental model of diabetes (35). In glomeruli of type 2 diabetic patients with nephropathy, Langham et al. (36) showed reduction of nephrin mRNA level, which was ameliorated by the use of an ACE inhibitor. These results propose that nephrin belongs to the as-yet-uncharacterized set of target genes for RAS modulation. In our study, we found no difference between the nephrinuric and nonnephrinuric patients in respect to use of antihypertensive agents. We expected the occurrence of nephrinuria in the Macro group to be higher compared with the other groups, but to our surprise, it was practically the same. It is interesting that 77% of the Macro patients were using RAS modifiers (ACE inhibitor or angiotensin II antagonist) and 18% other antihypertensive drugs, which may explain the results. The occurrence of nephrinuria in macroalbuminuric patients not using antihypertensive treatment, however, remains to be established.
Because nephrin is expressed by the pancreatic ß-cells (16), one may speculate that the urinary immunoreactive nephrin represents nephrin spilled out from the serum and thus possibly excreted by the pancreas. Our preliminary results from Western blots of serum samples from five nephrinuric and three nonnephrinuric study patients did not show any immunoreactive protein bands, so the urinary nephrin most likely originates from the kidneys (data not shown).
What is the biological significance of detecting cell typespecific proteins and their degradation products in urine? In addition to adding the dynamic information on podocyte metabolism, structure, and function, these proteins may be used to profile different patient groups and to optimize pharmacologic intervention. They may also give hints to the currently open questions of the pathogenesis of diabetic nephropathy. In conclusion, one-third of the normoalbuminuric type 1 diabetic patients showed immunoreactive nephrin in the urine. The occurrence of nephrinuria was closely the same in the other groups tested. Because all control subjects were nonnephrinuric, nephrinuria may be an early marker for damage of the glomeruli in type 1 diabetes.
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ACKNOWLEDGMENTS |
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The valuable comments and help of Marja Julin and Elsa Valtonen are acknowledged, as well as the technical assistance of Tarja Vesisenaho.
We acknowledge all of the physicians and nurses at each center participating in the collection of patients: Anjalankoski Health Centre: T. Uggeldahl, S. Koivula; Central Finland Central Hospital: A. Halonen, A. Koistinen, P. Koskiaho, M. Laukkanen, J. Saltevo, M. Tiihonen; Central Hospital of Kanta-Hame: P. Kinnunen, A. Orvola, T. Salonen, A. Vähänen; Central Hospital of Kymenlaakso: R. Paldanius, M. Riihelä, L. Ryysy; Central Hospital of Länsi-Pohja: P. Nyländen, A. Sademies; Central Ostrobotnian Hospital District: S. Anderson, B. Asplund, U. Byskata, T. Virkkala; City of Vantaa Health Centre (Myyrmäki): J. Laakso, A. Airas, K. Rautavaara; Helsinki University Central Hospital (Department of Medicine, Division of Nephrology): J. Fagerudd, S. Lindh, K. Pettersson-Fernholm, M. Rosengård-Bärlund, H. Rosvall, M. Rönnback, M. Saraheimo; Iisalmi Hospital: E. Toivanen; Jokilaakso Hospital, Jämsä: I. Pirttiniemi, A. Parta; Kainuu Central Hospital: S. Jokelainen, P. Kemppainen, A.-M. Mankinen, M. Sankari; Kerava Health Centre: H. Stuckey, P. Suominen; Kivelä Hospital, Helsinki: A. Aimolahti, E. Huovinen; Koskela Hospital, Helsinki: V. Ilkka, M. Lehtimäki; Kouvola Health Centre: E. Koskinen, T. Siitonen; Kuopio University Hospital: M. Laakso, L. Niskanen, I. Vauhkonen, T. Lakka, R. Ikäheimo, E. Voutilainen, P. Kekäläinen, L. Mykkänen, P. Karhapää, E. Lampainen, E. Huttunen, U. Tuovinen; Kuusankoski Hospital: E. Kilkki, L. Riihelä; Lohja Hospital: T. Salonen, M. Saari, T. Granlund; Länsi-Uusimaa Hospital, Tammisaari: J. Rinne, I.-M. Jousmaa; Mikkeli Central Hospital: A. Gynther, R. Manninen, P. Nironen, M. Salminen, T. Vänttinen; North Karelian Hospital: U-M. Henttula, A. Rissanen, H. Turtola, M. Voutilainen; Oulaskangas Hospital, Oulainen: E. Jokelainen, P.-L. Jylkkä, E. Kaarlela, J. Vuolaspuro; Päijät-Häme Central Hospital: H. Haapamäki, A. Helanterä, H. Miettinen; Pori City Hospital: K. Sävelä, P. Ahonen, P. Merensalo; Riihimäki Hospital: L. Juurinen, E. Immonen; Salo Hospital: J. Lapinleimu, M. Virtanen, P. Rautio, A. Alanko; Satakunta Central Hospital: M. Juhola, P. Kunelius, M.-L. Lahdenmäki, P. Pääkkönen, M. Rautavirta; Savonlinna Central Hospital: T. Pulli, P. Sallinen, H. Valtonen, A. Vartia; Seinäjoki Central Hospital: E. Korpi-Hyövälti, T. Latvala, E. Leijala; South Karelia Hospital District: T. Hotti, R. Härkönen, U. Nyholm, R. Vanamo; Tampere University Hospital: I. Ala-Houhala, T. Kuningas, P. Lampinen, M. Määttä, H. Oksala, T. Oksanen, K. Salonen, H. Tauriainen, S. Tulokas; Turku Health Centre: I. Hämäläinen, H. Virtamo, M. Vähätalo; Turku University Central Hospital: M. Asola, K. Breitholz, R. Eskola, K. Metsärinne, U. Pietilä, P. Saarinen, R. Tuominen, S. Äyräpää; Vasa Central Hospital: S. Bergkulla, U. Hautamäki, V.-A. Myllyniemi, I. Rusk.
Address correspondence and reprint requests to Harry Holthöfer, P.O. Box 63 FIN-00014, University of Helsinki, Finland. E-mail: harry.holthofer{at}helsinki.fi
Received for publication February 28, 2003 and accepted in revised form September 5, 2003
AER, albumin excretion rate; lnAER, loge transformation of AER; RAS, renin-angiotensin system
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REFERENCES |
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