a new role for the neuronal ubiquitin c-terminal hydrolase-l1 (uch-l1) in podocyte process formation...

Journal of PathologyJ Pathol 2009; 217: 452–464Published online 11 September 2008 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/path.2446

Original Paper

A new role for the neuronal ubiquitin C-terminalhydrolase-L1 (UCH-L1) in podocyte process formationand podocyte injury in human glomerulopathies

C Meyer-Schwesinger,1*,† TN Meyer,1† S Munster,1 P Klug,1 M Saleem,2 U Helmchen3 and RAK Stahl11Department of Internal Medicine, University Hospital Hamburg-Eppendorf, Hamburg, Germany2Children’s Renal Unit, Bristol Royal Hospital for Children, Bristol, UK3Department of Pathology, University Hospital Hamburg-Eppendorf, Hamburg, Germany

*Correspondence to:MD C Meyer-Schwesinger, RenalUnit, Department of InternalMedicine, University HospitalHamburg, Campus Forschung,Room 03.004, Martinistrasse 52,20246 Hamburg, Germany.E-mail: [email protected]

†These authors contributedequally to this work.

No conflicts of interest weredeclared.

Received: 27 March 2008Revised: 25 August 2008Accepted: 28 August 2008

AbstractGlomerular epithelial cell (podocyte) injury is characterized by foot process retraction,slit diaphragm reorganization, and degradation of podocyte-specific proteins. However, themechanisms underlying podocyte injury are largely unknown. The ubiquitin C-terminalhydrolase-L1 (UCH-L1) is a key modulator of ubiquitin modification in neurons. Like neu-rons, UCH-L1 expression was associated with an undifferentiated status in cultured humanpodocytes, whereas differentiation and arborization decreased UCH-L1 and monoUb expres-sion. Inhibition of UCH-L1 induced time and concentration-dependent process formationwith α-actinin-4 distribution to the cell membrane and processes. An immunohistochemicalapproach was used to evaluate whether UCH-L1 expression was associated with podocyteinjury in 15 different human glomerular diseases. Whereas normal kidneys expressed noUCH-L1 and little ubiquitin, a subset of human glomerulopathies associated with podocytefoot process effacement (membranous nephropathy, SLE class V, FSGS) de novo expressedUCH-L1 in podocyte cell bodies, nuclei, and processes. Interestingly, UCH-L1 expressioncorrelated with podocyte ubiquitin content and internalization of the podocyte-specific pro-teins nephrin and α-actinin-4. In contrast, minimal change glomerulonephritis, a reversibledisease, demonstrated minimal UCH-L1 and ubiquitin expression with intact α-actinin-4 butinternalized nephrin. Glomerular kidney diseases typically not associated with foot processeffacement (SLE class IV, ANCA+ necrotizing GN, amyloidosis, IgA nephritis) expressedintermediate to no UCH-L1 and ubiquitin. These studies show a role for UCH-L1 andubiquitin modification in podocyte differentiation and injury.Copyright 2008 Pathological Society of Great Britain and Ireland. Published by JohnWiley & Sons, Ltd.

Keywords: glomerulonephritis; podocyte injury; UCH-L1; PGP 9.5; ubiquitin modifica-tion; protein degradation and internalization

Introduction

Visceral epithelial cells (podocytes) are considered amajor regulator of the glomerular filtration barrier [1].Interdigitating podocyte foot processes (FPs) cover theglomerular basement membrane (GBM), forming fil-tration slits that are bridged by the extracellular slitdiaphragm. Podocyte injury is a key element of manyglomerular diseases with proteinuria and is character-ized by alterations in the molecular composition of theslit diaphragm or by reorganization of the foot pro-cess structure with fusion of filtration slits [2]. Theunderlying mechanisms are only partially understood.From morphological and biochemical studies it hasbecome evident that podocytes share a number of fea-tures with neuronal cells. Both cell types are highlyarborized, have a common cytoskeletal organization,

express a number of the same expression-restrictedproteins [1,3], and have a common machinery forprocess formation [4]. In neuronal cells, ubiquitinC-terminal hydrolase-L1 (UCH-L1) is a key enzymein regulating ubiquitin protein modification. Studiesdemonstrated that UCH-L1 plays an important rolein neuronal differentiation and function and processformation [5–9], and is associated with neurodegen-erative diseases and cancer [7,9–13].

Cellular malfunctions are frequently associated withaltered ubiquitin modification of proteins [14], a highlyconserved pathway to regulate cellular protein content,localization, and function [15]. For ubiquitin modifica-tion, proteins are tagged on lysine residues by additionof single ubiquitin molecules (mono-ubiquitination,monoUb) or poly-ubiquitin chains (poly-ubiquiti-nation, polyUb). The cytoplasmic pool of monoUb, the

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UCH-L1 in glomerular injury 453

key molecule for ubiquitination, is regulated by twoclasses of de-ubiquitinating enzymes: the ubiquitin C-terminal hydrolases (UCHs) and the ubiquitin isopep-tidases (UBPs) [16]. Three human UCH isoenzymeshave been cloned which exhibit a distinct tissue distri-bution [16]. UCH-L1 is mainly expressed in neuronaltissues during all stages of neuronal differentiation andin cells of the testis, ovaries, and kidney [17]. UCH-L1 regulates the intracellular pool of monoUb throughmonoUb stabilization [18] and by two opposing enzy-matic activities: the hydrolase activity increases themonoUb pool, while the ligase activity decreasesmonoUb [7]. Additionally, UCH isoforms hydrolysemono-ubiquitinated substrates [19], which are thennot targeted for degradation by the 26S proteasomepathway. This reversible modification is considered animportant regulatory strategy [16,20].

In the kidney, immunohistochemical studies demon-strated UCH-L1 expression in tubular epithelial cells,collecting duct cells, and parietal epithelial cells alongthe inner aspect of Bowman’s capsule [21,22]. Studieswith renal cells in culture or animal models of renaldisease show that UCH enzymes play a role in nephro-genesis, cell differentiation in tubulogenesis, and in theregulation of the cell cycle [14,23]. During nephroge-nesis of the rat kidney, UCH-L1 is expressed diffuselyin cells forming the comma-shaped body and laterin cells of the S-shaped body that gives rise to pari-etal epithelial cells. Importantly, UCH-L1 expressiondecreases in cells that later develop into podocytes.In the mature glomerulus of the rat, parietal epithelialcells exhibit UCH-L1, whereas podocytes are negative[21].

Here we demonstrate that UCH-L1 is de novoexpressed in podocytes in a subset of glomerulopathiesand correlates with changes of ubiquitin levels andwith internalization of podocyte-specific proteins. Inpodocyte culture, differentiation into an arborized cellis accompanied by down-regulation of UCH-L1 andmonoUb, and functional inhibition of UCH-L1 leadsto increased process formation. To our knowledge, thisis the first study demonstrating a role for an importantneuronal regulator of the monoUb pool and of proteinmodification in human podocyte process formation andin podocyte injury in human glomerulopathies.

Materials and methods

Antibodies

The primary antibodies used for the study are listed inthe Supporting information, Supplementary Table 1;all the secondary antibodies used were either biotiny-lated or HRP-conjugated affinity-purified donkeyantibodies (Jackson ImmunoResearch). Staining wasevaluated under an Axioskop and photographed withan Axiocam HRc using Axiostar software or with anLSM 510 beta microscope using LSM software (allZeiss).

Table 1. Pathological diagnosis and number of human biopsiesand podocytes analysed in this study

Pathological diagnosisn

biopsiesn

podocytes

Alport’s syndrome 11 968Amyloidosis AA 5 375Amyloidosis AL 4 679ANCA positive necrotizing GN 7 711Diabetic glomerulosclerosis 9 544Fibrillary GN 3 261Focal segmental GS (idiopathic) 9 731IgA nephritis (primary) 7 530IgA nephritis (Henoch–Schoenlein) 7 874Membranous GN (primary) 14 1541Membrano-proliferative GN type I 6 375Minimal change GN 12 1289Normal kidney 7 638SLE class V (membranous) 6 632SLE class IV (mesangio-proliferative) 6 1153Thin basement membrane disease 10 602

Podocyte cell culture

Human podocytes were cultured under permissiveconditions (33 ◦C, 5% CO2, RPMI 1640, 10% FCS,1× ITS) and for differentiation under non-permissiveconditions (37 ◦C) as previously described [24], eitheron uncoated (Sarstedt) or coated (laminin, collagen IV,collagen I, Becton Dickinson) cell ware. For dedif-ferentiation experiments, podocytes were switched to33 ◦C following 14 days of differentiation. For UCH-L1 inhibition, differentiated podocytes were treatedwith LDN-57 444 (Calbiochem) or DMSO. Processeswere counted from 60–100 solitary cells per groupin n = 3 experiments. A process is defined as mem-brane protrusion measuring over one eighth of thecell’s diameter in length.

Immunofluorescence

Podocytes were fixed (4% paraformaldehyde) andunspecific staining was blocked [2.5% horse serum(HS, Vector), 30 min, room temperature]. Primaryantibodies were incubated for 2 h at room temperatureand binding was detected with biotinylated secondaryantibodies (1 : 200 in 2.5% HS, 30 min, room tem-perature) and by FITC-avidin. After visualization off-actin, stains were mounted in hardset Vectashieldcontaining DAPI (Vector).

TUNEL assay

TUNEL assay was performed according to the manu-facturer’s instructions (Chemicon). Total cell numberand TUNEL-positive cells were counted at 200× mag-nification in ten visual fields per condition.

Immunoblot

Immunoblots were performed as described previously[25]. Briefly, samples were lysed in T-PER (Pierce)containing phosphatase inhibitors and denatured with

J Pathol 2009; 217: 452–464 DOI: 10.1002/pathCopyright 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

454 C Meyer-Schwesinger et al

4XLDS. Samples were separated on a 4–12% Bis-TrisNuPage gel (Invitrogen) in NuPage running buffer.Protein transfer was performed in transfer buffer(50 mM Tris base, 0.192 M glycine in doubly distilledH2O) in a Novex Mini Cell (Invitrogen). PVDF mem-branes (Millipore) were blocked (3% non-fat milk)prior to incubation with primary antibodies dilutedin Superblock blocking reagent (Pierce). Binding wasdetected by incubation with HRP-coupled secondaryantibodies (1 : 10 000, 3% NFM). Protein expressionwas visualized with ECL SuperSignal (Pierce), accord-ing to the manufacturer’s instructions, on a BiomaxLight Film (Kodak). Western blots were analysedusing software from ImageJ [26].

Reverse transcription-polymerase chain reaction(RT-PCR)

mRNA was isolated with the RLT plus kit (Qia-gen) according to the manufacturer’s instructions and1 µg was reverse-transcribed with Oligo dT Primer(Invitrogen) and MMLV reverse transcriptase (NEB).UCH-L1 and GAPDH (housekeeping gene) cDNAwas amplified (30 cycles) with exon spanning primers(sequence [21]) and separated on a 2% agarose gel(Serva).

Specimen collection

The study was conducted according to the Declarationof Helsinki principles, with approval from the localethics committee. Tissue samples were obtained from123 patients from the renal register of the UniversityHospital Hamburg between 2005 and 2007. The finalhistological diagnosis was used for stratification into15 different renal pathologies including normal kidney.

Immunohistochemistry

One-micrometre paraffin sections were deparaffinizedand antigen retrieval was performed by microwaveboiling (10 mM citrate buffer, pH 6.1) or by pro-tease digestion [protease XXIV (Sigma), 5 mg/ml orprotease XIV (Sigma), 10 mg/ml]. Unspecific bindingwas blocked (5% HS, 30 min, room temperature). Pri-mary antibody incubations (5% HS, overnight, 4 ◦C)were followed by incubation with biotinylated sec-ondary antibodies (1 : 400, 30 min, room temperature).Colour development was performed with the ABC-AP kit (Vector) or the TSA kit (NEN) according tothe manufacturers’ instructions with neufuchsin, andnuclei were counterstained with haematoxylin.

Evaluation of UCH-L1 immunoreactivity inpodocytes

UCH-L1 expression was analysed in at least threebiopsies per disease. Only biopsies with more thanthree glomeruli were included in the analysis. Podo-cytic UCH-L1 immunoreactivity was assessed by(1) the percentage of UCH-L1-positive podocytes of

all WT-1-positive podocytes from consecutive slides;and (2) the mean intensity of UCH-L1 staining, scaledarbitrarily from 0 (negative), 1 (faint), 2 (intermediate)to 3 (strong). The mean intensity was calculated foreach biopsy and is presented as the mean value of allbiopsies ± standard error of the mean (SEM).

Statistics

Values are means ± SEM; n refers to the numberof measurements. Paired Student’s t-test was used tocompare mean values within one experimental series.Data from two groups were compared by unpairedt-test. A p value of less than 0.05 was accepted asstatistically significant.

Results

Cultured human podocytes expressed UCH-L1and expression correlated with the differentiationstatus

UCH-L1 was detected by RT-PCR and immunoblotin lysates from cultured differentiated podocytes, inhuman kidney, and in rat brain lysates, whereas ratmesangial and glomerular endothelial cells were neg-ative (Figure 1A). Using the thermo-sensitive induc-tion of differentiation of podocytes, we investigatedwhether UCH-L1 expression was regulated with dif-ferentiation. Undifferentiated podocytes (Figure 1B,a–c: small, mostly triangular elongated cells) exhib-ited strong UCH-L1 expression in the cytoplasmand at the cell membrane along leading edges. Incontrast, UCH-L1 staining was weaker in differenti-ated podocytes (Figure 1B, d–f: large, flattened androunded cells) and mostly absent at the cell membrane.Quantification of UCH-L1 demonstrated a decreaseof expression with differentiation [(Figure 1C) char-acterized by an increase of the podocyte-specificactin-binding protein α-actinin-4 and an increase ofnephrin and synaptopodin immunofluorecence (Sup-porting information, Supplementary Figure 1A)] anda de novo increase of UCH-L1 expression afterre-switching differentiated podocytes from 37 ◦C to33 ◦C for 1 week. Following the re-switch, podocytesstarted to proliferate again and lost their large roundand flattened appearance (data not shown).

UCH-L1 prevented process formation

When cultured on coated cell ware, podocytes acquireda highly arborized morphology (Figure 2A). β-Catenin,an adherens junction protein and marker of podocytedifferentiation [24], increased with differentiationinto an arborized phenotype, whereas UCH-L1 andmonoUb were significantly down-regulated (Figure 2B).In order to assess whether UCH-L1 was functionallyassociated with alteration of morphology, podocytescultured on collagen I were treated with a specificinhibitor to UCH-L1, LDN 57 444 [11]. UCH-L1



Figure 1. UCH-L1 and monoUb expression depend on the differentiation status of cultured podocytes. (A) Upper panel: WBof UCH-L1 (13C4, ∼28 kD) in differentiated podocytes (PC), human kidney, rat brain, rat glomerular endothelial cells (EC), andrat mesangial cells (MC). Lower panel: RT- PCR of UCH-L1 and GAPDH (loading control) of the same samples. (B) f-actin (a,d) and UCH-L1 (b, c, e, f) double IF in undifferentiated (a–c) and differentiated (d–f) podocytes. (c) Double-headed arrows:plasma membrane; solid arrows: ruffles. Original magnification: 200× (a, b, d, e); 400× (c, d). (C) Upper panel: WB for UCH-L1,α-actinin-4, and β-actin in podocytes during differentiation and dedifferentiation (= switch, following 2 weeks of differentiation).Lower panel: quantification of UCH-L1 and α-actinin-4 expression (n = 4 experiments) normalized against β-actin. Values areexpressed as % expression of control (undifferentiated podocytes) ± SEM. ∗p < 0.05 compared with control; ∗∗p < 0.05 andn.s. = non-significant compared with differentiated podocytes day 21

inhibition led to a time and concentration-dependentformation of membrane protrusions (Figures 3Aand 3B), which were accompanied by redistributionof the FP-specific protein α-actinin-4 to the mem-brane (Figure 3C) as early as 20 min after treatment.The expression level of α-actinin-4 remained stable(Figure 3D), whereas the β-catenin content increased.

These data demonstrate that UCH-L1 down-regulation,extracellular matrix contact, and UCH-L1 functionalinhibition are accompanied by changes in podocytemorphology towards an arborized, differentiated phe-notype. Of note, the inhibition of UCH-L1 with LDN57 444 did not induce apoptosis (Supporting informa-tion, Supplementary Figure 1B).



Figure 2. UCH-L1 down-regulation is associated with arborization in podocytes. (A) Podocytes differentiated on uncoated (a) andcoated flasks (b–d). (B) Left panel: WB for UCH-L1, monoUb, β-catenin, and β-actin in differentiated podocytes on uncoatedor coated flasks. Right panel: Quantification of UCH-L1, monoUb, and β-catenin (n = 3 experiments) expression normalizedagainst β-actin. Values are expressed as % expression of control (podocytes differentiated on uncoated flasks) ± SEM. ∗p < 0.05compared with control

Patterns of UCH-L1 expression in podocytes

In cultured podocytes, UCH-L1 expression corre-lated with an undifferentiated phenotype. We there-fore analysed podocytic UCH-L1 expression in humanglomerular disease (Table 1). In the cortex of nor-mal biopsies, UCH-L1 was predominantly expressedin distal tubules (Figure 4A), macula densa, andnerve fibres in accordance with UCH-L1 as a neu-roendocrine marker (data not shown). All glomeru-lar cells were negative for UCH-L1 (Figure 4A, c).In contrast to normal podocytes, diseased podocytesexpressed UCH-L1 in distinct patterns (Figure 4B,a, c): (1) predominantly nuclear; (2) predominantlyperinuclear; and (3)cytoplasmic including podocyteprocesses. Staining of consecutive slides against thepodocyte-specific nuclear transcription factor WT-1demonstrated that all UCH-L1-positive cells were alsopositive for WT-1.

UCH-L1 in human glomerulopathies

UCH-L1 immunoreactivity in podocytes was detectedwith different intensities and prevalence in 15 dif-ferent disease entities (Figure 5). Among the protein-uric diseases, which are characterized by podocyte FPeffacement (membranous GN, SLE class V, FSGS,minimal change disease), the strongest expression ofUCH-L1 was observed in biopsies of patients withmembranous GN (Figure 6A, a–c). There, UCH-L1

expression was found in all the biopsies analysed,with the majority of podocytes involved in a char-acteristic nuclear and cytoplasmic pattern extend-ing into podocyte processes surrounding capillaries(Figure 6A, c). In SLE class V, which presents patho-logically as a membranous GN, a comparable UCH-L1expression pattern was observed in four out of sixbiopsies (Figure 6A, d, e). Interestingly, in biopsies ofpatients with SLE class IV, which presents patholog-ically as a proliferative glomerulonephritis, UCH-L1expression was low in podocytes (Supporting infor-mation, Supplementary Figure 2, f, g). In biopsies ofpatients with FSGS (Figure 6A, f–h), not all glomeruliwere UCH-L1-positive and within the glomerulus, notall podocytes were positive for UCH-L1. Interestingly,UCH-L1 expression was found predominantly withinnon-sclerotic regions of diseased glomeruli.

In contrast to the above findings, UCH-L1 expres-sion was only minimally detected in three other pro-teinuric glomerular diseases: minimal change disease;amyloidosis; and fibrillary GN (Figure 6B). In min-imal change disease, the vast majority of podocytesexpressed no UCH-L1 (Figure 6B, a, b) and werecomparable to the baseline values of biopsies frompatients classified as normal. Whereas six biopsieswere completely negative for podocytic UCH-L1, fiveout of 11 biopsies showed faint podocytic UCH-L1 expression in very few glomeruli. In renal biop-sies of patients with amyloidosis or fibrillary GN



Figure 3. Inhibition of UCH-L1 hydrolase activity alters podocyte morphology and α-actinin-4 immunolocalization. (A) f-actin IF ofdifferentiated podocytes treated with DMSO (a) or with 50 µM LDN 57 444 in DMSO (b) for 1 h; arrows: processes. (B) Processformation is concentration (left graph) and time-dependent (right graph) in LDN 57 444-inhibited solitary podocytes; untreated(−), DMSO-treated (0 µM). Values are expressed as number of processes per solitary podocyte ± SEM. ∗p < 0.05 compared withuntreated cells. (C) α-actinin-4 IF in DMSO and LDN 57 444-treated podocytes. Arrows: α-actinin-4 accumulation in membraneruffles and processes. Original magnification: 200× (a, c, e); 400× (b, d, f). (D) Upper panel: WB for β-catenin, podocin, α-actinin-4,and β-actin in differentiated podocytes treated with DMSO or LDN 57 444 for 1 h. Lower panel: quantification of β-catenin,podocin, and α-actinin-4 expression normalized against β-actin (n = 3 experiments). Values are expressed as % expression ofcontrol (podocytes treated with DMSO) ± SEM. ∗p < 0.05 compared with control

(Figure 6B, c–f), two proteinuric diseases character-ized by glomerular deposition of abnormal proteinsrather than by FP effacement, UCH-L1 expression inpodocytes was low.

In biopsies of patients with diabetic glomeru-losclerosis, membranoproliferative glomerulosclero-sis (MPGN) type 1, and necrotizing GN (Support-ing information, Supplementary Figure 2), podocytic

UCH-L1 expression was heterogeneous and low. Ofnote, in ANCA-positive necrotizing glomerulonephri-tis, most podocytes were negative for UCH-L1 expres-sion; however, crescents were composed of cellsexpressing UCH-L1 (Supporting information, Sup-plementary Figure 2, h–j). Whether these UCH-L1-expressing cells are of podocyte or parietal epithelialcell origin is not clear. Interestingly, in IgA nephritis



associated with Henoch–Schoenlein syndrome, UCH-L1 expression was increased compared with primaryIgA nephritis (Supporting information, Supplemen-tary Figure 3A) and localized to podocytes in areasof segmental necrosis, indicating that differences inUCH-L1 might be related to the severity of glomeru-lar involvement, ie necrosis. Since UCH-L1 expressionin podocytes was strongest in biopsies of diseases thatusually develop a nephrotic syndrome and are accom-panied by glomerular basement membrane alterations,we thought to analyse biopsies of other glomeru-lar pathologies with GBM alterations (Supportinginformation, Supplementary Figure 3B): Alport’s syn-drome and thin basement membrane disease (TBM).To our surprise, the prevalence of UCH-L1 expres-sion in Alport’s syndrome was the highest of thestudy and was comparable to the pattern and intensityof UCH-L1 expression in membranous GN or SLEclass V (Supplementary Figure 3B). In TBM, a diag-nosis based on morphometric measurements from elec-tron microscopic examination, the pattern of UCH-L1expression was comparable to that in Alport’s syn-drome, however more heterogeneous.

UCH-L1 expression correlated with ubiquitincontent

Immunohistochemical staining against ubiquitin wasperformed to assess whether UCH-L1 expression cor-related with changes in intracellular ubiquitin. Ubiqui-tin was expressed in tubuli of normal and diseased kid-neys (Figure 7A, a, d). In podocytes, ubiquitin expres-sion was low in normal biopsies, in which UCH-L1expression was also not detected in consecutive sec-tions (Figure 7A, a–c). In contrast, podocytes with denovo UCH-L1 expression such as membranous GNstained strongly for ubiquitin (Figure 7A, d–f). Anal-ysis of other glomerulopathies (Figures 7C and 7D)demonstrated a similar pattern: Whereas diseaseswith low UCH-L1 expression (amyloidosis, minimalchange disease or diabetic GN) had ubiquitin stain-ing comparable to normal biopsies (Figure 7C, a–c),diseases with de novo podocytic UCH-L1 expres-sion [SLE class V, FSGS, and Alport’s syndrome(Figure 7D, a–c)] exhibited an increase of cytoplasmicubiquitin. Therefore a striking correlation of UCH-L1 expression and cytoplasmic ubiquitin content wasnoted in specific glomerular diseases and in cell cul-ture. These data suggest that increased podocytic ubiq-uitin could have been supplied by UCH-L1 in thesediseases.

UCH-L1 expression correlated withinternalization of podocyte-specific proteins

In cultured podocytes, UCH-L1 expression corre-lated with an undifferentiated phenotype, while dif-ferentiation and UCH-L1 inhibition led to processformation with down-regulation and relocation ofUCH-L1 from the membrane and redistribution ofFP proteins to the membrane and processes. To

Figure 4. UCH-L1 expression patterns in normal anddiseased renal biopsies. (A) UCH-L1 in a normal kidney.(a) Black arrows: nuclear pattern in tubular cells; double-headedarrow: macula densa; ∗glomerulus. (b) Negative controlomitting primary antibody. (c) Normal glomerulus. (d) Enlargedarea; arrows: negative podocytes. (B) Patterns of UCH-L1expression in podocytes of two different patients withprimary FSGS. (a) Nuclear and cytoplasmic; (c) cytoplasmic.UCH-L1-expressing cells also express the podocyte-specificmarker WT-1 on consecutive sections (b, d, arrows).Double-headed arrow: podocyte with low UCH-L1 expression

assess whether UCH-L1 and ubiquitin expression inpodocytes correlated with altered protein homeosta-sis of podocyte-specific proteins in renal biopsies, thecellular localization of nephrin (Figure 8), podocin(not shown), and α-actinin-4 (Figure 9) was inves-tigated. Nephrin, podocin, and α-actinin-4 localizedwithin differentiated podocytes of normal biopsies in adistinct linear staining pattern (Figures 8 and 9, a, b).A similar staining pattern for all three proteins was



Figure 5. Number and intensity of UCH-L1-positivepodocytes in all analysed human biopsies. (A) Number ofUCH-L1-positive podocytes is expressed as the percentageof UCH-L1 and WT-1-positive podocytes of all WT-1-positivepodocytes/glomerulus per disease entity. (B) Mean intensity ofUCH-L1 staining per disease entity. TBM: thin basement mem-brane disease; IgA-HS: IgA nephritis with Henoch–Schoenleinsyndrome; diabetic GS: diabetic glomerulosclerosis; MC: mini-mal change disease

detected in glomerular diseases in which no podocyticUCH-L1 expression was observed; namely, amyloi-dosis, primary IgA nephritis, and ANCA-associatednecrotizing GN (Figures 8 and 9, e–j). Loss of linearstaining was detected in all glomerular diseases asso-ciated with de novo UCH-L1 expression and increasedpodocyte ubiquitin content: SLE class V, membra-nous GN, Alport’s syndrome and FSGS (Figure 8and 9, k–r); and is thought to correlate with inter-nalization of nephrin, podocin, and α-actinin-4 fromthe slit membrane to the cytoplasm [27] as a signof podocyte injury. The only exception was observed

in minimal change disease (Figures 8 and 9, c, d),in which despite the absence of UCH-L1 expres-sion, nephrin and podocin were internalized, whileα-actinin-4 remained in a linear staining pattern.

Discussion

Much attention has been drawn to the characteriza-tion of proteins of the podocyte cytoskeleton and ofthe slit membrane; however, no specific marker pro-tein has been identified that is absent in healthy butup-regulated in diseased podocytes and thus couldallow the histological differentiation between healthyand diseased podocytes, or allow a prediction aboutprognosis or the degree of podocyte damage. Ourstudy demonstrated that UCH-L1 could be such amarker. UCH-L1 was de novo expressed in specificdiseases which are difficult to differentiate with com-mon pathological tools (FSGS versus minimal changedisease) or which require electron microscopic anal-ysis (Alport’s syndrome, thin basement membranedisease). The present study noted a striking hetero-geneity of UCH-L1 expression in specific diseases,raising the intriguing possibility that the expressionpattern of UCH-L1 could differentiate disease enti-ties or prognosis. Whereas UCH-L1 expression wasuniform in primary membranous GN, expression wasmostly absent in minimal change disease and highlyheterogeneous in FSGS (which is known to resultfrom different pathophysiological causes). Further-more, UCH-L1 expression was observed in specificpatterns, such as predominant staining in podocyteprocesses (FSGS, membranous GN, SLE class V,Alport’s syndrome, TBM) or only within the podocytecell body (diabetes mellitus or IgA nephritis). Thesedifferent localizations within the podocyte may reflectdifferent requirements of the podocyte to modulatethe protein ubiquitin pool according to the primarydisease.

In vivo studies performed in rat embryos demon-strated that during glomerular development, UCH-L1 expression in podocytes inversely correlated withpodocyte differentiation [21]. Our data show thatUCH-L1 expression in cultured podocytes alsodepended on the differentiation status. Also, de novoexpression of UCH-L1 in cytoplasm and processesof injured podocytes in glomerular diseases such asSLE class V, membranous GN, Alport’s syndrome,and FSGS correlated with increased ubiquitin contentand with internalization of slit membrane proteins. IfUCH-L1 expression in vitro as well as in vivo can beregarded as a marker of podocyte dedifferentiation,these data suggest that podocyte dedifferentiation isalso present in many human glomerulopathies.

Podocyte and neuronal process formation sharemany common features [4,28] and UCH-L1 is involvedin neuronal process formation [5,6]. Podocyte FPeffacement, which may be comparable to neuronal



Figure 6. UCH-L1 immunoreactivity in nephrotic diseases. (A) Diseases with UCH-L1 immunoreactivity in podocytes. (a–c)Primary membranous glomerulonephritis; strong and homogeneous nuclear and cytoplasmatic UCH-L1 expression in podocytesextending into processes (c, double-headed arrows). Similarly, in systemic lupus erythematodes (SLE) class V (d, e). (f–h) Primaryfocal and segmental glomerulosclerosis with heterogeneous UCH-L1 expression [(g) predominantly nuclear or (h)cytoplasmatic].Original magnification: 400× (a, d, f) and 1000× (b, c, e, g, h); arrows indicate podocytes. (B) Glomerulopathies with nearly absentUCH-L1 immunoreactivity in podocytes. (a, b) Minimal change glomerulonephritis; absent UCH-L1 expression in podocytes as inAA amyloidosis (c, d). (e, f) Fibrillary glomerulonephritis; heterogeneous UCH-L1 expression in a few podocytes in a predominantlynuclear pattern. Original magnification: 400× (a, c, e) and 1000× (b, d, f); arrows indicate podocytes



Figure 7. Increased ubiquitin content in podocytes expressing UCH-L1 in renal biopsies. (A) Normal biopsy (a–c) demonstratesubiquitin expression in tubuli (double-headed arrows), whereas glomeruli and podocytes exhibit low ubiquitin content. UCH-L1expression on a consecutive slide of the same glomerulus (c). (B) In membranous GN (a–c), tubular ubiquitin content is similarto normal; glomerular and podocytic ubiquitin content is increased. UCH-L1 expression on a consecutive slide of the sameglomerulus (c). (C) Ubiquitin staining in diseases lacking UCH-L1 expression: AL amyloidosis (a), minimal change disease (MC, b),and diabetic glomerulosclerosis (GS, c). (D) Ubiquitin staining in diseases with high UCH-L1 expression: SLE class V (a), primaryFSGS (b), and Alport’s syndrome (c). Arrows indicate podocytes

process regression, was present in human glomeru-lopathies characterized by UCH-L1 expression suchas membranous GN. In injured podocytes in mem-branous GN, UCH-L1 was characteristically detectedin the cytoplasm and in primary podocyte processes.Interestingly, in cell culture, UCH-L1 localized to themembrane of undifferentiated podocytes and relocatedto the cytoplasm upon differentiation, indicating thatUCH-L1 might play a role in podocyte process patho-physiology. Similarly to neurons, cultured podocytesform processes under conditions of differentiation (ieculture on laminin) which resemble primary processes

in vivo [28–31]. They do not form typical FPs as inthe glomerulus; however, FP-specific proteins suchas nephrin and α-actinin-4 are expressed along themembrane and in cell–cell adhesions [32]. In ourexperiments, both differentiation-dependent decreaseof UCH-L1 expression and specific inhibition of UCH-L1 hydrolase activity promoted the development ofprocesses. Furthermore, inhibition of UCH-L1 hydro-lase activity led to a concentration and time-dependentincrease of process formation with redistribution ofα-actinin-4 and β-catenin to the membrane. Thesedata indicate that both proteins are regulated, at



Figure 8. Confocal analysis of nephrin distribution in renal biopsies. Normal kidney (a, b) and glomeruli of diseases associatedwith low to absent (c–j) or strong UCH-L1 expression in podocytes (k–q). Linear nephrin in normal (b), AL amyloidosis (f), IgAnephritis (h), and ANCA-associated necrotizing GN (j); disrupted nephrin in minimal change disease (MC, d), SLE class V (k, l),membranous GN (m, n), Alport’s syndrome (o, p), and FSGS (q, r). Original magnification: 200× (a, c, e, g, i, k, m, o, q) and 1000×(b, d, f, h, j, l, n, p, r); P = podocyte nuclei

least in part, by UCH-L1. It is noteworthy to men-tion that UCH enzymes are not primarily involvedin proteasomal degradation but can also reversiblyhydrolyse monoUb proteins [16,20], a process impor-tant for the internalization and degradation of plasmamembrane proteins [33–35]. It is therefore possi-ble that UCH-L1 influences podocyte morphology bymodification of podocyte-specific proteins such as α-actinin-4.

An additional effect of UCH-L1 activity wasrecently shown in neuronal progenitor cells. Trans-fection of these cells with UCH-L1 mutants unableto stabilize monoUB (D30A) or with hydrolysis-deficient mutants (C90S) demonstrated that processformation was dependent on monoUB stabilization [5].Our data show that UCH-L1 and ubiquitin expres-sion in podocytes correlated in renal biopsies and cellculture experiments, indicating that UCH-L1 might

increase the monoUb pool through either hydrolaseactivity or monoUb stabilization [18] in diseased orundifferentiated podocyes. We therefore speculate thatthe absence of processes in undifferentiated podocytescould be related to high UCH-L1 expression andmonoUb stabilization. Similarly, alteration of pro-cess morphology observed in renal biopsies in vivocould be dependent on UCH-L1 monoUb stabiliza-tion followed by protein ubiquitination and degrada-tion, at least in glomerulonephritis with irreversibledamage to the podocyte (as in cases of FSGS andmembranous GN). Strikingly, reversible FP efface-ment, which is present in most cases of minimalchange disease, presented with very low UCH-L1and ubiquitin expression, despite internalization ofnephrin and podocin. A likely explanation wouldbe that FP effacement in minimal change diseasedoes not involve ubiquitin-dependent modification of



Figure 9. Confocal analysis of α-actinin-4 distribution in renal biopsies. Normal kidney (a, b) and glomeruli of diseases associatedwith low to absent (c–j) or strong UCH-L1 expression in s (k–q). Linear α-actinin-4 in normal podocytes (b), minimal changedisease (MC, d), AL amyloidosis (f), IgA nephritis (h), and ANCA-associated necrotizing GN (j); disrupted α-actinin-4 in SLE classV (k, l), membranous GN (m, n), Alport’s syndrome (o, p), and FSGS (q, r). Original magnification: 200× (a, c, e, g, i, k, m, o, q)and 1000× (b, d, f, h, j, l, n, p, r); P = podocyte nuclei

FP-specific proteins and is therefore reversible. Inthe brain, UCH-L1 is involved in the regulation ofproteasomal degradation of abnormal proteins and isassociated with neurodegenerative disorders such asParkinson’s disease [7] and Alzheimer disease [8]. Inmany glomerular diseases, accumulation of proteins inthe subepithelial space or in the GBM (membranousGN, SLE class V), or altered GBM synthesis (mem-branous GN, SLE class V, Alport’s syndrome, diabeticglomerulosclerosis) has been described. Whether suchaccumulation drives UCH-L1 expression or whetherUCH-L1 expression leads to the accumulation of pro-teins in glomerulopathies remains to be explored. Fur-ther studies in cultured podocytes and animal modelsof glomerulopathies will be necessary to investigatethe specific role of UCH-L1 in podocyte injury. A bet-ter understanding of UCH-L1 regulation and functionmight lead the way to new diagnostic and prognostic

approaches in human glomerular disease and open anew avenue for therapeutic strategies.

Acknowledgements

We would like to thank Matthias Kretzler, MD, and SimoneBlattner, PhD (both Department of Internal Medicine, Univer-sity of Michigan, Ann Arbor, USA), for helpful discussions andfor providing the α-actinin-4 antibody used in these studies; andUrsula Kneissler (Department of Pathology, University Hos-pital Hamburg Eppendorf, Hamburg, Germany) and MariolaReszka (Department of Internal Medicine, University HospitalHamburg-Eppendorf, Hamburg, Germany) for excellent tech-nical assistance.

Supporting information

Supporting information may be found in the onlineversion of this article.



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a new role for the neuronal ubiquitin c-terminal hydrolase-l1 (uch-l1) in podocyte process formation...

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