Ubiquitin C-Terminal Hydrolase L1 is required for regulated protein degradation through the ubiquitin proteasome system in kidney

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a major deubiquitinating enzyme of the nervous system and associated with the development of neurodegenerative diseases. We have previously shown that UCH-L1 is found in tubular and parietal cells of the kidney and is expressed de novo in injured podocytes. Since the role of UCH-L1 in the kidney is unknown we generated mice with a constitutive UCH-L1-deficiency to determine its role in renal health and disease. UCH-L1–deficient mice developed proteinuria, without gross changes in glomerular morphology. Tubular cells, endothelial cells, and podocytes showed signs of stress with an accumulation of oxidative-modified and polyubiquitinated proteins. Mechanistically, abnormal protein accumulation resulted from an altered proteasome abundance leading to decreased proteasomal activity, a finding exaggerated after induction of anti-podocyte nephritis. UCH-L1–deficient mice exhibited an exacerbated course of disease with increased tubulointerstitial and glomerular damage, acute renal failure, and death, the latter most likely a result of general neurologic impairment. Thus, UCH-L1 is required for regulated protein degradation in the kidney by controlling proteasome abundance. Altered proteasome abundance renders renal cells, particularly podocytes and endothelial cells, susceptible to injury. Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a major deubiquitinating enzyme of the nervous system and associated with the development of neurodegenerative diseases. We have previously shown that UCH-L1 is found in tubular and parietal cells of the kidney and is expressed de novo in injured podocytes. Since the role of UCH-L1 in the kidney is unknown we generated mice with a constitutive UCH-L1-deficiency to determine its role in renal health and disease. UCH-L1–deficient mice developed proteinuria, without gross changes in glomerular morphology. Tubular cells, endothelial cells, and podocytes showed signs of stress with an accumulation of oxidative-modified and polyubiquitinated proteins. Mechanistically, abnormal protein accumulation resulted from an altered proteasome abundance leading to decreased proteasomal activity, a finding exaggerated after induction of anti-podocyte nephritis. UCH-L1–deficient mice exhibited an exacerbated course of disease with increased tubulointerstitial and glomerular damage, acute renal failure, and death, the latter most likely a result of general neurologic impairment. Thus, UCH-L1 is required for regulated protein degradation in the kidney by controlling proteasome abundance. Altered proteasome abundance renders renal cells, particularly podocytes and endothelial cells, susceptible to injury. Membranous nephropathy is the most common cause of nephrotic syndrome in adults, and one-third of patients progress to dialysis-necessitating terminal renal insufficiency. Membranous nephropathy (MN) is caused by autoantibodies directed against podocyte antigens such as the PLA2R1Beck Jr., L.H. Bonegio R.G. Lambeau G. et al.M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy.N Engl J Med. 2009; 361: 11-21Crossref PubMed Scopus (1504) Google Scholar and the recently identified THSD7A.2Tomas N.M. Beck Jr., L.H. Meyer-Schwesinger C. et al.Thrombospondin type-1 domain-containing 7A in idiopathic membranous nephropathy.N Engl J Med. 2014; 371: 2277-2287Crossref PubMed Scopus (536) Google Scholar We could recently demonstrate that altered protein degradation through the ubiquitin proteasomal system is a feature of irreversibly injured podocytes in MN.3Beeken M. Lindenmeyer M.T. Blattner S.M. et al.Alterations in the ubiquitin proteasome system in persistent but not reversible proteinuric diseases.J Am Soc Nephrol. 2014; 25: 2511-2525Crossref PubMed Scopus (23) Google Scholar Hence, understanding the role of protein homeostasis in the kidney is of great importance, and restoring altered protein degradation in disease could potentially prevent irreversible renal injury. The ubiquitin proteasome system (UPS) is one of the most prominent systems for intracellular protein degradation and is mainly responsible for the removal of short-lived proteins. The UPS comprises enzymes that ubiquitinate or deubiquitinate target proteins, and the 26S proteasome system, which degrades ubiquitinated proteins.4Hochstrasser M. Biochemistry. All in the ubiquitin family.Science. 2000; 289: 563-564Crossref PubMed Scopus (102) Google Scholar Ubiquitin is covalently attached to target proteins either as monomers (mono-ubiquitination) or as di-, oligo-, and polyubiquitin chains by a multi-enzymatic system consisting of E1 (ubiquitin-activating), E2 (ubiquitin-conjugating), and E3 (ubiquitin-ligating) enzymes. The mode of conjugation determines the fate of ubiquitinated proteins. The conjugation of ubiquitin to proteins is a reversible process that is tightly controlled by deubiquitinating enzymes. Deubiquitinating enzymes cleave monoubiquitin from proteins and disassemble polyubiquitin chains that are released from substrates before degradation in the multimeric protein complex called 26S proteasome, or in a more proteolytic effective form, the i26S immunoproteasome.5Seifert U. Bialy L.P. Ebstein F. et al.Immunoproteasomes preserve protein homeostasis upon interferon-induced oxidative stress.Cell. 2010; 142: 613-624Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar UCH-L1 is a small 27 kDa soluble protease,6Das C. Hoang Q.Q. Kreinbring C.A. et al.Structural basis for conformational plasticity of the Parkinson's disease-associated ubiquitin hydrolase UCH-L1.Proc Natl Acad Sci U S A. 2006; 103: 4675-4680Crossref PubMed Scopus (148) Google Scholar mainly expressed in neuronal tissues,7Ermisch B. Schwechheimer K. Protein gene product (PGP) 9.5 in diagnostic (neuro-) oncology. An immunomorphological study.Clin Neuropathol. 1995; 14: 130-136PubMed Google Scholar but also found in the testis8Kwon J. Wang Y.L. Setsuie R. et al.Two closely related ubiquitin C-terminal hydrolase isozymes function as reciprocal modulators of germ cell apoptosis in cryptorchid testis.Am J Pathol. 2004; 165: 1367-1374Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar and in tumors.9Fang Y. Fu D. Shen X.Z. The potential role of ubiquitin c-terminal hydrolases in oncogenesis.Biochim Biophys Acta. 2010; 1806: 1-6Crossref PubMed Scopus (97) Google Scholar, 10Kim H.J. Kim Y.M. Lim S. et al.Ubiquitin C-terminal hydrolase-L1 is a key regulator of tumor cell invasion and metastasis.Oncogene. 2009; 28: 117-127Crossref PubMed Scopus (125) Google Scholar Neural functions ascribed to UCH-L1 are in neuronal differentiation, regulation of synaptic function11Lansbury Jr., P.T. Improving synaptic function in a mouse model of AD.Cell. 2006; 126: 655-657Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar and structure,12Cartier A.E. Djakovic S.N. Salehi A. et al.Regulation of synaptic structure by ubiquitin C-terminal hydrolase L1.J Neurosci. 2009; 29: 7857-7868Crossref PubMed Scopus (109) Google Scholar and cell process formation. UCH-L1 is a member of the family of deubiquitinating cysteine proteases, which regulate monoubiquitin levels and thereby play an important role in ubiquitin protein modification. A role for UCH-L1 in the regulation of the intracellular monoubiquitin pool13Larsen C.N. Krantz B.A. Wilkinson K.D. Substrate specificity of deubiquitinating enzymes: ubiquitin C-terminal hydrolases.Biochemistry. 1998; 37: 3358-3368Crossref PubMed Scopus (345) Google Scholar and on K63-alternate ubiquitin linkage14Liu Y. Fallon L. Lashuel H.A. et al.The UCH-L1 gene encodes two opposing enzymatic activities that affect alpha-synuclein degradation and Parkinson's disease susceptibility.Cell. 2002; 111: 209-218Abstract Full Text Full Text PDF PubMed Scopus (690) Google Scholar has been shown. Additionally, UCH-L1 function appears to be not solely dependent on its hydrolase activity but also on its ability to bind to and stabilize ubiquitin.15Sakurai M. Ayukawa K. Setsuie R. et al.Ubiquitin C-terminal hydrolase L1 regulates the morphology of neural progenitor cells and modulates their differentiation.J Cell Sci. 2006; 119: 162-171Crossref PubMed Scopus (57) Google Scholar Mouse models with loss of UCH-L1 function have been described, whereby the gad mice (del. ex7/8)16Saigoh K. Wang Y.L. Suh J.G. et al.Intragenic deletion in the gene encoding ubiquitin carboxy-terminal hydrolase in gad mice.Nat Genet. 1999; 23: 47-51PubMed Scopus (0) Google Scholar and nm3419 mice (del. ex6 and partial incl. in6)17Walters B.J. Campbell S.L. Chen P.C. et al.Differential effects of Usp14 and Uch-L1 on the ubiquitin proteasome system and synaptic activity.Mol Cell Neurosci. 2008; 39: 539-548Crossref PubMed Scopus (64) Google Scholar both exhibit decreased monoubiquitin levels and a similar neurologic phenotype with early sensory ataxia followed by motor ataxia.18Mukoyama M. Yamazaki K. Kikuchi T. et al.Neuropathology of gracile axonal dystrophy (GAD) mouse. An animal model of central distal axonopathy in primary sensory neurons.Acta Neuropathol. 1989; 79: 294-299Crossref PubMed Scopus (62) Google Scholar In the human and rat kidney, UCH-L1 is found in tubular cells19Diomedi-Camassei F. Rava L. Lerut E. et al.Protein gene product 9.5 and ubiquitin are expressed in metabolically active epithelial cells of normal and pathologic human kidney.Nephrol Dial Transplant. 2005; 20: 2714-2719Crossref PubMed Scopus (21) Google Scholar and in rat parietal epithelial cells20Guhr S.S. Sachs M. Wegner A. et al.The expression of podocyte-specific proteins in parietal epithelial cells is regulated by protein degradation.Kidney Int. 2013; 84: 532-544Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 21Shirato I. Asanuma K. Takeda Y. et al.Protein gene product 9.5 is selectively localized in parietal epithelial cells of Bowman's capsule in the rat kidney.J Am Soc Nephrol. 2000; 11: 2381-2386Crossref PubMed Google Scholar and de novo in injured podocytes.22Meyer-Schwesinger C. Meyer T.N. Munster S. et al.A new role for the neuronal ubiquitin C-terminal hydrolase-L1 (UCH-L1) in podocyte process formation and podocyte injury in human glomerulopathies.J Pathol. 2009; 217: 452-464Crossref PubMed Scopus (63) Google Scholar, 23D'Andrea V. Malinovsky L. Berni A. et al.The immunolocalization of PGP 9.5 in normal human kidney and renal cell carcinoma.G Chir. 1997; 18: 521-524PubMed Google Scholar, 24Liu Y. Wu H. Wu J. et al.Detection of UCH-L1 expression by pre-embedding immunoelectron microscopy with colloidal gold labeling in diseased glomeruli.Ultrastruct Pathol. 2008; 32: 5-9Crossref PubMed Scopus (6) Google Scholar We have previously shown that UCH-L1 is de novo expressed in injured podocytes in human MN22Meyer-Schwesinger C. Meyer T.N. Munster S. et al.A new role for the neuronal ubiquitin C-terminal hydrolase-L1 (UCH-L1) in podocyte process formation and podocyte injury in human glomerulopathies.J Pathol. 2009; 217: 452-464Crossref PubMed Scopus (63) Google Scholar and in a rat model of MN.25Meyer-Schwesinger C. Meyer T.N. Sievert H. et al.Ubiquitin C-terminal hydrolase-l1 activity induces polyubiquitin accumulation in podocytes and increases proteinuria in rat membranous nephropathy.Am J Pathol. 2011; 178: 2044-2057Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar However, it is unclear how UCH-L1 is functioning at the cellular and molecular level in kidney. To assess the role of UCH-L1 in kidney, we generated a unique mouse model of UCH-L1 deficiency allowing the complete and tissue-specific knockout of UCH-L1 by Cre-lox technology. Here, we have evaluated the effect of complete UCH-L1-deficiency on murine kidney function and following the induction of an immune-complex nephritis, termed antipodocyte nephritis (APN). APN is induced by injection of polyclonal rabbit26Meyer T.N. Schwesinger C. Wahlefeld J. et al.A new mouse model of immune-mediated podocyte injury.Kidney Int. 2007; 72: 841-852Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar or sheep antibody27Meyer-Schwesinger C. Dehde S. Klug P. et al.Nephrotic syndrome and subepithelial deposits in a mouse model of immune-mediated anti-podocyte glomerulonephritis.J Immunol. 2011; 187: 3218-3229Crossref PubMed Scopus (38) Google Scholar directed against cultured murine podocytes. Mice exhibit linear glomerular binding of the injected anti-podocyte antibodies and develop severe nephrotic syndrome with subepithelial immune deposits.27Meyer-Schwesinger C. Dehde S. Klug P. et al.Nephrotic syndrome and subepithelial deposits in a mouse model of immune-mediated anti-podocyte glomerulonephritis.J Immunol. 2011; 187: 3218-3229Crossref PubMed Scopus (38) Google Scholar UCH-L1 was knocked out by crossing UCH-L1fl/fl mice with constitutive Cre-deleter mice. Analysis of UCH-L1 transcript levels in total kidney lysates confirmed complete loss of UCH-L1 mRNA and protein expression in UCH-L1-deficient mice in comparison with control littermates (Figure 1a and b). UCH-L1 protein levels were comparable between UCH-L1–deficient mice missing one Uchl1 allele (heterozygous mice) and wild-type littermates (Figure 1b). Therefore, both genotypes were used as control littermates for subsequent analyses. Immunofluorescence confirmed complete loss of renal UCH-L1 expression in UCH-L1–deficient mice. Wild-type mice demonstrated a constitutive UCH-L1 expression in tubulointerstitial and glomerular cells (Figure 1c). Specifically, UCH-L1 was expressed in nerve endings of renal arterioles (Figure 1d) and glomerular and tubulointerstitial endothelial cells (Figure 1e and f) and tubulointerstitial cells of nonvascular origin (Figure 1f). UCH-L1–deficient mice exhibit a profound neurodegenerative phenotype as published for other spontaneous UCH-L1–deficient knockout models.16Saigoh K. Wang Y.L. Suh J.G. et al.Intragenic deletion in the gene encoding ubiquitin carboxy-terminal hydrolase in gad mice.Nat Genet. 1999; 23: 47-51PubMed Scopus (0) Google Scholar, 17Walters B.J. Campbell S.L. Chen P.C. et al.Differential effects of Usp14 and Uch-L1 on the ubiquitin proteasome system and synaptic activity.Mol Cell Neurosci. 2008; 39: 539-548Crossref PubMed Scopus (64) Google Scholar We therefore analyzed blood pressure levels of UCH-L1–deficient mice at 25 weeks of age, when neurologic impairment was established. We noted a decrease of systolic blood pressure by around 35 mm Hg in comparison with control littermates (Figure 2a). Measurement of renin mRNA levels demonstrated a significant upregulation of renin in UCH-L1–deficient kidneys (Figure 2b). Furthermore, morphological examination demonstrated a significantly hypertrophied juxta-glomerular apparatus by periodic acid–Schiff staining and by specific labeling with lysosomal integral membrane protein (Limp)-2 (Figure 2c). These findings suggest a profound systemic hypotension secondary to neurologic impairment with renal hypoperfusion, leading to a compensatory increase of renin expression. Besides hypotension, we noted a neurogenic urinary retention in UCH-L1–deficient mice starting around 16 weeks of age with bladders filled with up to 2 ml of urine at the time of killing (Figure 3a and b). Of note, no signs of obstructive uropathy were seen macroscopically. The renal pelvis was not dilated. Resolving creatinine-adapted urinary proteins collected from 25-week-old mice by a silver gel demonstrated a preferential loss of albumin and of low molecular weight proteins (Figure 3c). Quantification of urinary albumin levels by enzyme-linked immunosorbent assay (ELISA) confirmed the finding that UCH-L1–deficient mice developed significant albuminuria with age (Figure 3d).Figure 3UCH-L1–deficient mice develop proteinuria and urine retention in the bladder. Urine of UCH-L1–deficient (KO) mice and control (Ctrl) littermates was collected over 25 weeks. (a) Representative micrographs of bladders in 25-week-old mice at the time of killing. Note filled bladder in UCH-L1–deficient mouse. (b) Quantification of bladder urine volume at time of killing in 9- to 11-, 16- to 20-, and 25- to 28-week-old mice. Values are expressed as mean bladder urine volume in μl ± SEM; n = 3–5 mice per group and time point, *P < 0.05 and **P < 0.01 to respective control, Mann-Whitney U test. (c) Silver stain of proteins from desalted and creatinine-adapted urine collected from 25-week-old mice and separated by SDS-PAGE. (d) Quantification of albuminuria by enzyme-linked immunosorbent assay in 9- to 11- and 16- to 25-week-old mice. Milligram albumin values were normalized to mg creatinine and plotted as mean ± SEM, n = 4 mice per group and time point, *P < 0.05 and **P < 0.01 to respective control, Mann-Whitney U test. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Due to the fact that UCH-L1 deficiency resulted in the development of proteinuria, we evaluated the integrity of the glomerular filtration barrier. However, we noted no significant morphologic changes. Synaptopodin (Figure 4a) and nephrin immunofluorescence (not shown) showed no major alterations of staining pattern. Electron microscopic evaluations exhibited a normal architecture of the glomerular filtration barrier including an intact fenestrated endothelium, regular glomerular basement membrane, and normal podocyte foot processes (Figure 4b). We searched for signs of glomerular cell stress by Western blotting and by immunofluorescence for oxidative-modified proteins. We noted increased levels of oxidative (carbonyl)-modified proteins in glomerular lysates of 25-week-old mice (Figure 4c) following derivatization 2,4-dinitrophenylhydrazine (DNPH) and Western blotting for 2,4-dinitrophenyl (DNP). Immunofluorescence for DNP (Figure 4c) or for 8-hydroxyguanosine (Figure 4d) (a DNA base modified by hydroxyl radicals) demonstrated that oxidative stress was most apparent in podocytes, glomerular and tubulointerstitial endothelial cells, and in tubuli of UCH-L1–deficient mice. In line with enhanced accumulation of oxidative modified proteins in glomerular cells, staining for superoxide dismutase (SOD)-2, a mitochondrial enzyme involved in detoxification of reactive oxygen species, demonstrated an enhanced expression of SOD2 in podocytes but not in glomerular endothelial cells of UCH-L1–deficient mice (Figure 4e). Taken together our data demonstrate that UCH-L1–deficient mice develop hypotension, neurogenic urine retention, and proteinuria without major morphologic alterations but with a significant accumulation of oxidative-modified proteins and DNA. Light microscopic evaluation exhibited normal tubulointerstitial and glomerular morphology in UCH-L1–deficient mice (Figure 5a). Because UCH-L1 is a central deubiquitinating enzyme of the neuronal UPS, we quantified renal and glomerular ubiquitin levels by Western blot. Interestingly, UCH-L1 loss resulted in increased levels of polyubiquitinated proteins in whole kidney and glomerular lysates (Figure 5b and c). Renal levels of monoubiquitin were comparable to levels in control mice, whereas glomerular monoubiquitin levels were significantly increased in UCH-L1–deficient mice. Because UCH-L1–deficient mice exhibited increased levels of polyubiquitinated proteins, we examined whether proteasomal activity was altered due to UCH-L1 deficiency. Proteasomal activity was assessed in total renal and glomerular lysates by measuring the chymotrypsin-like proteolysis of the synthetic proteasomal substrate Suc-LLVY-AMC. Proteolytic release of fluorometric AMC was significantly decreased in both whole kidney (Figure 6a) and glomerular lysates of UCH-L1–deficient mice (Figure 6b). The chymotrypsin-like activity of the proteasome is harbored within the beta5 subunit of the 26S proteasome and within the beta5i subunit of the proteolytic more effective i26S immunoproteasome. We therefore quantified the levels of the beta5 and beta5i subunits in whole kidney lysates (Figure 6c) and in glomerular lysates (Figure 6d) with subunit specific antibodies by Western blot. In both whole kidney and isolated glomeruli of UCH-L1–deficient mice, levels of the beta5i subunit were significantly decreased, whereas levels of the standard beta 5-subunit were increased. Analysis of mRNA levels to these and other proteasomal subunits from mRNA isolated from whole kidney suggested that decreased immunoproteasome subunit beta5i (PSMB8) was partly due to reduced transcript levels in UCH-L1–deficient mice (Figure 6e). mRNA levels to the nonproteolytic 26S subunits PSMA2, PSMD4, and PSMB5 (beta5) were unchanged in comparison with control littermates. Taken together these data suggest that UCH-L1–deficient mice exhibit an increased content of polyubiquitinated proteins due to decreased proteolytic proteasomal activity, as a result of an altered balance between the standard and the immunoproteasome. Frequently, impairment of the proteasomal system results in compensatory upregulation of the autophagosomal lysosomal system.3Beeken M. Lindenmeyer M.T. Blattner S.M. et al.Alterations in the ubiquitin proteasome system in persistent but not reversible proteinuric diseases.J Am Soc Nephrol. 2014; 25: 2511-2525Crossref PubMed Scopus (23) Google Scholar Indeed, we could detect a significant upregulation of Lamp-2–positive lysosomes in treatment-naïve UCH-L1–deficient kidneys, especially in tubular cells and of LC3 positive autophagosomes in podocytes (Supplementary Figure S1A–C). Upon induction of APN, a model of immune-complex glomerulonephritis mediated by sheep antibodies to podocytes,27Meyer-Schwesinger C. Dehde S. Klug P. et al.Nephrotic syndrome and subepithelial deposits in a mouse model of immune-mediated anti-podocyte glomerulonephritis.J Immunol. 2011; 187: 3218-3229Crossref PubMed Scopus (38) Google Scholar UCH-L1–deficient mice persistently exhibited elevated Lamp-2 levels in whole kidney lysate and isolated glomeruli of PI and APN treated mice in comparison to respective control group littermates (Supplementary Figure S1D and E). Immunofluorescence demonstrated that lysosomes were most prominently elevated in the tubular compartment of UCH-L1–deficient mice treated with PI or APN (Supplementary Figure S1F). Upon APN, lysosomal size increased. In UCH-L1–deficient mice lysosomes were increased and appeared disarranged in their relation to the brush border of proximal tubular cells.Figure 6Proteasomal abundance and activity is altered in kidneys of UCH-L1–deficient mice. Kidneys of 20- to 28-week-old UCH-L1–deficient (KO) mice and control (Ctrl) littermates were investigated for overall proteasomal activity. The synthetic proteasomal substrate suc-LLVY-AMC was added to lysates of (a) whole kidney and (b) isolated glomeruli and chymotrypsin-like proteolytic release of fluorescent AMC was measured after 60 minutes. Values are expressed as mean percentage change in absorbance ± SEM to control littermates, n = 3–7 mice per group, *P < 0.05 to control, Mann-Whitney U test. Western blotting for the proteasomal subunits beta5 and beta5i in (c) whole kidney and (d) isolated glomerular lysates. The graphs depict densitometric analyses of beta5 and beta5i content normalized to beta-actin as a loading control in n = 7–11 mice per group. Values are plotted as mean percentage change to control mice levels ± SEM, *P < 0.05 to control, Mann-Whitney U test. POI, protein of interest. (e) Quantitative polymerase chain reaction for the proteasomal subunits PSMA2, PSMD4, and PSMB5 (beta5) of the standard proteasome and PSMB8 (beta5i) and PSMB9 (beta1i) of the immunoproteasome. Values were normalized to ribosomal 18S mRNA as internal control and expressed as mean relative change to control littermates ± SEM, n = 5–13 per group and gene of interest (GOI), *P < 0.05 to control, Mann-Whitney U test. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) UCH-L1 is normally not expressed in healthy podocytes, but upon aging a slight UCH-L1 expression can be appreciated in podocytes of 2-year-old mice. Taking advantage of the cre-lox technology, we generated mice with UCH-L1 deficiency in podocytes (Figure 7a and b) and evaluated whether similar biochemical alterations as in constitutive UCH-L1–deficient mice could be observed in 2-year-old mice. Indeed, podocyte-specific UCH-L1–deficient mice developed a very mild phenotype with age, characterized by significantly decreased levels of the beta5i subunit of the proteasome (Figure 7c), slightly reduced chymotrypsin-like activity of the proteasome (Figure 7d), and albuminuria (Figure 7e). Of note, podocyte-specific UCH-L1 deficiency resulted in a significant accumulation of oxidative-modified proteins and of ubiquitinated, especially K48-polyubiquitinated, proteins in isolated glomeruli, reminiscent of the observations made in constitutive UCH-L1–deficient mice. Immunofluorescent analyses (Figure 7g–i) confirmed that accumulation of oxidative-modified DNA base 8-hydroxyguanosine (Figure 7g), of reactive oxygen species detoxifying enzyme SOD-2 (Figure 7h), and ubiquitinated, especially K48-polyubiquitinated, proteins (Figure 7i) occurred mostly in podocytes within glomeruli of podocyte-specific UCH-L1–deficient mice. Of note, glomerular endothelial cells also showed signs of reactive oxygen stress in podocyte-specific UCH-L1–deficient mice. Our previous work demonstrated a significant upregulation of UCH-L1 in kidney (particularly in podocytes) in human MN22Meyer-Schwesinger C. Meyer T.N. Munster S. et al.A new role for the neuronal ubiquitin C-terminal hydrolase-L1 (UCH-L1) in podocyte process formation and podocyte injury in human glomerulopathies.J Pathol. 2009; 217: 452-464Crossref PubMed Scopus (63) Google Scholar and in a rat model of MN.25Meyer-Schwesinger C. Meyer T.N. Sievert H. et al.Ubiquitin C-terminal hydrolase-l1 activity induces polyubiquitin accumulation in podocytes and increases proteinuria in rat membranous nephropathy.Am J Pathol. 2011; 178: 2044-2057Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar In order to evaluate the role of UCH-L1 in the course of immune-complex glomerulonephritis, we first analyzed whether and in which cells UCH-L1 was upregulated following induction of APN in 25-week-old wild-type mice. Quantification of UCH-L1 mRNA levels by qPCR (Figure 8a) demonstrated a significant upregulation of UCH-L1 transcript in whole kidney 14 days after induction of APN. Mirroring this finding, Western blotting exhibited increased UCH-L1 protein levels in whole kidney and in glomerular lysates (Figure 8b). Immunofluorescent analysis demonstrated a significant upregulation of UCH-L1 expression in tubulointerstitial cells of vascular and nonvascular origin, in atrophied and dilated tubuli, and in glomeruli. In glomeruli, UCH-L1 was specifically de novo upregulated in podocytes; additionally a prominent endocapillary UCH-L1 expression persisted (Figure 8c). In order to evaluate the role of UCH-L1 in immune-complex glomerulonephritis, we induced anti-podocyte nephritis in 20- to 25-week-old UCH-L1–deficient mice and control littermates. Survival of UCH-L1–deficient mice was significantly decreased following APN injection; the first mice died as early as 5 days following disease induction (Figure 9a). Of the surviving mice, levels of blood urea nitrogen were similarly elevated in control and UCH-L1–deficient mice treated with APN in comparison with pre-immune antibody treated control and UCH-L1–deficient mice (Figure 9b) suggesting acute renal failure. Basal glomerular filtration rate (GFR) measured by the FITC-sinistrin method28Schock-Kusch D. Sadick M. Henninger N. et al.Transcutaneous measurement of glomerular filtration rate using FITC-sinistrin in rats.Nephrol Dial Transplant. 2009; 24: 2997-3001Crossref PubMed Scopus (91) Google Scholar was comparable between control and UCH-L1–deficient mice and decreased slightly in the course of APN without significant difference between genotypes (Figure 9c). Light-microscopic evaluations exhibited tubulointerstitial injury with protein casts, tubular dilation, and tubular atrophy in APN-treated mice, which was significantly more pronounced in UCH-L1–deficient mice than in control littermates by semiquantitative scoring (Figure 9d and e). Immunofluorescence demonstrated a pronounced tubulointerstitial deposition of collagen type 4 in PI- and APN-treated UCH-L1–deficient mice (Figure 9f), whereas tubulointerstitial smooth muscle actin was only enhanced in APN-treated UCH-L1–deficient mice (Figure 9g and Supplementary Figure S2A) in comparison with APN-treated control littermates. Staining for kidney injury marker 1 (Kim1) (Figure 9h) and cleaved caspase 3 (Figure 9i and Supplementary Figure S2B), a marker for apoptotic cells exhibited injury of proximal and distal tubuli of APN-treated UCH-L1–deficient mice in comparison with APN-treated control littermates, with an apical Kim1 expression of tubular cells and apoptotic cells shed into the tubular lumen. Pre-immune antibody–injected mice did not show signs of tubular injury (Supplementary Figure S3). Overall, Kim1 and cleaved caspase 3 were not strongly expressed in the tubular cells of APN-treated mice. The strongest expression of cleaved-caspase 3 was in glomeruli of APN-treated UCH-L1–deficient mice. Anti-podocyte antibodies typicall