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

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Selective Impairment of Gene Expression and Assembly of Nephrin in Human Diabetic Nephropathy View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector 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 mi- croscopy. 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 do- main, 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, mini- mal 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 ex- pression, 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
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