Selective Impairment of Gene Expression and Assembly of Nephrin in Human Diabetic Nephropathy

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Kidney International, Vol. 65 (2004), pp. 2193–2200

Selective impairment of gene expression and assembly of nephrin in human diabetic nephropathy

ARIELA BENIGNI,ELENA GAGLIARDINI,SUSANNA TOMASONI,MAURO ABBATE,PIERO RUGGENENTI, RAGHU KALLURI, and GIUSEPPE REMUZZI

Mario Negri Institute for Pharmacological Research, Negri Bergamo Laboratories, Bergamo, Italy; Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts; and Division of Nephrology and Dialysis, Azienda Ospedaliera, Ospedali Riuniti, Bergamo, Italy

Selective impairment of gene expression and assembly of The nephropathy associated with diabetes is the lead- nephrin in human diabetic nephropathy. ing cause of end-stage renal disease (ESRD) worldwide Background. Recent disclosure of podocyte proteins has un- [1, 2]. In North America, 40% of patients adhering to raveled previously rather mysterious mechanisms that govern glomerular perm-selectivity in health and disease. Here we ad- dialysis transplantation programs in 1998 were diabetic. dressed the role of nephrin, CD2-associated protein (CD2AP), Related treatment costs were estimated at $51,000 per and podocin together with the integrity of the slit diaphragm year (i.e., about $12,000 more than the costs for nondia- in the pathogenesis of proteinuria of patients with diabetes and betics). nephropathy. Diabetic nephropathy, defined as urinary protein ex- Methods. Nephrin mRNA and protein expression were eval- > uated in parallel in adult diabetic patients by in situ hybridiza- cretion rate 0.5 g/24 hours (clinical proteinuria), man- tion and immunohistochemistry. For comparison, nondiabetic ifests within 15 to 20 years after the onset of disease in patients with minimal change nephrosis and normal control 20% to 40% of patients. Ultrafiltered proteins concur via patients were evaluated. CD2AP and podocin expression by intrinsic renal toxicity [3] to accelerate multifactorial pro- immunohistochemistry was also assessed. The filtration slit cesses responsible for progressive loss of glomerular fil- was analyzed by morphometry and transmission electron microscopy. tration rate (GFR), on average by 10 to 12 mL per minute Results. Extracellular nephrin mRNA and protein were per year in untreated diabetics [3]. Restoring the perms- markedly reduced in diabetic patients. No changes were found elective function of the kidney barrier to proteins limits in patients with minimal change versus controls. CD2AP and GFR decline that may even stabilize if proteinuria is re- podocin were comparable in all subjects. Ultrastructural analy- duced below 0.5 g/24 hours [4, 5]. While now 0.5 g/24 sis showed in diabetic patients a remarkable reduction in the percentage of electron dense slit diaphragms, despite a fre- hours or less is an acknowledged target of renoprotective quency of the filtration slits comparable to control patients. intervention in other conditions, such a goal is rather diffi- Conclusion. Down-regulation of nephrin and loss of the cult to achieve in diabetes [6]. The main reason lies in the electron dense structure of slit diaphragm indicate a novel relatively poor knowledge of mechanisms sustaining the mechanism accounting for proteinuria in diabetic nephropathy. glomerular defect. Major highlights have been offered by To the extent that glomerular protein trafficking contributes to renal disease progression, our findings may have clinical recent discoveries on podocytes and slit diaphragm com- relevance. Reduction of nephrin in the context of normal ex- ponent structures, namely nephrin and other functionally pression of CD2AP and podocin can be taken reasonably as a relevant molecules of the barrier. specific marker of renal disease in diabetes. Therapies targeted Nephrin is a transmembrane protein of the im- at correcting podocyte nephrin might be of value for diabetic munoglobulin superfamily, predominantly expressed by medicine. podocytes [7]. Another protein, CD2AP, acts as an adapter molecule to bind nephrin carboxy terminal domain, which serves to link the slit diaphragm to the podocyte cytoskeleton [8]. Mutations of the nephrin gene in severe congenital nephrotic syndrome of Finnish Key words: diabetes, nephrin, CD2AP, podocin, slit diaphragm, minimal change disease. type [9, 10] resulted in massive proteinuria in utero and nephrotic syndrome at birth. Animal studies found that Received for publication May 16, 2003 decreased nephrin expression was consistently associ- and in revised form September 15, 2003, and November 6, 2003 Accepted for publication January 9, 2004 ated with altered glomerular permeability to proteins [11, 12]. In severe passive Heymann nephritis, less glomeru- C 2004 by the International Society of Nephrology lar expression of nephrin paralleled the development of

2193 2194 Benigni et al: Nephrin in diabetic nephropathy

Table 1. Clinical parameters at the time of biopsy Age Gender U-Prot S-Creat SBP DBP Diagnosis years M/F g/day mg/dL mm Hg mm Hg Minimal change nephrosis 53 ± 10 4/2 5.2 ± 1.4 1.8 ± 0.5 118 ± 677± 4 Diabetic nephropathy 60 ± 4 10/5 4.1 ± 1 1.6 ± 0.2 143 ± 781± 3

Abbreviations are: U-Prot, urinary protein excretion; S-Creat, serum creatinine concentration; SBP, systolic blood pressure; DBP, diastolic blood pressure.

proteinuria and injury [11]. Others found similar results confounding conditions; HbA1c <8%; and informed con- in experimental diabetes and renal mass ablation [13, sent (including that of the parents for the 16-year-old 14]. Studies on nephrin expression in acquired human patient). nephroses led to controversial results due to the lack Speciﬁc inclusion criteria for cases were: proteinuria of simultaneous assessment of nephrin mRNA and pro- >0.5 g/24 hours for at least six months; and histo- tein expression [15–18]. Two very recent studies, sepa- logic diagnosis of diabetic glomerulopathy without other rately exploring the expression of nephrin mRNA or the concomitant glomerular disease. Inclusion criteria for relative protein product, found a reduction in diabetic proteinuric control patients were: proteinuria >0.5 g/ patients [19, 20]. Here, we evaluated nephrin mRNA 24 hours for at least six months; and histologic diagno- and protein expression in a more systematic way, to- sis of minimal change disease without other concomitant gether with other podocyte proteins, including CD2AP glomerular disease. Inclusion criteria for nonproteinuric and podocin, in patients with diabetic nephropathy in control patients were: proteinuria <0.5 g/24 hours; no comparison with nondiabetic proteinuric patients with clinical or histologic evidence of glomerular disease; and minimal change and normal control patients. We further nephrectomy for kidney adenocarcinoma. investigated the ﬁltration slit architecture by transmis- Table 1 summarizes clinical features. Twenty-four– sion electron microscopy, with special attention of the hour urinary proteins and serum creatinine were electron dense structure of slit diaphragms, along with measured using an autoanalyzer (CX5; Beckman Instru- the morphometric assessment of the slit pore frequency ments, Inc., Fullerton, CA, USA). and width.

Reagents METHODS Goat polyclonal antibody raised against a peptide map- Patients ping near the amino terminus of nephrin of human ori- gin and rabbit polyclonal antibody to a recombinant pro- This observational study considered all consecutive pa- tein corresponding to amino acids 350–639 mapping at tients admitted for diagnostic biopsy to the Renal Unit of the carboxy terminus of human CD2AP was from Santa the “Ospedali Riuniti” of Bergamo since the study start Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit (January 2001) until November 2002, at closure of the polyclonal anti-podocin antibody were obtained as pre- database. viously described [21]. Rabbit polyclonal antibody di- The predefined inclusion criteria for potentially el- rected against the intracellular domain of mouse nephrin igible subjects were: age ≥16 years; admission to the [22] was kindly provided by Dr. L.B. Holzman. A 346-bp Renal Unit in the same period; kidney biopsy for fragment of mouse nephrin cDNA (positions 1155–1501) clinical reasons; no previous steroid or immunosuppres- coding for a portion of the extracellular domain was con- sive therapy for at least six months; no previous treat- structed by polymerase chain reaction (PCR) amplifica- ment with (or withdrawal since at least two months tion of the 2400-bp fragment of mouse nephrin cDNA [11] of) angiotensin-converting enzyme (ACE) inhibitors, an- using specific primers. The PCR product was gel-purified, giotensin II receptor antagonists, statins, nonsteroidal digested, and subcloned into pBluescript SK (Stratagene, anti-inflammatory drugs, and other treatments that could Florence, Italy). potentially affect urinary protein excretion, nephrin expression, and any parameter that, in the Investigators’ judgment could confound the findings; serum creatinine <4 mg/dL (in order to exclude aspecific changes that In situ hybridization might reflect progressive structural damage by mech- Mouse nephrin antisense and sense riboprobes were la- anisms sustained by intracapillary hypertension and beled by in vitro transcription using digoxigenin-labeled protein traffic); no evidence of renovascular disease, ob- uridine triphosphate (Roche, Milan, Italy) according to structive uropathy, systemic disease, and other possibly the manufacturer’s protocol. The sequence of nephrin Benigni et al: Nephrin in diabetic nephropathy 2195 riboprobe showed 82% homology to the human se- glomerular basement membrane (GBM) length. GBM quence. Hybridization was performed as previously de- length and slit diaphragm width (SDW) were calculated scribed [11] on 3 lm-thick sections of Dubosq-Brazil using computer-assisted morphometric unit. GBM pro- fixed, paraffin-embedded renal tissue. Sets of biopsies file and SDW were manually outlined using a line auto- comprising samples from each patient group were pro- matically measured in screen pixels. Exact enlargement cessed simultaneously.Glomeruli (N = 7) for each sample was calculated using digitized images of a calibration grid were analyzed. Negative control included hybridization (Ernest F. Fullam, Inc., Latham, NY, USA). SDW was with sense probe. Evaluation was done without knowl- calculated, as previously described [25], as the harmonic edge of the study groups. mean (in nanometers) of the distance between cell membrane of two adjacent foot processes. The percentage of Immunohistochemical analysis measured slit diaphragms with a filamentous structure was also determined. Nephrin, CD2AP, and podocin expression was de- tected by immunoperoxidase analysis. Dubosq-Brazil fixed, paraffin-embedded kidney sections (3-lm) were Statistical analysis deparaffinized, rehydrated, and incubated for 30 min- Results are reported as mean ± SE. Differences be- utes with 0.3% H2O2 in methanol to quench endogenous tween groups were analyzed by Kruskal-Wallis test. peroxidase. Tissues were treated with proteinase-K (20 Linear regression analysis was performed between im- lg/mL, Sigma-Aldrich, Milan, Italy) for 10 minutes at munohistochemistry scores for nephrin and degree of 37◦C, followed by microwave (twice for 5 minutes in cit- proteinuria. A P value <0.05 was considered significant. rate buffer 10 mmol/L, pH 6 at operating frequency of 2450 MHz and 600-W power output) and citrate buffer (15 minutes) incubations to increase the reactivity of anti- RESULTS body to antigen. Antibodies against extra- and intracellu- Patients lar nephrin domains, CD2AP and podocin, were diluted The study included 15 cases with diabetes (type 1: N = 1:100, 1:300, 1:100, and 1:1000, respectively. Samples from 1; type 2: N = 14) and biopsy-proven diabetic glomeru- each group were processed simultaneously.Negative con- lopathy, six proteinuric controls with biopsy-proven trols were obtained by omitting the primary antibody on minimal change disease, and five nonproteinuric con- adjacent sections. Seven glomeruli on average in each trols undergoing nephrectomy for kidney adenocarci- sample were analyzed by an investigator who was un- noma without histologic evidence of glomerular disease. aware of biopsy groups. Signal intensity was graded on Hypertension was controlled by drugs such as diuretics, a scale of 0 to 3 (0, no staining; 1, weak; 2, moderate; 3, beta-blockers, and vasodilators that did not directly inter- strong intensity). fere with glomerular barrier function, proteinuria, and, conceivably, nephrin expression. Metabolic control of di- < Electron microscopy abetes was reasonably good (HbA1c 8%). Proteinuria was comparable in cases and proteinuric control patients Biopsy specimens were fixed in 2.5% glutaralde- (Table 1). hyde in 0.1 mol/L cacodylate buffer, pH 7.4, and postfixed with 1% tannic acid, 1% glutaraldehyde in 0.1 mol/L phosphate buffer, pH 7.4 [23]. Tannic acid In situ hybridization for nephrin mRNA contributes to increase the contrast and definition of Nephrin expression, typically localized at the periph- the isoporous substructure of the slit diaphragm [23]. eral capillary loops of the tuft, was absent or markedly Kidney fragments were then processed, and ultrastruc- reduced in glomeruli from diabetic patients (Fig. 1A) tural evaluations were performed by transmission elec- in respect to control patients (Fig. 1C and E). By con- tron microscopy (Morgagni 268D; Philips, Brno, Czech trast, nephrin mRNA staining was comparable in patients Republic) [24]. with minimal change nephrosis (Fig. 1B) and control patients. In all groups, a mild staining was only occasion- Morphometric analysis ally observed in parietal epithelial cells and tubuli, as we observed in severe experimental nephrosis [11]. No back- The filtration slit was analyzed by morphometry and ground signal was observed with nephrin sense riboprobe transmission electron microscopy in four patients per (Fig. 1D). group. An average of 45 (35–71) images per patient was digitized (final magnification × 56,000) and processed with the image-processing software (NIH Image version Immunoperoxidase staining for nephrin 1.6). A total number of 1141 slits was analyzed. Slit Results of the immunolocalization of the extracellular frequency was evaluated as number of slits per lmof portion of nephrin were consistent with those found for 2196 Benigni et al: Nephrin in diabetic nephropathy

Fig. 2. Representative photomicrographs of nephrin extracellular domain by the immunoperoxidase method in patients with diabetes (A) or minimal change nephrosis (B), and in control kidney (C). No spe- ciﬁc signal was obtained omitting the primary antibody (D). Original magniﬁcation, ×250.

Fig. 1. Representative pictures of nephrin mRNA expression by in situ hybridization in patients with diabetes (A) or minimal change nephrosis (B), and in control kidney (C and E). No speciﬁc signal was obtained with the sense nephrin probe (control kidney) (D). Arrows indicate podocyte nephrin expression. Original magniﬁcation, ×250 (A-D), and ×1000 (E).

nephrin mRNA. The staining was significantly reduced or even absent in some glomeruli from patients with diabetes (score: 0.7 ± 0.1, P < 0.05) (Fig. 2A) compared to control patients (3 ± 0) (Fig. 2C). In patients with minimal change nephrosis the score was fairly comparable to control patients (2.7 ± 0.2) (Fig. 2B). The staining intensity was variable along capillary walls. Staining was absent in control sections upon omission of primary antibody (Fig. 2D). A different trend was observed using a specific anti- Fig. 3. Representative photomicrographs of nephrin intracellular do- body for nephrin intracellular domain, which revealed main by the immunoperoxidase method in patients with diabetes (A) comparable capillary wall staining in patients with di- or minimal change nephrosis (B), and in control kidney (C). No specific signal was obtained omitting the primary antibody (D). Original abetes, minimal change nephrosis, and control patients magnification, ×250. (Fig. 3). The pattern was epithelial with linear distribution along the peripheral loops. Abnormal staining was found in extensively sclerotic areas, which did not stain. No signal was observed in negative control experiments (Fig. 4A and E), minimal change nephrosis (Fig. 4B and (Fig. 3D). F), and control patients (Fig. 4C and G), all maintain- ing strong positivity along capillary loops. Sclerotic areas were negative. No signal was observed omitting the pri- Immunoperoxidase for CD2AP and podocin mary antibodies (Fig. 4D and H). The appreciable differ- A prominent glomerular localization with a linear dot- ences between the extracellular portion of nephrin and ted line of reactivity, giving a preferentially epithelial- CD2AP or podocin staining in diabetic patients indicate like staining, was observed for both CD2AP and podocin. that changes in nephrin protein were selective, rather No differences were seen between patients with diabetes than part of a generalized phenomenon. Benigni et al: Nephrin in diabetic nephropathy 2197

Fig. 4. Immunoperoxidase staining for CD2AP and podocin in patients with diabetes (A and E) or minimal change nephrosis (B and F), and in control kidney (C and G). No signal was obtained omitting the primary antibody (D and H). Original magniﬁcation, ×250. 2198 Benigni et al: Nephrin in diabetic nephropathy

DISCUSSION Diabetics with nephropathy enrolled in the present study had remarkably less renal expression of extracellular nephrin than control patients. Changes in nephrin gene and protein were associated with podocyte ultrastructural abnormalities (i.e, reduced percentage of electron dense diaphragms taken as an index of intact filtration machinery), and loss of filamentous images within slit diaphragms—reported here for the first time in human diabetes. Patients with minimal change nephrosis had no abnormalities in nephrin mRNA and protein expression or loss of electron dense diaphragms within the intimate structure of filtration slits, despite extensive effacement. Differences between diabetic patients and control patients were clear-cut, achieving statistical significance regardless of small sample size. Low expression of nephrin has been recently observed in diabetic patients. However, lack of comparison with proteinuric nondiabetics, and of assessment of other slit diaphragm molecules prevented conclusions to be drawn on the specificity and significance of these observations [19, 20]. Findings of reduced nephrin mRNA and protein expression in human diabetes in the present study are consistent with reported abnormalities in diabetic rats shown in the long term [28, 29]. Collectively, our data would indicate that the slit diaphragm change resulting from selective reduction in extracellular nephrin may contribute to the glomerular permeability defect of diabetes. It is conceiv- Fig. 5. Representative transmission electron micrographs of the ultrastructure capillary wall in patients with diabetes (A) and in control able that the nephrin loss would eventually lead to per- kidneys (B). (C) An enlarged detail of (B) representing the distance turbed function of interacting slit diaphragm molecules, between two adjacent foot processes used to quantify the slit diaphragm resulting in further increases in proteinuria. Alterna- width is shown. Arrows indicate electron dense diaphragms in the filtration slits. Original magnification, ×36,000. tively, decreased nephrin could be a consequence of increased protein ultrafiltration, which, however, appears difficult to reconcile with preserved nephrin in nondia- Electron microscopy studies betics at comparable levels of proteinuria. Of additional We investigated the filtration slit architecture by trans- interest here is the finding of unaltered nephrin levels in mission electron microscopy. In diabetes, the frequency minimal change disease, which could be interpreted to of filtration slits did not change in comparison with con- suggest that the preservation of nephrin may specifically trol patients (1.9 ± 0.15 vs. 1.97 ± 0.08 slits/lm GBM). play a role in preventing this disease from progressing, In patients with minimal change disease, the frequency of in contrast to other forms of glomerular disease, possibly filtration slits was significantly reduced to 1.09 ± 0.06 slits/ by maintenance of nephrin-dependent signaling in the lm GBM in association with the effacement of foot pro- podocyte. cesses (P < 0.05). Tannic acid fixation increased the Discovery in the last few years of slit diaphragm pro- definition of the electron dense slit diaphragms. The per- teins uniquely expressed by podocytes has contributed centage of slit diaphragms displaying filamentous image enormously to the current understanding of the tridimen- was significantly reduced in diabetic patients (24% ± 6%, sional structure of the filtration barrier. In such context, P < 0.05) in respect to minimal change and control pa- animal models of targeted disruption of single protein or tients, averaging 50% ± 5% and 63% ± 6%, respectively. functional inactivation of a specific domain were instru- The latter percentages compare reasonably well in few mental. Thus, mice homozygous for inactivated nephrin other available studies [9, 26, 27]. Slit pore width was com- gene develop massive proteinuria and die at birth. Null parable in diabetes (43.95 ± 1.2 nm) or minimal change mice exhibited foot process effacement and no slit di- (42.2 ± 2.1 nm) and control patients (38.56 ± 2.5 nm). aphragms [30]. Alterations in the slit diaphragm organi- Figure 5 shows representative pictures of the capillary zation can be also induced by infusion of a monoclonal wall ultrastructure. antibody, recently found to recognize the extracellular Benigni et al: Nephrin in diabetic nephropathy 2199 domain of nephrin [31] and to cause massive proteinuria were comparable to control. Alterations of the filtration [32]. slit architecture can be a mechanism of proteinuria in di- Why should nephrin extracellular domain expression abetes. Nephrin may represent the therapeutic target of be impaired in diabetes? The reduction at protein level treatments aimed at preventing proteinuria, in the hope underlying the slit diaphragm abnormality reflects re- of extinguishing renal disease and possibly limiting car- duced mRNA, here assessed using a specific probe for diovascular complications of diabetes. a portion of the extracellular domain. The fact that intracellular nephrin is normally expressed would suggest ACKNOWLEDGMENTS a mechanism of alternative splicing of nephrin gene in Reproduction of Figures 1–4 in color was made possible by the gener- diabetes. Nephrin-specific mRNA is spliced in the hu- ous sponsorship of AstraZeneca S.p.a., Basiglio, Milano. Prof. Antonino man kidney glomeruli with a variant called nephrin a 1 Lembo and Marlisa Capitanio are greatly acknowledged for invaluable collaboration in obtaining kidney specimens. The Authors are deeply lacking exon 24 coding for the transmembrane domain endebted to Dr. Lawrence Holzman for kindly providing antibodies. [33]. This alternative splicing cannot explain our present We thank Dr. Andrea Remuzzi for help in ultrastructural morphome- findings. It is possible that in patients with diabetes addi- try. Dr. Gagliardini is the recipient of a fellowship from the “Aiuto per la ricerca sulle malattie rare” (ARMR), Bergamo, Italy, in memory of tional splicing sites might exist, leading to the formation Dr. Luigi and Tilde Bianchi. The study received support by a grant from of nephrin molecules lacking the extracellular domain. the Associazione Trenta Ore Per La Vita (Rome, Italy). Theoretically, an additional explanation for our findings Reprint requests to Elena Gagliardini, Biol.Sci.D., Mario Negri In- could be the presence of an additional initiation site in the stitute for Pharmacological Research, Negri Bergamo Laboratories, Via promoter of nephrin gene in diabetes. That this could oc- Gavazzeni 11, 24125 Bergamo, Italy. cur is supported by studies that found the existence of dif- E-mail: [email protected] ferent regulatory elements in the nephrin gene promoter responsible for the presence of different isoforms [34]. REFERENCES Moreover, a recent observation in cultured glomerular 1. 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