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Mitochondrial Autophagy Promotes Cellular Injury in Nephropathic Cystinosis

Poonam Sansanwal,* Benedict Yen,† William A. Gahl,‡ Yewei Ma,§ Lihua Ying,* Lee-Jun C. Wong,§ and Minnie M. Sarwal* *Department of Pediatrics, Stanford University, Stanford, California; †Department of Pathology, Veterans Affairs Medical Center, San Francisco, California; ‡Section on Human Biochemical Genetics, Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland; and §Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas

ABSTRACT The molecular and cellular mechanisms underlying nephropathic cystinosis, which exhibits generalized proximal tubular dysfunction and progressive renal failure, remain largely unknown. Renal biopsies from patients with this disorder can reveal abnormally large mitochondria, but the relevance of this and other ultrastructural abnormalities is unclear. We studied the ultrastructure of fibroblasts and renal proximal tubular epithelial cells from patients with three clinical variants of cystinosis: Nephropathic, intermediate, and ocular. Electron microscopy revealed the presence of morphologically abnormal mitochondria and abnormal pat- terns of mitochondrial autophagy (mitophagy) with a high number of autophagic vacuoles and fewer mitochondria (P Ͻ 0.02) in nephropathic cystinosis. In addition, we observed increased apoptosis in renal proximal tubular epithelial cells, greater expression of LC3-II/LC3-I (microtubule-associated protein 1 light chain 3), and significantly more autophagosomes in the nephropathic variant. The autophagy inhibitor 3-methyl adenine rescued cell death in cystinotic cells. Cystinotic cells had increased levels of beclin-1 and aberrant mitochondrial function with a significant decrease in ATP generation and an increase in reactive oxygen species. This study provides ultrastructural and functional evidence of abnormal mitophagy in nephropathic cystinosis, which may contribute to the renal Fanconi syndrome and progressive renal injury.

J Am Soc Nephrol 21: 272–283, 2010. doi: 10.1681/ASN.2009040383

Ϫ Ϫ Cystinosis is an inherited disorder caused by muta- cystine load in the Ctns / mouse kidney4 and pro- tions in the CTNS gene, which encodes cystinosin, a gressive renal injury occurs despite cystine depletion lysosomal transmembrane protein involved in cystine therapy. In untreated patients, with the generalized export to the cytosol.1 A large number of genetic vari- accumulation of cystine in various organs, patients ants have been characterized in CTNS; nevertheless, develop hypothyroidism, photophobia myopathy poor clinical correlation exists between genotyped and retinal blindness, chronic renal failure, pulmo- mutations and the different clinical phenotype of in- nary dysfunction, and central nervous system calcifi- fantile nephropathic cystinosis with proximal Fanconi cations and symptomatic deterioration.5 To improve tubulopathy2 and renal failure in the first decade of life3 or adult and ocular cystinosis, which have mild or Received April 9, 2009. Accepted October 18, 2009. 4 no renal involvement. The molecular and cellular ba- Published online ahead of print. Publication date available at sis of disease heterogeneity and mechanisms underly- www.jasn.org. ing renal Fanconi syndrome and renal tubulopathy Correspondence: Dr. Minnie Sarwal, Department of Pediatrics, are not well understood. Although cystine accumula- G306, 300 Pasteur Drive, Stanford, CA 94304. Phone: 650-723- tion is toxic to the milieu of the cells, renal injury in 7903 (nephrology), 650-498-5542 (transplant); Fax: 650-497-8614 (clinical), 650-723-4517 (Sarwal lab); E-mail: msarwal@stanford. nephropathic cystinosis may not simply be caused just edu or Dr. Poonam Sansanwal, Department of Pediatrics, G378, by cystine accumulation, because renal injury is not 300 Pasteur Drive, Stanford, CA 94304. Phone: 650-724-3320 seen either in other human forms of cystinosis or in (Sarwal lab); Fax: 650-723-4517; E-mail: [email protected] the murine cystinosis knockout model despite high Copyright ᮊ 2010 by the American Society of Nephrology

272 ISSN : 1046-6673/2102-272 J Am Soc Nephrol 21: 272–283, 2010 www.jasn.org BASIC RESEARCH

nal proximal tubular epithelial (RPTE) cells and skin fibroblasts extracted from patients with three clinical phenotypes of cystinosis— nephropathic (in which the Fanconi syndrome and renal failure is mandatory), intermediate (in which Fanconi syndrome and renal insuffi- ciency are mild and of later onset), and ocular (in which there is no renal involvement and corneal crystals are the main complaint)9—to explore the specific injury mechanism in ne- phropathic cystinosis. Our data demonstrate enhanced apoptosis, abnormal mitochondria, mitophagy, reduced mitochondrial ATP gener- ation, and increased ROS generation in nephro- pathic cystinosis. We further demonstrate that specific inhibition of autophagy results in sig- nificant attenuation of cell death in nephro- pathic cystinosis. Our findings suggest that mi- tochondrial autophagy may be a critical mechanism of renal injury in nephropathic cystinosis.

Figure 1. Morphologic anomalies in nephropathic cystinotic fibroblasts are shown. RESULTS (A and B) MitoTracker Red CMXROS staining reveals abnormal mitochondrial staining and distribution in cystinotic fibroblasts. Live cells (1 ϫ 106 cells/ml) were incubated Morphologic Analysis of Cystinotic with MitoTracker Red (50 nM) for 45 min under growth conditions, followed by fixation Fibroblasts by Fluorescence Imaging with 3.7% (vol/vol) formaldehyde. Mitochondria were visualized by confocal micros- copy, and the representative images are shown. (C and D) Immunostaining specific for With the aim to study the morphology of mi- a mitochondrial protein, ATP5H, revealed deteriorated mitochondrial morphology in tochondria in the nephropathic cystinotic cystinotic fibroblasts. Cells (1 ϫ 106 cells/ml) were fixed in 4% formalin, permeabilized, cells, we stained four nephropathic cystinosis and immunostained with antibodies for ATP5H. Slides were viewed by confocal fibroblast samples with MitoTracker Red and microscopy, and the representative images are shown. As seen, MitoTracker Red is analyzed them by confocal microscopy. Nor- already diffuse in the cytoplasm, suggesting loss of mitochondrial membrane poten- mal fibroblasts showed intense and definite tial in cystinosis. Magnifications: ϫ63 in A and B; ϫ100 in C and D. staining, whereas a diffused staining was ob- served in the cystinotic cells (Figure 1, A and on current treatment options for cystinosis, it is critical to under- B). Moreover, in the normal fibroblasts, mitochondria were stand the basis of generalized and multisystem tissue injury from organized as extended tubular structures, whereas, in the lysosomal cystine accumulation, as well as the specific cellular and cystinotic fibroblasts, the mitochondrial network was al- molecular injury mechanisms in the kidney that result in early tered to rounded, nonfilamentous, fragmented structure. Fanconi syndrome and subsequent renal failure, despite the use of We further verified the mitochondrial morphologic aberra- aggressive cystine depletion therapy.6 tions in nephropathic cystinotic cells by evaluating the pat- Autophagy is the process by which organelles and bits of cyto- terns of immunostaining for ATP5H, a mitochondrial plasm are sequestered and subsequently delivered to lysosomes membrane protein. The immunofluorescence (IF) images for hydrolytic digestion.7 Increasing evidence indicates that for ATP5H were in agreement with the MitoTracker images autophagy of mitochondria occurs selectively, and the term “mi- and clearly revealed a deteriorated fragmented mitochon- tophagy” has been suggested for this process.8 As a major source drial morphology in cystinosis as opposed to an intact fila- of reactive oxygen species (ROS), mitochondria are especially mentous tubular network in normal fibroblasts (Figure 1, C prone to ROS damage. Oxidative stress and various disease pro- and D). cesses cause mitochondrial damage and dysfunction. Timely elimination of aged and dysfunctional mitochondria is essential to Ultrastructural Morphologic Alterations in Cystinosis protect cells from the harm of disordered mitochondrial metab- Electron Microscopy of Cystinotic RPTE Cells. olism and release of proapoptotic proteins. The mechanism of To characterize better the morphologic aberrations of mi- mitochondrial turnover is predominantly autophagic sequestra- tochondria in cystinosis, we used electron microscopy (EM) tion and delivery to lysosomes for hydrolytic degradation. to image the ultrastructural alterations in cystinotic cells. We used individual samples of nephropathic cystinotic re- The renal proximal tubules are the major site of injury in

J Am Soc Nephrol 21: 272–283, 2010 Autophagy in Cystinosis 273 BASIC RESEARCH www.jasn.org nephropathic cystinosis; therefore, we performed EM on served structures with normal morphology (Figure 2B), RPTE cells obtained from patients with nephropathic cysti- whereas the cystinotic cells presented mitochondria with nosis and compared them with the normal RPTE cells. We condensed matrix (Figure 2C) and abnormally shaped dark analyzed eight nephropathic cystinotic RPTE cell cultures cristae (Figure 2D) in addition to the abundant AVs and by EM. Figure 2A represents low-power EM images showing autolysosomes (ALs; Figure 2, E and F). abundant autophagic vesicles (AV) in cystinotic cells. In Careful EM examination of cystinotic cells at a higher magni- normal cells, the mitochondria were present as well pre- fication revealed various stages of degradation of mitochondria by

Figure 2. Electron microscopic evaluation of cystinotic cells is shown. (A) EM of human normal and nephropathic cystinotic RPTE cells at low magnification. Arrows indicate AVs. (B through F) EM of normal RPTE (B) and nephropathic cystinotic RPTE cells (C through F). Abnormal mitochondria in cystinosis are shown by arrows in C through F and an inset in C. AVs and ALs are shown in E and F. Bars ϭ 1 ␮m. (G through N) Progressive mitochondrial autophagy in nephropathic cystinotic RPTE cells. Structurally abnormal mitochondria (G) are surrounded by typical double membrane of autophagosomes (H and I) or fuse with vacuoles (J and K), followed by fusion with lysosomes (L) to generate ALs (M and N). Bars ϭ 1 ␮m in G through J and L through N and 0.5 ␮m in K. (O) Quantification of mitochondria in cystinotic RPTE cells. Number of mitochondria per cell in cystinosis was significantly low as compared with normal cells, whereas no significant decrease was observed in CDME-treated normal cells. (P through S) Electron microscopy on normal human fibroblasts (P) and cystinotic fibroblasts (Q through S). Q and R insets show magnified version of abnormal mitochondria indicated by arrows. Inset in S shows magnified autophagosomes in cystinosis. Bars ϭ 2 ␮m. (T) Quantification of mitochondria in cystinotic fibroblasts. Number of mitochondria per cell in nephropathic cystinosis was significantly low as compared with normal cells, whereas no significant decrease was observed in CDME-treated normal cells. No significant change was observed in the number of mitochondria in intermediate and ocular phenotype compared with the normal controls. CDME-treated normal fibroblasts and RPTE cells did not exhibit the presence of AV, but morphologically abnormal mitochondria were observed. Only the representative EM images are shown. Magnifications: ϫ20,500 in B through J and L through N; ϫ44,000 in K; ϫ5900 in A and P through S.

274 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 272–283, 2010 www.jasn.org BASIC RESEARCH autophagy (Figure 2, G through N). Figure 2G demonstrates mor- correlates with the number of autophagosomes.14 LC3-II is the phologically abnormal mitochondria with dark cristae and con- only known protein that specifically associates with autopha- densed matrix. The abnormal mitochondria were often seen in gosomes and not with any other vesicular structures. We used close association with sequestering membranes during the initial this property of LC3 to monitor the dynamics of autophagy in stages of mitophagy (Figure 2H). The sequestering membranes cystinosis. This lipid-conjugated, final form of LC3, designated were observed to form a cup-shaped membranous structure that LC3-II, migrates faster than LC3-I in SDS-PAGE. Conse- eventually enveloped the aberrant mitochondria (Figure 2I). In quently, Western blot analysis with anti-LC3 antibody gives addition, mitochondria seemed to be closely associated with the two bands with different relative molecular weights (18 kD for vacuoles (Figure 2J). These vacuolar membrane formations have LC3-I and 16 kD for LC3-II). The intensity of the LC3-II previously been described to occur in mitophagy.10,11 Also, the bands, when compared with the control cells, was higher in all close contacts that looked like fusion between the vacuoles and of the nephropathic cystinotic RPTE and fibroblasts cell lysates mitochondria were observed (Figure 2K). The double-membrane that were analyzed by Western blot (Figure 3A, top). Densi- vesicles sequestering mitochondria, called autophagosomes, were tometry of LC3 bands, normalized to glyceraldehyde-3-phos- seen fusing with lysosomes to form ALs (Figure 2, L through N). phate dehydrogenase (GAPDH), in both fibroblasts and RPTE Selective degradation of organelles has been described to occur via cells demonstrated that there was a significant increase in the microautophagy or macroautophagy.12,13 Evidence of the vacuo- LC3-II/LC3-I ratio in the nephropathic cystinotic cells com- lar membranes fusing with mitochondria and a complete se- pared with the controls (P ϭ 0.039 and 0.021, respectively; quence of autophagosome formation engulfing mitochondria Figure 3A, bottom). Cystine dimethyl ester (CDME)-treated strongly suggest that both microautophagy and macroautophagy normal fibroblasts and RPTE cells did not exhibit any signifi- of mitochondria are taking place in cystinotic RPTE cells. Quan- cant difference in the levels of LC3-II (data not shown). This is tification of mitochondria per cell (Figure 2O) demonstrated a in agreement with the electron micrographs of CDME-treated significant decrease in number of mitochondria in nephropathic cells, which demonstrate the absence of AVs. cystinotic RPTE cells as compared with the normal cells (P ϭ Increased autophagy in cystinotic cells was further con- 0.016). firmed by Western blot for another autophagy marker, be- clin-1. This mammalian orthologue of the yeast Apg6/Vps30 EM of Cystinotic Fibroblasts. gene has a key role in autophagy.15–18 It regulates the autoph- We also performed EM on eight fibroblast cell cultures obtained agy-promoting activity of Vps3419 and is involved in the re- from patients with cystinosis (all three phenotypes) and com- cruitment of membranes to form autophagosomes. Beclin-1 pared them with the normal fibroblasts. We observed well-pre- can also bind to Bcl-2, an important regulator of apoptosis.20 served mitochondrial structures in normal fibroblasts (Figure As shown in Figure 3B, increased beclin-1 expression was ob- 2P), whereas giant (Figure 2Q) and spherical mitochondria with served in nephropathic cystinotic RPTE cells compared with abnormal cristae (Figure 2R) were present in nephropathic cysti- normal RPTE and HK-2 controls. These results demonstrate notic fibroblasts. EM also revealed the presence of numerous au- that beclin-1 is upregulated in cystinotic kidney and may be a tophagosomes in nephropathic cystinotic fibroblasts (Figure 2S). critical regulator of autophagy in cystinosis. Interestingly, we observed very few AVs and mostly morphologi- cally normal mitochondria in the fibroblasts from ocular and in- Evaluation of LC3 in Nephropathic Cystinotic RPTE Cells by IF. termediate phenotypes (data not shown). Quantification of mito- To assess the enhanced autophagy in cystinosis, we performed chondria (Figure 2T) demonstrated that there was a significant IF analysis to compare the distribution of endogenous LC3 in decrease in the number of mitochondria per cell in the nephro- four nephropathic cystinotic RPTE cell cultures. As expected, pathic cystinotic fibroblasts (P ϭ 0.0005) when compared with LC3 was mainly cytosolic with diffused staining in control the normal fibroblasts. cells, although occasional LC3-labeled autophagosomes could also be detected (Figure 3C). In contrast, numerous LC3-pos- Specific Mechanism of Cell Injury in Cystinosis itive structures with enhanced fluorescence and punctate Demonstration of Increased Autophagy in Cystinosis staining pattern were observed in cystinotic cells, suggesting Expression analyses of autophagy related proteins, LC3 and the translocation of LC3 to autophagosomes and thus indicat- beclin-1, were conducted to investigate the enhanced autoph- ing constitutive activation of basal autophagy in these cells agy in cystinosis. (Figure 3C). Quantitative analysis revealed that the LC3-posi- tive dots per cell were significantly greater in cystinotic cells Evaluation of Autophagy Markers LC3 and Beclin-1 in Cystinotic than in control cells (P ϭ 2.50E-06; Figure 3D). Altogether, our Fibroblasts and RPTE Cells by Western Blot. results indicate an enhancement of autophagy in cystinosis. To characterize further the enhanced autophagy in cystinosis, we analyzed the expression status of LC3 and beclin-1 in the Demonstration of Increased Mitophagy in Nephropathic Cysti- cystinotic cells. LC3 is a mammalian homologue of yeast nosis by IF Imaging. Apg8p, and during the formation of autophagosomes, the We further investigated mitophagy in nephropathic cystinotic LC3-I isoform is converted into LC3-II, the amount of which RPTE cells by immunostaining for LC3 (autophagy marker),

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Figure 3. Modification of endogenous LC3 and beclin-1 in nephropathic cystinosis indicating activated autophagy. (A) Fibroblasts and RPTE cell extracts were subjected to PAGE througha4to20%gel, and separated proteins were immunoblotted using a rabbit polyclonal anti-LC3 antibody and subsequently detected with goat anti-rabbit peroxidase and ECL reagent (Amersham Biosciences). The same immunoblots were stripped off and reprobed with a mouse monoclonal anti-GAPDH antibody. LC3 bands were normalized and quantified by Image J, and the LC3-II/LC3-I ratio was plotted. In fibroblasts, the increase in LC3-II signal was detected only in the nephropathic cystinotic fibroblasts and not in intermediate and ocular cystinotic fibroblasts. (B) Nephropathic cystinotic RPTE cell extracts were examined by Western blot specific for beclin-1 and compared with normal RPTE and HK-2 controls. The same immunoblot was stripped off and reprobed with a mouse monoclonal anti-GAPDH antibody. (C) Nephropathic cystinotic RPTE cells immunostained with anti-LC3 antibody and visualized under a confocal microscope revealed a vesicular punctate pattern in contrast to normal RPTE cells, which exhibit more diffuse cytosolic expression of LC3. Bar ϭ 47.62 ␮m. (D) Quantification of LC3 immunostaining was done by counting and averaging the LC3-positive dots per cell in at least five view fields per specimen under confocal microscope, suggesting enhanced autophagy in cystinosis. The figure is representative of multiple experiments. Magnification, ϫ63 in C.

LAMP2 (lysosomal marker), and ATP5H (mitochondrial showed a largely nonoverlapping pattern in normal control marker), as shown in Figure 4, A through F. Merged images of cells (Figure 4I). Cystinotic cells showed overlapping struc- individual immunostaining are shown in Figure 4, G through tures, indicative of the formation of ALs (Figure 4M). Also, N. We attempted to ascertain autophagy of mitochondria, as distribution of LAMP2 and ATP5H exhibited overlapping pat- observed in electron micrographs, by evaluating co-localiza- tern in cystinosis (Figure 4L), whereas no such co-localization tion of ATP5H and LC3. Our data indicated co-localization of was observed in the normal control cells (Figure 4H). Next, as LC3 and ATP5H in cystinotic cells, thereby confirming the evident in Figure 4, J and N, when the images for all three sequestration of mitochondria inside autophagosomes (Figure markers (LC3, LAMP2, and ATP5H) were merged, the cysti- 4K). Because autophagy is a constitutive cellular event, our notic cells exhibited much more enhanced co-localization as data indicated a basal co-localization of LC3 and ATP5H in compared with the control cells, indicating autophagy of mi- control cells (Figure 4G). When autophagy is induced, auto- tochondria in cystinosis. phagosomes fuse with lysosomes to form ALs, the site of ulti- To validate further mitophagy in nephropathic cystinotic mate degradation of their cargo; therefore, we examined the cells, we investigated the co-localization of ATP5H and LC3 co-localization of LAMP2 and LC3 to gain insight into this at a higher magnification. We immunostained the normal final step of autophagy. The ubiquitously expressed LAMP2 is and nephropathic cystinotic RPTE cells for LC3 and ATP5H localized primarily in the late endosomes and lysosomes.21 and captured the fluorescence images at a higher magnifi- This enrichment of LAMP2 in late AVs is similar to that ob- cation (ϫ100). We observed a weak and diffused cytoplas- served for LAMP1.22 The distribution of LC3 and LAMP2 mic signal of LC3 in normal control cells with a minimal

276 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 272–283, 2010 www.jasn.org BASIC RESEARCH

Figure 4. Fluorescence immunostaining for autophagic marker (LC3), lysosomal marker (LAMP2), and mitochondrial marker (ATP5H) in nephropathic cystinotic RPTE cells is shown. The intracellular distributions of LC3, LAMP2, and ATP5H were studied by indirect IF. (A through F) Cells (1 ϫ 106 cells/ml) were fixed in 4% formalin, permeabilized, and co-immunostained with antibodies for LC3, LAMP2, and ATP5H. (G through N) Localization patterns of LC3:LAMP2, LC3:ATP5H, LAMP2:ATP5H, and LC3:LAMP2:ATP5H are shown in nephropathic cystinotic and normal RPTE cells. A significant extent of co-localization is observed in cystinotic kidney cells. Bar ϭ 47.62 ␮m. (O through Q) High-power images for co-localization of mitochondrial marker (ATP5H, blue) with LC3 (red) in autophagosomes in cystinotic RPTE cells. (Q) White arrowhead in the zoomed image indicates a ring-like shape and a co-localized ATP5H-LC3 signal in cystinosis. Bar ϭ 30 ␮m. Magnification, ϫ63 in A through N; ϫ100 in O through Q. co-localization with ATP5H (Figure 4O). In contrast, LC3 complex II substrate (succinate) with complex I inhibitor, rote- signals in cystinosis were often found to be circular, consis- none. We detected a substantial reduction of ATP synthesis in tent with their localization on autophagosomal membrane cystinotic cells with complex I substrate (P ϭ 0.01), whereas ATP (Figure 4P). The merged images for LC3 and ATP5H in level measured in the presence of complex II substrate was not cystinosis (Figure 4Q) clearly indicated circular LC3 signals significantly different in cystinotic and control cells (Figure 5A). sequestering ATP5H, suggesting autophagy of mitochon- These results indicate a correlation of the observed reduction of drial structures in these cells. Thus, our fluorescence mi- ATP synthesis with an impairment of complex I activity in cysti- croscopy data confirmed enhanced autophagy of mitochon- notic cells. dria in nephropathic cystinotic RPTE cells. In mitochondria and submitochondrial particles as well as in

intact cells, respiration produces ROS such as H2O2 and superox- Functional Significance of Mitophagy in Nephropathic ide anion, especially when respiration is inhibited or otherwise Cystinosis disordered.23–26 Accordingly, We measured ROS generation in Mitochondrial Dysfunction in Cystinosis. the cystinotic cells. DCFH-DA was used for ROS detection. We further analyzed mitochondrial function by assaying the rate DCFH-DA is cleaved intracellularly by nonspecific esterases to of ATP synthesis and ROS production in nephropathic cystinotic form DCFH, which is further oxidized by ROS to form the fluo- RPTE cell cultures. Accordingly, we assessed electron transfer ca- rescence compound DCF.27 As shown in Figure 5B, ROS levels pacities of nephropathic cystinotic RPTE cells and measured ATP were significantly higher in the cystinotic cells (P ϭ 0.003). synthesis in the presence and absence of oligomycin, an inhibitor of mitochondrial ATP synthase, to evaluate mitochondrial ATP Autophagy is Pro-death in Nephropathic Cystinosis. production only. The ATP synthesis rate was measured in the Autophagy has also been more recently linked to the death presence of either complex I substrates (glutamate and malate) or process itself. Furthermore, apoptosis and autophagy are not

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the pathogenesis of nephropathic cystino- sis remains unclear. Thus, we sought pos- sible factors causing ATP depletion and perturbation of mitochondrial function, a potential cause for renal Fanconi in cysti- nosis. Here, for the first time, we describe a striking degree of mitochondrial autoph- agy specific to the nephropathic phenotype that could possibly be an important event leading to ATP depletion and renal Fan- coni with subsequent renal injury and cell death in nephropathic cystinosis. Abnormal autophagy and mitochon- drial aberrations were also recently ob- served in other types of lysosomal storage Figure 5. Determination of mitochondrial ATP synthesis rate and ROS generation is diseases.34 This is the first study in which shown. (A) Mitochondrial ATP synthesis rate was measured in the presence and involvement of autophagy in nephropathic absence of oligomycin. By providing glutamate and malate, ATP synthesis dependent cystinosis has been investigated. To date, on complexes I, II, III, IV, and V was stimulated. Using the complex I inhibitor rotenone, EM is the gold standard to monitor the for- succinate ATP production dependent on complexes II, III, IV, and V was stimulated. mation of autophagosomes, the morpho- Significantly decreased mitochondrial ATP generation was observed in nephropathic logic hallmark of autophagy. Here, EM cystinotic RPTE cells with complex I substrate. (B) ROS production was determined by showed an increased autophagic activity in dichlorodihydrofluorescein diacetate fluorescence, and the data are expressed as nephropathic cystinotic cells that displayed fluorescence (arbitrary units) per milligram of protein. ROS levels were significantly higher in the cystinotic cells when compared with the normal control cells. CDME- abundant vacuolization, a widely known treated control cells did not reveal any significant change in ATP synthesis and ROS morphologic indicator for autophagic cell generation. Experiments were conducted on four nephropathic cystinotic cells cultures, death. Indeed, Western blot and IF evalua- and the averages are plotted. tion of LC3-II, a widely used marker for autophagy, also revealed an increased ex- mutually exclusive pathways; they may act in synergy or pression only in the most severe35 nephropathic phenotype of counter to each other.28 To examine whether abnormal induc- cystinotic fibroblasts and RPTE cells. Vesicles of autophagic tion of autophagy correlated with decrease in cell survival in pathway appear as electron-dense dark bodies in electron mi- cystinotic cells, we measured the viability of four nephropathic crographs.36 Previously reported “mysterious” dark cells of cystinotic RPTE cell cultures in the presence and absence of unexplained origin that are unique to cystinotic kidneys may 3-MA, an autophagy inhibitor. The viability of cystinotic cells be due to accumulation of vesicular compartments of autoph- increased significantly when treated with 3-MA (P ϭ 1.35E-07; agic origin.37 Figure 6A). We further stained the 3-MA–treated and un- The next question we addressed was whether this abnormal treated cystinotic RPTE cells with Annexin V and analyzed induction of autophagy observed in the nephropathic cysti- them using flow cytometry. Cystinotic cells had a higher per- notic cells was specific to an organelle in the cells. Our EM and centage of Annexin V–positive cells (Figure 6B, top), with a IF co-localization data established that the mitochondria in significant reduction (approximately 14%) in apoptosis when cystinotic cells were specifically targeted to autophagy. Inter- rescued with 3-MA (Figure 6B, bottom). estingly, IF results exhibited higher levels of perinuclear stain- ing for LAMP2 in cystinosis. It was previously shown that LAMP2 may be involved in lysosomal biogenesis and/or the DISCUSSION fusion between autophagosomes and lysosomes required for the final catabolism of autophagic material.15,38,39 Thus, in- The pathogenesis of proximal renal tubular dysfunction and creased LAMP2 expression in cystinosis also suggests en- Fanconi syndrome in nephropathic cystinosis is poorly under- hanced active autophagy in these cells. Studies in mouse mod- stood. The most frequent renal symptom in mitochondrial cy- els of two LSDs showed that the autophagic delivery of bulk topathies is Fanconi syndrome, and why the proximal tubules cytosolic contents to lysosomes is impaired as a result of de- seem particularly sensitive to mitochondrial injury and its con- creased ability of lysosomes to fuse with autophagosomes34; sequences is not clear.29 An obvious answer is that it requires however, in the cystinotic cells, we observed that there is a high levels of energy in the form of ATP. In cystinosis, data significant degree of co-localization between lysosomes and from fibroblasts and animal models suggested decreased ATP autophagosomes as evident by LC3 and LAMP2 immunostain- levels but intact mitochondrial electron transport chain activ- ing of these cells. This suggests that fusion of AV and lysosomes ity30–33; however, the involvement and role of mitochondria in may be efficient in cystinosis.

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impact on the viability of the cells as a result of direct toxicity from CDME, independent of cys- tine accumulation.44,45 CDME is rapidly (within minutes) converted to cystine in lysosomes and leaves fairly rapidly from normal cells; therefore, drawing conclusions on cystinosis cellular injury on the basis of CDME-loading models may not be accurate. Our results suggest that the mitophagy observed in cystinosis may be independent of the effect of simply cystine accumulation in cystinotic cells. Increased apoptosis has been reported in cul- tured cystinotic fibroblasts and RPTE cells.40,42,46 Apoptosis is thought to involve the activation of caspases as well as a stereotyped pattern of mito- chondrial alterations, namely release of cyto- chrome c and mitochondrial membrane potential dissipation that contribute to the acquisition of the apoptotic morphology. Apoptosis rate deter- mined by caspases-3 activity in cystinotic RPTE cells has been reported to be significantly in- creased both with and without an apoptosis trig- ger.42 Basal level of apoptosis in cystinotic RPTE cells, as measured by Annexin V staining, was ob- served to be increased in this study as well. It has been shown that protein kinase C␦ forms disul- fide bonds specifically with cystine that is released from lysosomes in cultured cystinotic fibroblasts and RPTE cells during apoptosis.46 Also, loss of mitochondria integrity, as measured by cyto- chrome c release, has been shown to occur down- stream of lysosomal permeability in cystinotic cells.46 Accumulating evidence suggests that dif- Figure 6. Effect of autophagy on cell survival and apoptosis. (A) Nephropathic ferent intracellular organelles contribute syner- cystinotic RPTE cells and normal control RPTE cells were seeded in 12-well gistically to the initiation of apoptosis by specific plates overnight and then treated with 3-MA. After 8 h, viable cells were determined by Trypan Blue Exclusion assay. Percentage of viable cells increased stress inducers. It is evident from the electron mi- significantly in 3-MA–treated nephropathic cystinotic RPTE cells. The plot is crographs that the mitochondria in cystinotic representative of three independent experiments performed in triplicate of each cells are abnormal in structure and vary in size. sample. (B) Increase apoptosis in nephropathic cystinosis, with a reduction in The abnormal giant mitochondria have previ- apoptosis when treated with 3-MA, as seen by a significant decrease in the ously been reported in cystinotic kidney.47 It has percentage of Annexin V-FITC–stained cystinotic RPTE cells with 3-MA treat- been shown that there is a disorder in ATP gener- ment. Shown is one representative experiment of four cystinotic RPTE cell ation and ROS generation in cystinotic cells.30,31 cultures, each performed in triplicate. Our data show that the mitochondria in cysti- notic kidney cells are dysfunctional and indepen- Interestingly, EM and IF analysis of CDME-treated normal dent of the loading effect of cystine, with increased ROS and fibroblasts and RPTE cells exhibited the presence of morpho- deficient mitochondrial ATP generation, which may result logically abnormal mitochondria, but no AVs were observed, from an impairment of complex I activity in nephropathic which was also confirmed by LC3-II Western blot of CDME- cystinosis.48 Mitochondrial dysfunction is instrumental in treated cells. In fact, many dead cells were observed, indicating triggering apoptosis/autophagy.49 These alterations in mito- that CDME treatment was leading to cell death, as analyzed by chondrial function likely also result in endoplasmic reticu- EM and IF. Many studies have used a CDME in vitro model to lum (ER) stress or vice versa; ER stress was previously demon- study cystinosis in the past, for its ability artificially to load strated in various lysosomal storage diseases, including lysosomes with cystine30,40–43; however, the use of a CDME- nephropathic cystinosis.50 ER stress can further induce an au- loading model in studying pathogenesis of cystinosis can be tophagic response by stimulating the assembly of the preauto- questioned, because loading with CDME has a direct and acute phagosomal structures.51 Interestingly, it was recently shown

J Am Soc Nephrol 21: 272–283, 2010 Autophagy in Cystinosis 279 BASIC RESEARCH www.jasn.org that caspase-4 expression, which is known to be activated by (Woburn, MA), rabbit polyclonal anti-human GAPDH antibody ER stress, increases specifically in proximal tubules in cysti- purchased from Abcam (Cambridge, MA), rabbit polyclonal anti- notic kidneys52 and that ER stress induces autophagy in renal human beclin-1 antibody purchased from Cell Signaling Technology proximal tubular cells.53 Both lysosomal and mitochondrial (Beverly, MA) and mouse monoclonal anti-human ATP synthase destabilization may contribute to the initiation stage of apo- subunit d antibody purchased from Mitosciences (Eugene, OR). Sec- ptosis under ER stress. A number of studies revealed interest- ondary antibodies used were peroxidase-conjugated goat ant-rabbit ing cross-talk between the apoptotic and autophagic pathways, IgG and FITC-conjugated rabbit anti-mouse IgG purchased from with the identification of several proteins that can play a role in Jackson ImmunoResearch Laboratories (West Grove, PA). For co- both responses.54,55 Moreover, these pathways are not mutu- immunostaining, the secondary antibodies used were Alexa Fluor 555 ally exclusive; they may be both synergistic and antagonistic donkey anti-rabbit IgG, Alexa Fluor 647 donkey anti-mouse IgG, and under different conditions. On the basis of our data, autophagy Alexa Fluor 488 donkey anti-goat IgG purchased from Invitrogen. and apoptosis seem to be synergistic in nephropathic cystino- sis. Autophagy may act as an enabler of apoptosis, participating Electron Microscopy in certain morphologic and cellular events that occur during Cells were harvested gently using Trypsin EDTA, washed with PBS, the injury associated with nephropathic cystinosis. Specific fixed at 4°C for 2 h with 2% glutaraldehyde in neutral phosphate and selective inhibition of autophagy in vitro results in a sig- buffer, postfixed in osmium tetroxide, and embedded in Epon. Sec- nificant attenuation of apoptosis in nephropathic cystinosis. tions were cut at 80 nm, stained with lead citrate and uranyl acetate, Inhibition of autophagy may thus be an attractive target for the and examined under an FEI (Hillsboro, OR) Tecnai10 electron mi- attenuation of tissue injury and, specifically, renal injury in croscope. For quantification of mitochondria, the number of mito- nephropathic cystinosis. chondria per cell was scored. The electron micrographs obtained from multiple distinct low-powered fields (ϫ4200 to ϫ5900) were used to count the number of mitochondria per cell in at least five CONCISE METHODS different view fields for each cell culture sample, and the average num- ber of mitochondria per cell culture was calculated. Data are pre- Ϯ Cells sented as means SD. Ten primary fibroblast cultures were used for this study; two were form normal fibroblasts (GM02651 and GM00316; Coriell Cell Re- IF Imaging and MitoTracker Staining positories, Camden, NJ) and eight were from cystinosis patients at- MitoTracker Red CMXROS kit (Molecular Probes) was used for Mi- tending the National Human Genome Research Institute at the Na- toTracker Red staining of the cells. MitoTracker Red is a marker that tional Institutes of Health (Bethesda, MD). Eighteen RPTE cultures accumulates in the mitochondria regardless of the mitochondrial were used; one lot of normal primary RPTE cells (Cambrex Bio- membrane potential. Briefly, 1 ϫ 106 cells/ml cells were incubated in sciences, East Rutherford, NJ), the HK-2 cell line (American Type the prewarmed growth medium containing the MitoTracker Red Culture Collection, Manassas, VA), and 16 primary cultures from probe (40 nM) for 45 min. After staining, cells were washed with cystinotic RPTE cells isolated from the urine of patients with nephro- prewarmed growth medium without MitoTracker Red. The growth pathic cystinosis (a gift from Dr. Lorraine Racusen56). The study was medium was then replaced with freshly prepared 4% paraformalde- controlled by institutional review board approvals from the National hyde to fix the cells and incubated at 37°C for 20 min, after which the Institutes of Health and Stanford University. cells were rinsed several times with PBS and mitochondria were visu- The fibroblast cells were cultured in MEM with Earle’s salts, sup- alized at ϫ63 (oil) magnification using a Leica SP2 AOBS Confocal plemented with 15% FBS, 2 mM Lglutamine, 2ϫ concentration of Laser Scanning Microscope equipped with Leica software. nonessential AA, 100 ␮g/ml penicillin, 100 U/ml streptomycin, and For immunostaining of mitochondrial ATP5H protein, cells were 0.5 ␮g/ml Fungizone (all from Invitrogen Corp., Carlsbad, CA) at plated on chamber slides, washed twice in PBS, fixed in 4% formalin

37°C in a 5% CO2 atmosphere. The medium was changed every 3 d, (30 min), and permeabilized with 0.5% Triton X-100 (30 min). Cells and cultured cells were released by 0.05% Trypsin-EDTA (Invitrogen) were incubated in blocking buffer (PBS [pH 7.2] and 3% BSA) for 1 h, and passaged. All RPTE cells were cultured in renal epithelial growth then washed twice in PBS followed by incubation with the primary medium, made according to the manufacturer’s instructions (Cam- antibody for 2 h at room temperature. FITC-conjugated secondary brex). All cells were passaged with trypsin (0.05%) and were cultured antibody was used to detect bound primary antibody for1hatroom in a 95% air/5% CO2 Thermo Forma incubator (Waltham, MA) at temperature. Similarly, for LC3 immunostaining and co-immuno- 37°C. All of the experiments were performed between passages 2 and staining of LC3, LAMP2, and ATP5H, cells were processed and co- 5. For in vitro mimicking of cystinosis, the cells were treated with 1 immunostaining was performed by incubation with the primary an- mM CDME (Sigma) for 30 min.44 tibodies (LC3, LAMP2, and ATP5H) for2hatroom temperature followed by washing and incubation with secondary antibodies (Al- Antibodies exa Fluor 555, Alexa Fluor 488, and Alexa Fluor 647, respectively) for The primary antibodies used were goat polyclonal anti-human 2 h at room temperature. Slides were viewed using a Leica SP2 AOBS LAMP2 purchased from Santa Cruz Biotechnology (Santa Cruz, CA), Confocal Laser Scanning Microscope, and the images were analyzed rabbit polyclonal anti-human LC3 antibody purchased from MBL by Leica Confocal software (version 2.5). For quantification of au-

280 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 272–283, 2010 www.jasn.org BASIC RESEARCH tophagy in LC3-immunostained RPTE cells, the number of LC3-pos- cells. The rate of ATP synthesis was expressed as nanomoles of ATP pro- itive dots per cell was scored. The fluorescence images were used to duced per minute per milligram of protein. count the LC3-positive dots per cell in at least five different view fields for each cell strain, and the average number of dots per cell strain was Cell Viability Measurement calculated. Data are presented as means Ϯ SD. Cells were seeded in 12-well plates and then incubated in the presence or absence of 10 mM 3-MA (Sigma) for 8 h. Cell death was deter- mined by the Trypan blue exclusion assay. The cells were scraped and Protein Extraction and Immunoblotting resuspended in the Trypan blue solution (0.4%) and counted in a Cells were harvested and lysed in CellLytic-M reagent (Sigma) sup- hemacytometer under a light microscope, and percentage of viable plemented with protease inhibitor cocktail (Pierce) according to the cells was calculated. At least three independent experiments were con- manufacturer’s instructions. Cell extracts were quantified using ducted, and each sample was tested in triplicate. QuantiPro BCA Assay Kit (Sigma), and an equal amount of total protein was subjected to SDS-PAGE. Cell extracts were mixed with ϫ Flow Cytometry 6 Laemmli loading buffer and heated to 95°C for 5 min before Apoptosis of nephropathic cystinotic RPTE cells treated with or with- electrophoresis at 120 V for 1 to2hon4to20%SDS-polyacrylamide out autophagy inhibitor (3-MA) was assessed by flow cytometry, an- gels. For immunoblotting, proteins were transferred to Immobilon alyzing the Annexin V–stained cells (Annexin Apoptosis Detection polyvinylidene difluoride membrane (Millipore, Billerica, MA) for 75 Kit; Santa Cruz Biotechnology) with FACSCalibur (Becton Dickin- min at 20 V in a MiniProtean III transfer tank (BioRad, Hercules, son,) cytometry. Cells were seeded in six-well culture plates. After CA). Membranes were then air-dried before rewetting with 70% 15 h, the cells were treated with 10 mM 3-MA for 8 h. After harvesting, methanol followed by blocking with PBS supplemented with 0.1% the cells were washed, incubated for 15 min with Annexin V–FITC, (vol/vol) Tween-20 and 5% (wt/vol) milk. All primary antibody in- and analyzed by flow cytometry. The percentage of Annexin cubations were done in PBS supplemented with 0.1% (vol/vol) V–stained cells was assessed using the CellQuest Pro software (Becton Tween-20 and 1% (wt/vol) milk for a minimum of1hatroom tem- Dickinson). At least three independent experiments were performed, perature followed by washing with PBS-Tween (PBS supplemented and each sample was tested in triplicate. with 0.1% Tween). Peroxidase-conjugated secondary antibodies were diluted 1:10,000 in PBS-Tween, incubated with the blot for a mini- Statistical Analysis mumof1hatroom temperature, then washed with PBS-Tween and Unless otherwise indicated, data are presented as means Ϯ SD of three developed using ECL Plus detection reagent (Amersham). Band or more experiments. The t test was used to compare two means. quantification was performed using the ImageJ software (National Statistical significance was defined at the level of P Ͻ 0.05. Institutes of Health).

Determination of ROS ACKNOWLEDGMENTS The cells were seeded and grown in medium for 24 h in 24-well plates (2 ϫ 104/well). ROS production was measured after loading with 5 This work was supported by grants from Cystinosis Foundation Ire- ␮mol/L DCFH-DA for 30 min.57 After washing, cells were incubated land, Health Research Board Ireland, and the Intramural Research in medium without phenol red. Fluorescence measurements (excita- Program of the National Human Genome Research Institute, Na- tion and emission wave lengths of 485 and 535 nm, respectively) were tional Institutes of Health. carried out with a Tecan Infinite M200 (Mannedorf, Switzerland). We gratefully acknowledge Dr. Racusen’s gift of cystinotic RPTE Fluorescence values were normalized for protein concentration. cells for the study.

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