ADAM10 is expressed in human podocytes and found in urinary vesicles of patients with glomerular kidney diseases

Background The importance of the Notch signaling in the development of glomerular diseases has been recently described. Therefore we analyzed in podocytes the expression and activity of ADAM10, one important component of the Notch signaling complex. Methods By Western blot, immunofluorescence and immunohistochemistry analysis we characterized the expression of ADAM10 in human podocytes, human urine and human renal tissue. Results We present evidence, that differentiated human podocytes possessed increased amounts of mature ADAM10 and released elevated levels of L1 adhesion molecule, one well known substrate of ADAM10. By using specific siRNA and metalloproteinase inhibitors we demonstrate that ADAM10 is involved in the cleavage of L1 in human podocytes. Injury of podocytes enhanced the ADAM10 mediated cleavage of L1. In addition, we detected ADAM10 in urinary podocytes from patients with kidney diseases and in tissue sections of normal human kidney. Finally, we found elevated levels of ADAM10 in urinary vesicles of patients with glomerular kidney diseases. Conclusions The activity of ADAM10 in human podocytes may play an important role in the development of glomerular kidney diseases.


Background
The important role of podocytes in the development of many glomerular diseases are documented in renal disorders like minimal change disease, focal segmental glomerulosclerosis and membranous nephropathy [1]. Adhesion molecules like the integrin α 3 β 1 and dystroglycan are the major receptors studied today, which connect the podocytes to the glomerular basement membrane (GBM) [2]. During development L1 adhesion molecule is known to be regulated in the renal epithelium and is involved in kidney branching morphogenesis [3]. L1 adhesion molecule exists in a transmembrane form, but can also be processed into a soluble form about 200 kDa by a disintegrin and metalloproteinase (ADAM10) [4,5]. Furthermore, L1 adhesion molecule can be cleaved in vitro in the third fibronectin III domain by trypsin [6], plasmin [7] or the proprotein convertase PC5A [8], resulting in a 140 kDa and 80 kDa fragment. Interestingly, different patterns of proteolytic cleavage of L1 during nephrogenesis have been observed, but the significance of this cleavage remains unclear [3]. In addition, a 200 kDa soluble form of L1 adhesion molecule was found in patients with acute tubular necrosis and may represent a marker of distal nephron injury [9]. In the developing rat kidney ADAM10 was highly expressed in the late ureteric bud [10]. Recently we have characterized in detail the tubular and glomerular ADAM10 expression in the human kidney [11,12]. Interestingly, we found in renal allograft biopsies with histopathological diagnosis of acute interstitial rejection increased tubular ADAM10 expression, which was accompanied by high numbers of infiltrating Tcells [12]. It is known, that ADAM10 is involved in the cleavage of growth factors, adhesion molecules and cell surface receptors like Notch and their ligands Delta and Jagged [13]. In this context, two recent publications have highlighted the importance of the Notch signaling pathway in podocytes for the development of glomerular diseases. Waters et al reported, that ectotopic Notch activation in developing podocytes leads to glomerulosclerosis [14]. In addition, increased expression of the intracellular domain of Notch-1 was found in podocytes of patients with diabetic nephropathy and focal segmental glomerulosclerosis [15].
To characterize the expression of ADAM10 and its substrates L1 adhesion molecule in more detail, we analyzed their expression in a human podocyte cell line and in human renal tissue. We demonstrate that ADAM10 and L1 are expressed in human podocytes. In differentiated podocytes we detected increased amounts of mature ADAM10 and high levels of soluble L1. In addition, injuring podocytes with puromycin induced ADAM10 mediated cleavage of L1. Furthermore podocytes isolated from urines of patients with glomerular kidney diseases expressed constitutively ADAM10. Isolating urinary vesicles from healthy donors and patients with inflammatory kidney diseases, revealed increased amounts of ADAM10 expression in patients with glomerular kidney diseases.

Chemicals
Interferon-γ (IFN-γ) was purchased from Peprotech (Frankfurt, Germany), hyperfilms and the enhanced chemiluminescence (ECL) reagents were ordered from Amersham Pharmacia Biotech Europe GMBH (Freiburg, Germany), all cell culture nutrients were from Invitrogen/Life Technologies (Karlsruhe, Germany). The ADAM10 specific inhibitor GI254023X was assayed for inhibition of recombinant human ADAM17 and ADAM10 ectodomains as described before [16].

Cell Culture
Human condititionally immortalized podocytes (HPC) were isolated and cultivated as previously described [17]. Prior to stimulation, cells were incubated for 16 h in RPMI 1640 medium, supplemented with 0.1 mg/ml of fatty acid-free bovine serum albumine.

Experimental subjects
We examined the urines of a group of 7 individuals composed of 5 patients with glomerular diseases (diagnosis of patients are depicted in Table 1) and 2 healthy subjects.

Isolation of cells from human urines
Freshly voided urine of healthy donors and patients with glomerular kidney diseases were centrifuged at room temperature at 700 g for 10 min. The supernatant was removed by careful aspiration, the cell pellet was resuspended in 10 ml podocyte medium. The cell suspension was placed into culture flasks and incubated at 37°C in 5% CO 2 .

Antibodies
Mouse mAb (L1-11A) to the ectodomain of human L1 adhesion molecule and polyclonal L1 were provided from Prof. Dr. Altevogt (Heidelberg, Germany). Monoclonal antibody to the extracellular part of ADAM10 was from R&D Systems (Wiesbaden-Nordenstadt, Germany). Polyclonal anti-ADAM10 antibody from eBioscience (San Diego, USA) was used for Western blot and immunofluorescence staining. Polyclonal antibodies against nephrin and podocin were kindly provided from Dr. Shuyu Ren (Bern, Switzerland). Monoclonal antibodies for β1 and α3 integrin subunits were from Chemicon (Hampshire, United Kingdom, England). WT1 antibody for immunofluorescence analysis was purchased from Santa Cruz (Heidelberg, Germany).

Preparation of supernatants for the detection of soluble molecules
These assays were described previously [4,18]. Briefly, cell monolayers in serum-free medium were exposed to 5 μg or 10 μg puromycin to induce shedding. The ADAM10 specific metalloproteinase inhibitor GI254023X was added 15 min before treatment. Cell-free supernatants were TCA precipitated, protein samples were boiled with non-reducing sodium dodecyl sulfate (SDS) sample buffer and investigated by western blot analysis.

Cytofluorography
The cells were stained with saturating amounts of mAbs, either hybridoma supernatants or purified antibodies, and phycoerythrin (PE)-conjugated goat antibodies to mouse immunoglobulins. For intracellular FACS staining, cells were fixed with 1% paraformaldehyd for 15 min at RT. Cells were washed in PBS and permeabilised with 1% Triton X-100/PBS. Primary antibodies were diluted in 1%Triton X-100/PBS and added for 30 min at 4°C to the cells. After washing the cells twice with 1%Triton-X-100/PBS, fluorescence coupled secondary antibodies were added for 20 min at 4°C in the dark. After extensive washing with 1%TX-100/PBS, stained cells were analyzed by a FACScan cell analyzer (Becton & Dickinson, Heidelberg, Germany) using Cellquest software (Becton & Dickinson, Heidelberg, Germany).

Fluorescence microscopy (cells)
Cells were grown on coverslips and fixed with 4% paraformaldehyde/PBS or with methanol and fluorescence staining was carried out as previously described [19].

Transfection of siRNA
Twenty-four hours before transfection 5 × 10 4 cells were seeded in 6-well plates. Transfection of siRNA was carried out using Oligofectamine (InVitrogen, Karlsruhe, Germany) and 10 nM siRNA duplexes (MWG Biotech AG, Ebersberg, Germany) per well. All cells were assayed 48 h after the transfection.

Isolation of the human glomeruli
The glomeruli were isolated from the human kidney tissue according to the method of Striker and Striker [20] with minor modifications. The cortical tissue was first gently minced with a razor blade and then pushed through a steel sieve of 250-μm pore size by using a spatula. The passthrough was then filtered through a 150-μm pore size sieve and, finally, the glomeruli were collected by rinsing with PBS/1%FCS from the surface of a third sieve of 100-μm pore size. The preparation was examined under a light microscope for purity; regularly nearly 100% pure glomeruli were obtained.

Isolation of urinary vesicles
15 ml of freshly voided urine of healthy volunteers and patients with glomerular kidney diseases were used to isolate urinary vesicles with serial centrifugation steps as described previously [19].

Surface expression of ADAM10 and L1 is reduced during differentiation of podocytes
We analyzed the protein expression of ADAM10 and L1 adhesion molecule with FACS-analysis in undifferentiated and 9 days differentiated human podocytes. Interestingly, undifferentiated podocytes showed strong ADAM10 and L1 surface expression ( Fig. 1A and 1B, green line). In contrast, in differentiated podocytes the surface expression of ADAM10 and L1 was significantly reduced ( Fig. 1A and 1B, red line). In addition, we detected increasing amounts of mature ADAM10 in lysates of differentiated podocytes (Fig. 1C), which correlated with higher amounts of soluble L1 (Fig. 1D) and L1-32 (Fig. 1E), the cellular counterpart of soluble L1.

ADAM10 is involved in the cleavage of L1 adhesion molecule
Podocyte injury occur in many glomerular diseases [21]. To injure podocytes we treated the cells with different concentrations of puromycin. Interestingly, increasing amounts of puromycin induced L1-32 in podocytes ( Fig. 2A), which was accompanied by an increased amount of soluble L1 (Fig. 2B). In addition with a specific metalloproteinase inhibitor GI254023X (Fig. 2C) and ADAM10 specific siRNA (Fig. 2D) we could significantly reduce the release of L1 adhesion molecule. Interestingly, the puromycin induced cleavage of L1 was only partially inhibited by ADAM10 siRNA, whereas the constitutive release of L1 was almost completely blocked. The efficient knockdown of ADAM10 is represented in Fig. 2D.

Urinary cells from nephrotic kidney patients express ADAM10, L1, alpha3 and nephrin
Viable podocytes are detectable in the urine of patients with glomerular kidney diseases [22]. Therefore we isolated urinary podocytes from patients with glomerular diseases. As demonstrated by FACS analysis (Fig. 3A) cells isolated from the urine of a patient expressed significant amounts of ADAM10 at the cell surface. Interestingly, urinary podocytes expressed mainly the mature form of ADAM10 and low levels of full-length L1 (Fig. 3B). By RT-PCR (Fig. 3C lower panel), Westernblot (Fig. 3C upper panel) and immunofluorecense (Fig. 3D) of podocyte specific marker proteins (integrin α 3 β 1 or podocin) we confirmed that cells isolated from the urine are podocytes. In addition, by intracellular FACS staining using ADAM10 and WT1 as a specific marker for podocytes we confirmed that podocytes express ADAM10 (Fig. 3E). To determine if L1 is expressed in urinary and glomerular podocytes we performed immunofluorescence and westernblot analysis. As shown in Fig. 3F urinary podocytes only expressed low levels of L1, but L1 expression was induced after the treatment of the cells with proinflammatory cytokine IFN-γ (Fig. 3F). In addition, L1 expression was also detectable in lysates of glomeruli of normal human kidney (Fig. 3G).

Podocytes in human renal tissue express ADAM10
In glomeruli of human renal tissue we detected ADAM10 expression by immunohistochemistry ADAM10 expression (data not shown). To confirm, that podocytes are expressing ADAM10, double immunofluorescense analysis with a podocyte specific marker (WT1) was performed. ADAM10 expression was detectable in WT1 expressing podocytes (Fig. 4A). In addition, we isolated glomeruli out of the human kidney and investigated glomerular lysats by western blot. ADAM10 protein expression was detectable in glomeruli lysats (Fig. 4A left lane).

ADAM10 is found in the urine and urinary vesicles of patients with glomerular kidney diseases
Exosomes in the urine are known to be a rich source for potential biomarkers [23]. Therefore we analyzed urine and urinary vesicles isolated from healthy volunteers and patients with glomerular diseases for the expression of ADAM10 and L1 adhesion molecule. We detected elevated levels of ADAM10 in urine and in urinary vesicles of patients with glomerular diseases compared to healthy volunteers (Fig. 4B). To investigate if increased amounts of ADAM10 is due to elevated levels of urinary vesicles we probed the membranes with CD9 an exosome specific marker. As shown in Fig. 4B patients with high amounts of vesicular ADAM10, demonstrated lower levels of CD9. Furthermore, we detected only in exosomes of untreated and ionomycin (induces the release of exosomes) treated human podocytes the mature form of ADAM10, whereas in the supernatants of the cells the immature form of ADAM10 could be seen (Fig. 4B). Notably, no differences in L1 expression was observed in urine and urinary vesicles of patients compared to healthy controls (data not shown).

Discussion
In this work we demonstrated the expression of ADAM10 and L1 adhesion molecule in human podocytes. The importance of ADAM10 and L1 adhesion molecule in develop-mental processes are manifested in knockout models. ADAM10 knockout mice die before embryonic day 10 as a result of major defects in epithelial tissues [24]. L1 knockout mice show severe malformation of the nervous system, underlyning the importance of this molecule in the developing nervous system [25].
In the kidney it has been suggested, that L1 acts as a guidance molecule in the development of distal tubules and collecting ducts [3]. L1 knock out mice develop diverse renal malformations in addition to neurological abnormalities [26]. In contrast to previous published data [27] we detected L1 expression not only in tubular cells but also in immortalized human podocyte cell line and in primary podocytes isolated from urine of patients with glomerular disease. In the urine of patients with acute tubular necrosis (ATN) high levels of soluble L1 was detectable and the authors strongly suggest that urinary L1 could be a potential biomarker of distal injury during acute kidney injury (AKI) [9]. Beside urine and serum of patients, exosomes of body fluids may provide an avenue for the discovery of biomarkers useful for the early detection of kidney diseases and for the monitoring of treatment. We did not find significant differences in the amount of L1 in urine and urinary vesicles of healthy volunteers and patients with glomerular kidney diseases (data not shown). In contrast elevated levels of ADAM10 were detectable in urine and urinary vesicles of patients with glomerular kidney diseases. Although we have analyzed only few urine samples, this finding should be further investigated with higher numbers of urine samples from different renal diseases. Interestingly, in the urine of bladder cancer high levels of ADAM12 were detectable, suggesting ADAM12 as a promising biomarker for bladder cancer [28]. Another important substrate of ADAM10 is the Notch receptor which has also a crucial role in podocyte development. Interestingly, we found increased amounts of mature ADAM10 during differentiation of podocytes, suggesting ADAM10 as a differentiation marker for podocyte development. Importantly, a recent publication demonstrated the involvement of the Notch pathway in the development of glomerular disease [15]. In summary our finding that ADAM10 is expressed in podocytes and found in elevated levels in the urine of patients with glomerular diseases needs further investigation to clarify the involvement of this molecule in the development of glomerular kidney diseases and its usefulness as a new biomarker for glomerular injury. Human podocytes were treated for 6 h and 24 h with 5 μg/ml and 10 μg/ml puromycin (Puro), supernatants were collected and after TCA-precipitation, equal amounts of protein samples were loaded on a SDS-PAGE. Membranes were probed with L1-11A, an antibody against the ectodomain of L1. (C) Human podocytes were pretreated 30 min with 3 μM ADAM10 inhibitor GI254023X (GI) before incubating cells for 6 hours with 10 μg/ml puromycin (Puro). Supernatants were analyzed for soluble L1 by western blot analysis. (D) Western Blot analysis of soluble L1 after the transfection of ADAM10 specific siRNA in the presence or absence of 5 μg/ml puromycin (24 hour treatment). As a negative control a scrambled siRNA was used (A10 = ADAM10, sc = Scrambled, Puro = Puromycin). Efficient knockdown of ADAM10 was controlled by westernblot with ADAM10 specific antibody (A10 = ADAM10, sc = scrambled) and equal loading of the samples were determined by β-actin westernblot.