Keratins are novel markers of renal epithelial cell injury

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Keratins are novel markers of renal epithelial cell injury see commentary on page 738 Sonja Djudjaj1,2,7, Marios Papasotiriou1,3,7, Roman D. Bu¨low2, Alexandra Wagnerova2,5, Maja T. Lindenmeyer6, Clemens D. Cohen6, Pavel Strnad4, Dimitrios S. Goumenos3, Ju¨rgen Floege1 and Peter Boor1,2,5 1 Division of Nephrology, Rheinisch-Westfälische Technische Hochschule Aachen, Aachen, Germany; 2Institute of Pathology, RheinischWestfälische Technische Hochschule Aachen, Aachen, Germany; 3Department of Nephrology, University Hospital of Patras, Patras, Greece; 4Department of Internal Medicine 3 and Interdisziplinäres Zentrum für Klinische Forschung, Rheinisch-Westfälische Technische Hochschule Aachen, Aachen, Germany; 5Institute of Molecular Biomedicine, Comenius University, Bratislava, Slovakia; and 6Division of Nephrology and Institute of Physiology, University Zürich, Zürich, Switzerland

Keratins, the intermediate filaments of the epithelial cell cytoskeleton, are up-regulated and post-translationally modified in stress situations. Renal tubular epithelial cell stress is a common finding in progressive kidney diseases, but little is known about keratin expression and phosphorylation. Here, we comprehensively describe keratin expression in healthy and diseased kidneys. In healthy mice, the major renal keratins, K7, K8, K18, and K19, were expressed in the collecting ducts and K8, K18 in the glomerular parietal epithelial cells. Tubular expression of all 4 keratins increased by 20- to 40-fold in 5 different models of renal tubular injury as assessed by immunohistochemistry, Western blot, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The up-regulation became significant early after disease induction, increased with disease progression, was found de novo in distal tubules and was accompanied by altered subcellular localization. Phosphorylation of K8 and K18 increased under stress. In humans, injured tubules also exhibited increased keratin expression. Urinary K18 was only detected in mice and patients with tubular cell injury. Keratins labeled glomerular parietal epithelial cells forming crescents in patients and animals. Thus, all 4 major renal keratins are significantly, early, and progressively up-regulated upon tubular injury regardless of the underlying disease and may be novel sensitive markers of renal tubular cell stress. Kidney International (2016) 89, 792–808; http://dx.doi.org/10.1016/ j.kint.2015.10.015 KEYWORDS: chronic kidney disease; renal pathology; tubular cells ª 2016 International Society of Nephrology

Correspondence: P. Boor, Institute of Pathology and Division of Nephrology, University Hospital Aachen, Pauwelsstrasse 30, D-52074 Aachen, Germany. E-mail: [email protected] 7

MP and SD contributed equally to this study.

Received 9 March 2015; revised 25 September 2015; accepted 22 October 2015; published online 6 February 2016 792

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ubular epithelial cells (TECs) are the most abundant, highly differentiated renal cells essential for normal kidney function. TEC injury is a common finding in a multitude of renal diseases and a critical determinant of a progressive disease course and development of renal fibrosis.1 TECs react sensitively to stress by profound and uniform phenotypic changes (termed tubular atrophy), which are found in virtually all biopsies of patients with chronic kidney diseases with fibrosis. These changes include cell simplification with loss of epithelial features and functions and de novo expression of mesenchymal markers accompanied by remodeling of the cell cytoskeleton.2–4 Keratins are the largest subgroup of intermediate filaments that constitute the cytoskeletal component of most eukaryotic cells.5 Of the 54 known human keratins, 24 are nonhair keratins, and these are further subdivided into type I (K9–K28) and type II (K1–K8; K71–80) keratins. Keratins are expressed in an organ and epithelial cell-specific manner, which renders them excellent markers in diagnostic pathology.6 Keratins are obligatory heteropolymers, in other words, for the assembly of mature filaments, at least 1 type I and 1 type II keratin in a 1:1 stoichiometry must be expressed in a cell.7 Within the cell, they tether the nucleus, span through the cytoplasm and are attached to the cytoplasmic plaques of the epithelial cell–cell junctions called desmosomes. Keratins are involved in the maintenance of cell shape, mechanical stability, locomotion, and intracellular organization and transport.8 Beyond their role as cytoskeletal proteins, keratins were proposed to act as cellular stress proteins, resembling the stress-induced heat-shock proteins.9,10 Keratins are up-regulated and functionally involved in various disease states, such as in skin diseases, cancer, and in multiple experimental animal models of liver and pancreas injury.7,11,12 Keratin mutations have been linked to at least 60 different human disorders.10 Apart from the use of K7 in pathological diagnostics of renal cell carcinoma subtypes, surprisingly little is known about their role in the kidneys. The currently available data suggest that in healthy human kidneys 4 keratins are mainly expressed—K7, K8, K18, and K19. K8 and K18 are constitutively expressed in all epithelial cells of the nephron including the parietal epithelial cells of Kidney International (2016) 89, 792–808

S Djudjaj et al.: Keratins in renal diseases

the Bowman capsule, whereas K7 and K19 are localized to the parietal epithelial cells, the thin limbs of Henle, and the collecting ducts.13,14 The regulation of keratin expression during human kidney injury is largely unknown. The only 2 available studies suggested that keratins might be both up-14 and down-regulated15 during injury, and no data are available in animal models. Here, we analyze the expression and regulation of keratins in healthy and diseased murine kidneys and in human renal biopsies.

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RESULTS Keratin expression in healthy mouse kidneys

In healthy glomeruli, parietal epithelial cells expressed K8 and K18, but in contrast to human renal tissue,13,14 no K7 or K19 expression was found (Figure 1a–d). In the tubulointerstitium, the expression of the 4 keratins was confined to TECs in all kidneys examined and was restricted to cortical and medullar collecting ducts with no obvious expression in other parts of the tubules (Figure 1e–l). Double

Figure 1 | Keratin expression in murine normal renal tissue. Parietal epithelial cells of the Bowman capsule were (a,b) positive for K8 and K18 (arrowheads), whereas (c,d) K7 and K19 were not expressed in glomeruli or parietal epithelial cells. (e–h, respectively) K8, K18, K7, and K19 were only expressed in the collecting ducts (thin arrows), whereas proximal and distal tubules were negative (thick arrows). (i–l) A more prominent expression in the medulla was found, which is consistent with higher porportions of collecting ducts. (m) K8 and K18 were colocalized in renal tubular epithelial cells. As exemplified for (n) K18 and (o) K7, colocalization with aquaporin-2 (AQP2), as a specific marker of collecting ducts, confirmed their localization only in collecting ducts in healthy animals. A subpopulation of single cells within the collecting ducts was found to be negative for keratin expression in healthy kidneys. (p) Costaining with ATP6V1B1 indicated that the keratin-negative cells are intercalated cells. (a–l) Bars ¼ 20 mm; (m–p) bars ¼ 30 mm. Kidney International (2016) 89, 792–808

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immunofluorescence staining with aquaporin-2, a marker for collecting ducts, confirmed the restricted expression of keratins in the collecting ducts (Figure 1m–o). A rare subpopulation of single cells within the collecting ducts was found to be negative for keratin expression in healthy kidneys. Costaining with ATP6V1B1 indicated that these keratin-negative cells are intercalated cells (Figure 1p). Regulation and cellular localization of keratins in UUO

Next, keratin regulation was analyzed in a widely used model of primary obstruction-induced tubulointerstitial fibrosis, the unilateral ureteral obstruction (UUO), at the initial stages (days 1 and 3), fully established disease (days 5 and 7), and an advanced stage with marked tubular atrophy and nephron loss (day 10). Representative histology (periodic acid–Schiff staining) confirmed the expected injury pattern and tubular cell damage (Supplementary Figure S1). Tubular expression of all 4 keratins was significantly up-regulated already on the first day after UUO induction and continued to increase until the seventh day with an up to 25-fold increase in percentage of keratin-positive area (Figure 2a–j). The expression was mainly found in the collecting ducts. Even in advanced disease stages, that is, on day 10, keratin expression remained highly up-regulated compared with the contralateral kidneys or early stages. Quantification of keratin-positive area in single tubular cells showed a significant increase in cytoplasmic area occupied by keratins in UUO compared with healthy kidneys, suggesting an increase in keratin expression in single cells (Figure 2k–o). In healthy murine renal tissue, all keratins were localized mainly on the basolateral side of the tubular cells in the medulla and also apicolateral in cortical collecting ducts (Figure 2p). Already early after UUO, keratin localization changed to a circumferential and in part cytoplasmic pattern, excluding the nuclei (Figure 2p). This altered localization was confirmed with laser scanning microscopy (Supplementary Figure S2). The rare subpopulation of keratin-negative cells seemed to increase numerically during the disease (Figure 2p marked with an arrowhead). The importance of this finding is currently not clear. Staining for the tubular cell injury marker lipocalin 2/neutrophil gelatinase-associated lipocalin (NGAL), which is not expressed in healthy kidneys (Figure 3a0 ), showed that during injury all NGALþ cells expressed K18, but only a subset of K8þ cells expressed NGAL (Figure 3b0 ). Immunofluorescence staining with tubular-specific markers confirmed the up-regulation of keratins in collecting ducts. Albeit weaker, it was also observed de novo in distal tubules (Figure 3c–h). Using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analyses, all 4 keratins were significantly, early, and progressively up-regulated up to 40-fold, closely resembling and confirming the data obtained by immunohistochemistry (Figure 4a-d). Western blot analyses of high salt extracts, representing the insoluble and thereby the most abundant form of keratins, further confirmed our data showing an up-regulation of all 4 keratins up to 30-fold (Figure 4e–i). 794

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Regulation of keratins in different models of renal diseases

As for UUO, representative histology (periodic acid–Schiff staining) showed the expected tubular cell injury in all models analyzed (Supplementary Figure S1). In a model of crystalinduced renal injury, that is, adenine nephropathy, an early (day 5), established (day 14), and advanced (day 28) disease stages were analyzed. Compared with healthy control animals, all keratins were significantly up-regulated already on day 5, continued to rise until day 14 with an up to 20-fold increase, and remained stable thereafter as shown with immunohistochemistry, qRT-PCR and Western blot (Supplementary Figure S3A–Q). The latter was performed in total protein lysates, which include both the insoluble and soluble forms of keratins, and the results closely resembled those obtained by immunohistochemistry and qRT-PCR as well as Western blot of insoluble keratin fraction (high-salt extracts) in UUO. Similar to UUO, a de novo expression in distal tubules and an altered subcellular localization occurred (Supplementary Figure S3A–D). Twenty-one days after unilateral ischemia-reperfusion injury, tubular expression of keratins was also significantly increased up to 4-fold in immunohistochemistry (Supplementary Figure S4A–H), and this finding was confirmed by qRT-PCR and Western blot of total protein lysates (Supplementary Figure S4I–Q). In a model of toxic tubular injury, that is, folic acid nephropathy, compared with healthy mice, diseased kidneys had a significant and up to 16-fold increased positive-stained area of all 4 keratins on protein level using immunohistochemistry or Western blot of total protein lysates (Supplementary Figure S5A–L and Q–U), and up to 10-fold increase on mRNA level (Supplementary Figure S5M–P). Finally, we analyzed a model of progressive glomerulonephritis with proteinuria and secondary tubulointerstitial injury with fibrosis, namely the Col4a3-deficient (Alport) mice. We analyzed the early stage of Alport mice at the age of 4 weeks and the late stage at the age of 8 weeks. At the early stage we did not observe any obvious histological damage of the tubuli nor any inflammation or fibrosis (Supplementary Figure S1). In line with this, we found only a slight increase in keratins at this stage and only K7 showed significant increase (Supplementary Figure S6A–H). As in all of the abovementioned models, a significant increase in expression of all 4 keratins was found in late stage Alport mice that displayed prominent tubular injury (Figure 5a–u). Analyses of keratin expression in publicly available datasets

The analyses of available microarray data (European Bioinformatics Institute, Cambridge, UK; http://www.ebi.ac.uk) from normal murine kidney tissues revealed a basal expression of the 4 renal keratins we analyzed, in other words, the expression was above the cutoff value of 0.5. The analyses of datasets comparing differential gene expression between healthy and diseased kidneys showed a constant up-regulation of all 4 keratins during disease in all datasets identified (Supplementary Figure S7). Kidney International (2016) 89, 792–808

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Figure 2 | Keratin expression, quantification, and intracellular localization after unilateral ureteral obstruction (UUO). (a) Compared with healthy kidney tissue, (b) a gradual up-regulation of K8 tubular expression already on day 1 of UUO was observed, which further (c,d) increased thereafter and (e,f) peaked between day 7 and day 10. At day 10, albeit weak, de novo K8 expression in tubuli other than collecting ducts was found (K18, K7, and K19 showed the same staining pattern as K8 in all analyzed time points, pictures not shown). (g–j) Quantification by morphometric image analysis confirmed these findings for K8, K18, K7, and K19. The peak of keratin expression was consistently found on day 7 of UUO. (k–o) Quantification of keratin-positive area in single tubular cells as outlined with the dashed line in image o showed a significant increase in cytoplasmic-positive area compared with healthy kidneys, suggesting an increase in keratin expression in single cells. (p) K8, K18, K7, and K19 were localized mainly at the basolateral side of epithelial cells in healthy murine kidneys (arrow). All 4 keratins, exemplified for K18 and K19, showed an up-regulation of expression with a shift to circumferential (arrows) and cytoplasmic distribution after UUO-mediated injury. A subpopulation of dispersed cells (arrowheads) within the collecting ducts was negative for K18 and K19 in injured collecting ducts. (a–f) Bars ¼ 50 mm; (k–m) bars ¼ 15 mm. *P < 0.05, ***P < 0.001.

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Figure 4 | Confirmation of keratin up-regulation. Compared with untreated kidneys, substantial up-regulation of (a) Krt8, (b) Krt18, (c) Krt7, and (d) Krt19 mRNA expression was found as soon as day 1 of unilateral ureteral obstruction (UUO). Keratin mRNA expression continued to increase during the progression of the UUO model. Compared with healthy kidneys, (e) Western blot analysis and quantification confirmed the increased expression of (f–i) K8, K18, K7, and K19 in kidneys from mice with UUO at day 5. HSE, high-salt extraction. *P < 0.05, **P < 0.01, ***P < 0.001.

Phosphorylation of keratins

To analyze a major post-translational modification of keratins, that is, phosphorylation, a serine-34 phospho-epitope– specific keratin 18 (K18 pS34) immunofluorescence showed only very weak phosphorylation in healthy kidneys (Figure 6a–c), whereas a striking increase in injured tubuli was observed in diseased UUO kidneys (Figure 6d–f). Interestingly, some tubular cells showed a strong and nearly complete colocalization of K18 and the phosphorylated K18, whereas other neighboring tubular cells showed virtually no phosphorylation (Figure 6d’–f ’). Increase of phosphorylation

was independent of initial injury and similar and significant in all models (Figure 6g–m). Virtually no phosphorylation on serine-74 of K8 (K8 pS74) was observed in healthy kidneys. After injury, K8 phosphorylation was increased, but was less pronounced compared with K18 phosphorylation and showed only few scattered positive cells (Figure 6n–q). Keratin expression in normal human kidneys

As described previously,14 our data confirmed the human expression of K8 and K18 within all tubular cells of the

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Figure 3 | De novo expression of keratins in distal tubules. In healthy murine kidneys, K18 was expressed only by (a) collecting ducts. (a0 ) Tubular injury marker neutrophil gelatinase-associated lipocalin (NGAL) was not detectable as also seen in (a00 ) merged picture. During unilateral ureteral obstruction (UUO), (b) K18 expression increased and (b0 ) NGAL was de novo expressed by injured tubules as also shown in (b00 ) merged pictures. K18 was not only expressed by injured NGAL-positive tubules (arrowheads), but also in tubules, which were negative for NGAL (thin arrow). In healthy kidneys all four keratins, here exemplified for K18, were not expressed by (c-c0 ) proximal or (d-d0 ) distal tubules and were only localized in (e-e0 ) the collecting duct (arrowhead). (f–h) After UUO, keratins were not only up-regulated in single cells of collecting ducts (arrowhead in h) but de novo expressed by some distal tubules (arrowhead in g and thin arrow in h). (g) Other distal tubules were still negative for keratins (thin arrows). (c0 –h0 ) show higher magnification of (f–h). Bars ¼ 50 mm. AQP2, aquaporin-2; d, day; THP, Tamm-Horsfall protein. Kidney International (2016) 89, 792–808

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nephron, whereas the pattern of K7 and K19 expression was more restricted. The keratins colocalized with each other, in particular in the collecting ducts as shown using the specific marker aquaporin-2 (Figure 7a–c). Whereas K7 and K19 were mostly confined to the collecting ducts, K8 and K18 was found in all human tubular segments as exemplary shown for the colocalization of proximal tubules marker CD13 with K18 (Figure 7d–f). Immunohistochemistry for K8 combined with periodic acid–Schiff staining further confirmed these findings (Figure 7g–i). Keratin expression in human renal diseases

Keratin expression was analyzed in renal biopsies from patients with multiple myeloma (cast nephropathy; n ¼ 9) as a disease with primary tubular injury, and in diabetic nephropathy (DN; n ¼ 5) and systemic lupus erythematosus (n ¼ 8), which might have both primary and secondary tubular injury. We found an increased intensity of the staining of all 4 keratins in the majority of tubules, in particular in those with injury and myeloma casts. Of note, the tubular casts were also positive for keratin staining (Figure 8m–p). The quantification of staining intensity was somewhat challenging due to much more abundant basal keratin staining in humans than in mice. Nonetheless, positively stained keratin area was increased in patients compared with healthy control subjects with most prominent increase in cast nephropathy (Figure 8q–t). To further confirm this finding, we analyzed the mRNA expression of keratins in microdissected cortical tubulointerstitium from renal biopsies of patients with cast nephropathy (n ¼ 15), systemic lupus erythematosus (n ¼ 13) or DN (n ¼ 9) and compared them with healthy living donors used as control subjects (n ¼ 6). KRT7 and KRT18 were significantly increased in DN and systemic lupus erythematosus. Although none of the results for KRT8 and KRT19 reached statistical significance, we observed a trend toward increased rather than decreased mRNA expression in DN and systemic lupus erythematosus patients (Figure 8u–x). Keratin expression in parietal epithelial cells and crescents

In the glomeruli of healthy human kidneys, most parietal epithelial cells in the Bowman capsule were positive for all 4 keratins, whereas no cells of the glomerular tufts were positive (Figure 9a–d). Patients with crescentic necrotizing glomerulonephritis exhibited increased keratin positivity of parietal epithelial cells and cells forming the crescents (Figure 9e–h). The majority of cells forming the crescents were positive for K8 and K18 with an overlapping staining pattern, whereas only a subset of these cells were positive for K19 and even

fewer for K7 (Figure 9e-h). Cellular crescents also developed in the Alport mice and cells forming these extracapillary proliferative lesions showed strong positive staining for K8 and 18, but not K7 and 19 (Figure 9i–l). Urinary keratin 18

To assess the potential of keratins as markers of tubular injury, K18 in urine samples of mice and humans with renal injury was analyzed using Western blot. In urine of healthy animals, no K18 was detectable, whereas all animals with adenine nephropathy or Alport mice showed detectable K18 bands, the full-length K18 (48kD) and a shorter fragment (28kD), the latter being more pronounced (Figure 10a). Similar findings were observed for the established tubular injury marker NGAL, albeit K18 seemed to be more prominent, in particular the 28kD fragment. Healthy human volunteers showed no detectable urinary K18 bands, whereas patients with acute kidney injury showed bands of both full-length and more prominently also of the 28kD fragment of K18, closely resembling data from mice (Figure 10b). Urinary NGAL behaved similarly to urinary K18, albeit slightly less pronounced and not detected in 1 acute kidney injury patient. DISCUSSION

The key novel finding of this study is that all major renal keratins, K7, K8, K18, and K19, are significantly up-regulated upon TEC injury. This up-regulation occurs already very early during disease, is common to all types of stress models, further increases with disease progression, and is reflected by increased urinary keratin levels, which might serve as a noninvasive biomarker of tubular cell stress. As early as day 1 of the UUO and day 5 in the adenine nephropathy model, when no or only minor damage or fibrosis is observed histologically, keratins were already highly up-regulated. Thus, keratins might serve as early and sensitive markers of TEC injury. As the disease and the kidney injury progressed, the expression of keratins further increased. This suggests that the extent of keratin up-regulation correlates with the extent of injury. We have used different progressive kidney injury models with very distinct mechanisms of induction and tubular cell stress reflecting a great number of human kidney diseases. In all of these models, the upregulation of all 4 keratins occurred in a stereotypical manner and remained high even in advanced disease stages, characterized by marked parenchymal atrophy and tubular loss. Our data are in line with previous studies showing that K8 and K18 but also K19 are up-regulated in various liver diseases.9,16 The keratin up-regulation in murine models of

= Figure 5 | Keratin expression increased in Alport mice with progressive glomerulonephritis. K8, K18, K7, and K19 immunohistochemical tubular expression in (a–d) healthy control animals in comparison to (e–h) Alport kidneys and (i–l) their quantification showed a significant increase of K8, K18, K7, and K19 expression in Alport kidneys, compared with healthy control animals. (m–p) Significant up-regulation of Krt8, Krt18, Krt7, and Krt19 mRNA expression was found in Alport mice in comparison to wild-type control mice. Compared with healthy kidneys, (q) Western blot analysis and (r–u) quantification confirmed the increased expression of K8, K18, K7, and K19 in kidneys from Alport mice. Bars ¼ 50 mm. *P < 0.05, **P < 0.01, ***P < 0.001. Kidney International (2016) 89, 792–808

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Figure 6 | K18 phosphorylation at serine 34 and K8 phosphorylation at serine 74 were increased in all models. In healthy mice, (a) normal expression of K18, and (b) virtually no positive staining for K18 at serine 34, was observed (c shows merging of a and b, and a0 –c0 show higher magnification of a–c). During unilateral ureteral obstruction (UUO), (d) a substantial up-regulation of K18 and (e) hyperphosphorylation at serine 34 K18 was found (f shows merging of d and e, and d0 –f0 show higher magnification of d–f). (f) Some cells showed no phosphorylation, whereas others showed a significant marked hyperphosphorylation (arrowhead). (m) Phosphorylation of K18 increased significantly and independently from the initial injury (g and g0 ) in comparison to healthy animals after (h,h0 ) ischemia-reperfusion (I/R) day 5, (i,i0 ) in folic acid nephropathy (FAN) day 28, (j,j0 ) 8-week-old Alport mice, and in adenine nephropathy (k and k0 ) day 5 and (l and l0 ) day 28. Albeit weaker, K8 showed de novo phosphorylation at S74 in (o–q) all analyzed models compared with (n) healthy control subjects. (a–f) Bars ¼ 20 mm; (g–q) bars ¼ 50 mm. *P < 0.05. d, day. 800

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Figure 7 | Keratin expression in normal human tubuli. Consecutive sections from healthy renal tissue showed a colocalization of K8 with (a) K19 and (b) K7. (c) Collecting duct specific-marker aquaporin-2 ([AQP2], arrow) confirmed the localization of K19 in collecting ducts. Similarly, AQP2-positive collecting duct (arrowhead in d and e) co-localized with (d) K7 and (e) K18. Note the expression of K18 also in AQP2-negative proximal and distal tubules. (f) This was further confirmed by co-localization of K18 with CD13 as a marker of proximal tubules. (g–i) Immunohistochemistry for K8 combined with periodic acid–Schiff staining further confirmed a strong expression in the collecting ducts (thin arrows in g and i) and a lower expression in proximal tubules (thick arrow in g). Of note, parietal epithelial cells were also K8-positive (arrowheads in h). Bars ¼ 20 mm. DAPI, 40 ,6-diamidino-2-phenylindole.

kidney injury was due to increased number of cells expressing keratins, as shown by de novo keratin expression in distal tubules, but possibly also due to increased expression in cells of the collecting ducts, as shown by the analyses of single-cell keratin expression. The protein and mRNA data showed very similar increase in all models, albeit some differences in the extent of keratin up-regulation were found, suggesting possible involvement of translational regulation of keratin expression. Collectively, these data suggest that keratins might serve as sensitive and early markers of renal tubular injury. Although preliminary, our data in both animals with primary and secondary tubular damage and patients with acute kidney injury suggested that urinary K18 might be a potential and specific marker of tubular cell injury. Confirmatory to our results, compared with healthy control subjects, total serum K18 concentration increased in patients with chronic kidney disease (stages 3–5) and was also significantly Kidney International (2016) 89, 792–808

increased in the urine in advanced stages (chronic kidney disease stage 5).17 In fact, serum K8, K18, and K19 fragments are established tumor and liver injury markers.10 Whether urinary keratins might prove diagnostically useful will, however, require specifically designed clinical trials. The transcriptional regulation of keratins has not been studied in great detail, but nuclear factor kB, tumor necrosis factor-a, and interleukin-6 were suggested as possible regulators of keratin expression and were shown to be upregulated early in renal disease models.9 Whether they might be responsible for keratin up-regulation during tubular cell injury or whether other factors, such as biomechanical forces are involved,9 remains to be delineated. The second major finding is that upon injury, keratins showed an altered cellular and intracellular localization. In physiological conditions, keratins were localized basolaterally in collecting ducts and this changed to a circumferential and 801

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Figure 9 | Keratin expression in the parietal epithelial cells in healthy human glomeruli, in patients with rapidly progressive glomerulonephritis (RPGN) with crescents and in Alport mice with crescents. (a–d) Weak expression of K8, K18, K7, and K19 was found in parietal epithelial cells in healthy human renal tissue (arrows). (e–h) A marked increase in K8, K18, K7, and K19 expression was found in parietal epithelial cells and cells forming the glomerular crescents of patients with RPGN (arrows). Whereas most of the cells forming the crescents were similarly positive for K8 and K18, only a subset of cells expressed K19 and even less cells expressed K7. In Alport mice, (i,j) crescents were positive for K8 and K18, whereas (k,l) no expression of K7 or K19 was found. Bars ¼ 50 mm.

cytoplasmic pattern upon injury. Similarly, upon pancreatic injury, the up-regulation of K19 and K20 was accompanied by their incorporation into cytoplasmic filaments, whereas they revert to the typical apicolateral distribution upon healing.7 It was suggested that K8 and K18 localize preferentially to cytoplasmic filaments, whereas K7 and K19 are primarily found in the membrane-proximal apicolateral compartment.18 However, we did not find any differences in intracellular localization between all analyzed keratins neither in healthy nor in diseased kidneys. In all our murine animal models, we observed a de novo and cytoplasmic expression of keratins in proximal and distal tubuli. This is in accordance with findings in human renal tissue, where compared with physiological conditions, during injury, epithelial cells may

express de novo different keratin chains, for example, K7 and K19 in injured proximal tubular cells.13,14 Such de novo expression might be particularly useful when considering keratins as markers of cell stress. During specific disease conditions, keratins might form intracellular inclusions such as the Mallory-Denk bodies in the liver or in renal cell carcinoma.19,20 However, in all of the non-neoplastic kidney diseases analyzed in mice and humans, we did not observe any keratin inclusions in tubular cells. The third novel finding is that during kidney injury, keratins became highly phosphorylated, at least at the serine-34 of K18. Similarly, we found virtually no phosphorylation of K8 at serine-74 in healthy kidneys, whereas it was detected in various models of tubular injury, albeit less prominently than

= Figure 8 | Keratin expression in normal and diseased human renal tissue. (a–d) K8, K18, K7, and K19 expression in healthy kidneys showed a typical tubular pattern. (a–t) Quantification of immunohistochemistry for keratins in lupus nephritis, diabetic nephropathy (DN), multiple myeloma showed an up-regulation of all 4 keratins. In patients with multiple myeloma cast kidneys, (m) K8 and (p) K19 showed an increased tubular expression. In addition, the intratubular casts were also positive (arrow in m and p). (s) Due to lack of available tissue for serial sections, we were not able to analyze K7 in systemic lupus erythematosus (SLE). (u–x) Analyses of mRNA expression of KRT8, KRT18, KRT7, and KRT19 in microdissected tubulointerstitium from human renal biopsies of healthy living kidney donors (LD), SLE, DN, and cast nephropathy (Cast) showed an increased expression in all diseases, however, due to variability reaching statistical significance only for KRT18 and KRT7. Bars ¼ 20 mm. *P < 0.05, **P < 0.01. NA, not analyzed. Kidney International (2016) 89, 792–808

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Figure 10 | Urinary K18 constitutes a potential marker of kidney injury. (a) Western blot analyses of urine of healthy animals showed no K18, whereas all animals with adenine nephropathy or Alport mice showed detectable K18 bands corresponding to the full-length K18 (48kD) and a shorter fragment (28kD). Similar findings were observed for tubular injury marker neutrophil gelatinase-associated lipocalin (NGAL). (b) In urine of healthy human volunteers no K18 was detectable, whereas in patients with acute kidney injury (AKI) bands of both full-length and even more prominently of the 28kD fragment of K18 were clearly detectable. Urinary NGAL was similar to urinary K18, albeit slightly less pronounced and not detected in one AKI patient.

K18. A wide range of post-translational keratin modifications have been described, including phosphorylation, ubiquitylation, sumoylation, or acetylation.21 The bestdocumented modification is phosphorylation, and keratins have several known phosphorylation sites. Keratin phosphorylation regulates their organization, interaction with associated proteins, and degradation. Keratins were proposed to act as “phosphorylation sinks” providing cellular protection from increased kinase activity during cellular injury.5,22 In particular, K8 and K18 undergo hyperphosphorylation in response to various stress situations.7 Our data support this finding as we found virtually no phosphorylation of K8 or K18 in physiological conditions, whereas a prominent phosphorylation was found upon injury. The fourth important novel finding is that keratins are expressed and seem to be up-regulated during glomerulonephritis in parietal epithelial cells and cells of the extracapillary proliferates, that is, crescents. This up-regulation was found both in mice and humans. We have previously shown that parietal epithelial cells are the major constituent of cellular crescents and that their activation leads to glomerular scarring, that is, focal segmental glomerulosclerosis.23–27 Our data support this, because in healthy glomeruli, keratins are only expressed on parietal epithelial cells but not on podocytes or any other cells of the glomerular tuft, and the majority of cells forming the crescents are K8- and K18-positive. K7 and K19 seem to mark only some of the cells forming the crescents in humans, suggesting the existence of phenotypically distinct cell subsets within crescents.26 Interestingly, in mice, K7 and K19 were expressed neither in parietal epithelial cells nor in crescents. Taken together, our data suggest that keratins might also be used as markers of extracapillary proliferates and perhaps of parietal epithelial cell activation. We observed only a few species differences in renal keratin expression. In healthy humans, K8 and K18 was found throughout the nephron, whereas in healthy mice it was only found in the collecting ducts. The tubular K7 and K19 expression was similar in mice and humans as was the upregulation of all 4 keratins during stress. As a model of direct tubular stress in humans, we have analyzed keratin expression in biopsies of patients with multiple myeloma, representing an acute disease mediated by paraproteins and 804

tubular cells injury. We found that in particular tubules with paraprotein casts, keratin expression was increased. The casts themselves were also positive for keratin expression, suggesting that keratins released from necrotic TECs are a likely component of these casts. Because keratins themselves are rather poorly soluble, it might be hypothesized that they might even play a role in precipitation of the casts. Albeit less extensively, keratins were also increased in kidneys of diabetic nephropathy and systemic lupus erythematosus patients. Most importantly, compared to not-detectable K18 in urine of healthy volunteers, patients with acute kidney injury showed prominent K18 bands. Taken together, similarly to animal models, tubular injury in patients is also associated with increased renal and urinary keratins. Western blot analyses of keratins revealed a single band with expected molecular weights in adenine-, folic acid–, or ischemia-reperfusion–induced injury models. In kidneys of UUO and Alport mice K7, K8, and K18, but not K19, and in urine of both humans and mice, 2 distinct bands were found. Whether this finding has pathophysiological or diagnostic implications remains to be analyzed. Such double bands are relatively common in various stress situations and can be both due to partial proteolysis or post-translational modifications of keratins such as phosphorylation, in our case, the former being more likely in particular in the urine.28,29 One of the suggested mechanisms driving progressive renal disease and fibrosis is the epithelial-to-mesenchymal transition, during which tubular cells lose their epithelial and acquire mesenchymal characteristics.30–32 Keratins are highly specific markers for epithelial cells and their loss was suggested as a characteristic finding of epithelial-to-mesenchymal

Table 1 | Characteristics of patients with acute kidney injury

Patients Sex (male/female) Age (yr) Serum creatinine at sampling (mg/dl)

Healthy control subjects

AKI

3 1/2 34.3  0.6 0.9  0.1

6 5/1 76  5.7 4.25  2.4

AKI, acute kidney injury. Data are given as n or mean  SD. Kidney International (2016) 89, 792–808

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Table 2 | List of antibodies used for Western blot, immunohistochemistry, and immunofluorescence staining Target

Clone/label

Host

Application

Supplier

K7 K7 K8 K8 K18 K19 K19 K18 pS34 K8 pS74 CD13 AQP2 THP ATP6V1B1 LCN2/NGAL b-Actin Mouse IgG Rabbit IgG Rat IgG Mouse IgG Rat IgG Goat IgG Mouse IgG Rabbit IgG Rabbit IgG Goat IgG Rat IgG

OV-TL12/30 OV-TL12/30 EP1628Y Ks8.7 RCK 106

Mouse Mouse Rabbit Mouse Mouse Rat Mouse Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Goat Rabbit Sheep Goat Rabbit Sheep Donkey Rabbit Sheep Goat Donkey Donkey Rabbit

IHC; IF; WB IHC; IF IHC; IF WB IHC; IF; WB IHC; IF; WB IHC; IF IF IF IF IF IF IF IF; WB WB IHC IHC IHC WB WB WB IF IF IF IF IF

Thermo Scientific (Waltham, MA) Dako (Carpinteria, CA) Thermo Scientific Progen (Zhongshan, Guandong, China) Progen DSHB (Iowa City, IA; developed by R. Kemler) Thermo Scientific St. Johns Laboratory (Owerri, Imo State, Nigeria) St. Johns Laboratory Abcam (Cambridge, UK) Abcam Santa Cruz (Santa Cruz, CA) Abcam R&D Systems (Minneapolis, MN) Sigma-Aldrich (St. Louis, MO) Vector Biolabs (Malvern, PA) Vector BioLabs Vector BioLabs Vector BioLabs Dako Dako Life Technologies (Carlsbad, CA) Life Technologies Life Technologies Life Technologies Life Technologies

KS19.1

EPR4058 H135 EPR16400

Biotin Biotin Biotin HRP HRP HRP Alexa 555 Alexa 488 Alexa 647 Alexa 546 Alexa 555

AQP2, aquaporin-2; DSHB, Developmental Studies Hybridoma Bank; IF, immunofluorescence staining; IHC, immunohistochemistry; K, keratin; LCN2, lipocalin 2; NGAL, neutrophil gelatinase-associated lipocalin; THP, Tamm-Horsfall protein; WB, Western blot.

transition and chronic kidney injury.33–35 Our data unequivocally suggest the opposite, namely that injured tubules up-regulate keratins even during advanced disease stages, characterized by prominent fibrosis and tubular atrophy. Other marker proteins that were suggested to be lost during fibrosis and epithelial-to-mesenchymal transition are the cell– cell junction proteins, in particular E-cadherin.31,33 Similar to our data, a previous study showed that E-cadherin is in fact up-regulated during fibrosis in the UUO model.36 We propose that keratins should not be viewed simply as epithelial cell markers used “in opposition” to mesenchymal markers, such as vimentin, but should rather be viewed as stressresponsive proteins in tubular cells.9,10,37 Keratins were shown to constitute versatile cytoprotective proteins,9,10 although no functional data are yet available for the kidneys. At present, it can only be speculated which functional roles keratins play in kidney homeostasis and during disease. Future studies should aim at analyzing the functional role of keratins in tubular regeneration, apoptosis, cell death, and mechanical stability. The stereotypical and

robust up-regulation during various kidney diseases suggests a renoprotective role. This is in line with the suggested role of keratins as stress-induced proteins.9 A similar up-regulation of keratins during stress is found in the liver, in which it has been shown previously that K8 and K18 have a cytoprotective function and ensure mechanical stability in hepatocytes.38 Our study focused on the previously described, and likely the most abundant renal keratins, K7, K8, K18, and K19. It is likely that as in other organs, such as the stomach and intestines,39 other keratins might be expressed in the renal tubular cells. Expression, regulation, and potential diagnostic utility of such keratins clearly deserve further studies. Which particular keratin might perform best for diagnostic purposes also remains to be seen, but K18 is the best-established keratin-based marker of liver and intestinal injury and our own data and a previous study on K18 in chronic kidney disease patients suggest this isoform to be of particular interest.17,40,41 In conclusion, we found that keratins are highly upregulated upon tubular cell stress independently of the underlying injury in both murine and human kidneys. Therefore

Table 3 | List of primers used for real-time polymerase chain reaction Gene

Sequence (sense)

Sequence (antisense)

Krt8 Krt18 Krt7 Krt19 Gapdh

5’-GGACATCGAGATCACCACCT-3’ 5’-CAAGTCTGCCGAAATCAGGGAC-3’ 5’-ACGGCTGCTGAGAATGAGTT-3’ 5’-CGGTGGAAGTTTTAGTGGGA-3’ 5’-GGCAAATTCAACGGCACAGT-3’

5’-TGAAGCCAGGGCTAGTGAGT-3’ 5’-TCCAAGTTGATGTTCTGGTTTT-3’ 5’-CGTGAAGGGTCTTGAGGAAG-3’ 5’-AGTAGGAGGCGAGACGATCA-3’ 5’-AGATGGTGATGGGCTTCCC-3’

All genes were murine. Kidney International (2016) 89, 792–808

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rather than markers of differentiated tubular cells, keratins might serve as novel, broad, and sensitive markers of renal epithelial cell stress.

MATERIALS AND METHODS Animal experiments All animal experiments were approved by the local and government review boards. The animals were held in cages with constant temperature and humidity with ad libitum access to tap water and standard chow. First, twenty-four 10-week-old male C57BL6/N mice underwent UUO. Renal tissue (for histological evaluation, protein and RNA isolation) from both the obstructed and contralateral kidney was obtained when the mice were killed, on day 1 (n ¼ 5), day 3 (n ¼ 4), day 7 (n ¼ 5), day 5 (n ¼ 5), and day 10 (n ¼ 5) after UUO. Second, adenine nephropathy was induced in 12 male 10-weekold C57BL6/N mice by administration of an adenine-rich diet (supplemented with 0.25% adenine; Altromin, Lippe, EasternWestphalia, Germany). Mice were killed on day 5 (n ¼ 4), day 14 (n ¼ 4), and day 28 (n ¼ 4) after diet start.42,43 Third, folic acid nephropathy was performed in 8 female 10week-old C57BL6/N mice by weekly i.p. injection of folic acid (250 mg/kg of body weight and dissolved in NaHCO3) or solvent alone. All mice were killed after 8 weeks. Fourth, unilateral ischemia reperfusion injury was performed in 7 male FVB/N mice. Renal tissue from both the ischemic and healthy contralateral kidney was obtained when the mice were killed on day 21 after ischemia reperfusion. Fifth, 6 male Col4a3-/- (Alport) mice44 and wild-type littermates, all on Sv126 background, were analyzed at 4 and 8 weeks of age. In all experiments, kidneys were taken and immediately processed for further analyses. If not analyzed immediately, all samples apart from fixed tissues for histology and immunohistochemistry were stored at –80  C. Human renal tissue and urine Six patients with rapidly progressive glomerulonephritis, 5 with DN, 8 with systemic lupus erythematosus, and 9 with multiple myeloma were included in the study. Tumor-distant kidney tissues from nephrectomy specimens of patients with renal cell carcinoma without any other renal disease and normal histopathology as well as nephrectomies of normal kidneys due to other reasons (e.g., trauma) were used as control tissues (n ¼ 5). Urine was collected from 3 human healthy volunteers and 6 patients with acute kidney injury stage II or III, among them 5 patients due to prerenal and 1 patient due to postrenal causes.45 Volunteer and patient data are summarized in Table 1. All patients gave their informed consent or were analyzed in a retrospectively anonymized manner. The study protocol is in accordance with the Helsinki Declaration of 1975, as revised in 2000, and has been approved by the Patras and Aachen University Hospital committee on human research. Immunohistochemistry and immunofluorescence Tissue for light microscopy and immunohistochemistry was fixed in methyl Carnoy solution (mice only) or formalin (mice and humans) and embedded in paraffin. Indirect immunoperoxidase and immunofluorescence procedures were performed on 1-mm sections as described previously.46 The primary and secondary antibodies used in this study are listed in Table 2. 806

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The evaluation of immunohistochemistry was performed on whole slide images scanned by NanoZoomer 2.0-HT (Hamamatsu Photonics, Shizuoka Prefecture, Japan) and using the ImageJ version 1.48 software (National Institutes of Health, Bethesda, MD; http:// imagej.nih.gov/ij/). The percentage of positively stained area in each tissue was calculated separately in 20 interstitial fields at 200 magnification, representing almost the entire cortical area as described previously.47,48 Furthermore, single cell immunohistochemical expression was evaluated in all cells of selected collecting ducts. Immunohistochemistry was performed on negative control subjects as described previously (Supplementary Figure S8).49 RNA extraction and analyses Total RNA was extracted from renal cortex using the RNeasy Mini Kit (Qiagen, Hilden, Germany). From total RNA, cDNA was synthesized and real-time qRT-PCR was performed as described previously.49 Sequences of primers and probes used are listed in Table 3. Messenger RNA for each sample was normalized to glyceraldehyde3-phosphatedehydrogenase (Gapdh) as a housekeeping gene. Results are given as relative mRNA expression normalized to the expression in contralateral healthy kidneys. Western-blot analyses High-salt extraction was performed in the UUO experiment to enrich keratin in cortex tissue lysates by taking advantage of their poor solubility in nonionic detergents as described previously.50 Whole cortex lysates were performed as described previously.41 For Western blot and quantification, all animals were analyzed and representative pictures of 3 animals per group are shown (Figures 4, 5, and 10). Denatured protein samples were separated by electrophoresis in 10% and 4% to 12% sodium dodecyl sulfate–periodic acid gel, transferred to nitrocellulose membranes, blocked with 2% (weight/ volume) bovine serum albumin in phosphate-buffered saline, washed with tris-buffered saline, and incubated with primary antibodies diluted in tris-buffered saline overnight at 4  C. Primary antibodies (Table 2) were detected using horseradish peroxidase– conjugated antibodies and visualized by ECL (Roche, Basel, Switzerland). b-Actin quantification was performed on the same nitrocellulose blots to normalize for loading incongruities. Band intensities were quantified by Scion Image software. Quantitative real-time PCR of renal biopsies with diverse human kidney diseases Human kidney biopsies were collected in a multicenter study (European Renal cDNA Bank-Kroener-Fresenius Biopsy Bank, ERCB; see Cohen et al.51 for participating centers) and were obtained from patients after informed consent and with the approval of the local ethics committees. After renal biopsy, the tissue was transferred to a ribonuclease inhibitor and microdissected into glomerular and tubular fragments. Total RNA isolation from microdissected glomeruli, reverse transcription and real-time RT-PCR were performed as reported earlier.52 Predeveloped TaqMan reagents were used for human KRT7, KRT8, KRT18, and KRT19, as well as the reference genes, 18S rRNA and GAPDH (Applied Biosystems, Carlsbad, CA). The expression of the candidate gene was normalized to the reference genes. The mRNA expression was analyzed by standard curve quantification. Reanalyses of public arrays All comparisons were performed using the GEO2R Program (NCBI, Bethesda, MD; http://www.ncbi.nlm.nih.gov/geo/geo2r/). Five freely Kidney International (2016) 89, 792–808

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available arrays were analyzed: GSE36496 (UUO vs. SHAM in wildtype mice), GSE35226 (Tg26 HIV transgenic vs. wild-type), GSE52004 (bilateral ischemia reperfusion injury vs. SHAM in wild-type mice), GSE642 (diabetic db/db mice vs. nondiabetic db/m littermates), and GSE19817 (genetically hypertensive vs. normotensive and hypotensive vs. normotensive). Statistical analyses All values are expressed as means  SD. For comparison of 2 groups, t test or Wilcoxon U test were used where appropriate; for comparison between multiple groups, analysis of variance with Bonferroni post hoc test was used. All tests were 2-tailed and statistical significance was defined as P < 0.05. DISCLOSURE

All the authors declared no competing interests. SOURCE OF FUNDING

This work was supported by research grants from the SFB/Transregio 57 “Mechanisms of organ fibrosis” (to PB, JF, and PS), BO 3755/2-1 (to PB), all from the German Research Foundation (Deutsche Forschungsgemeinschaft), the Else-Kröner Fressenius Stiftung (EKFS 2012_A216 to PB), the German Ministry of Education and Research (BMBF Consortium STOP-FSGS number 01GM1518A to PB), and a research grant from the Hellenic Society of Nephrology to MP. PB and PS are supported by the Interdisciplinary Center for Clinical Research (Interdisziplinäre Zentrum für Klinische Forschung) within the Faculty of Medicine at the (Rheinisch-Westfälische Technische Hochschule) Aachen University (K7-3). CDC and MTL are supported by the ElseKröner Fresenius Foundation. Active members at the time of the study are listed in Martini et al.53 ACKNOWLEDGMENTS

The authors thank all participating centers of the European Renal cDNA Bank—Kroener–Fresenius Biopsy Bank and their patients for their cooperation. The technical assistance of Cherelle Timm, Claudia Gavranic, and Simon Otten is gratefully acknowledged. The K18-P S343 antibody was kindly provided by Dr. M. Bishr Omary (University of Michigan Medical School, Ann Arbor, MI). SUPPLEMENTARY MATERIAL Figure S1. Periodic acid–Schiff (PAS) staining of all analyzed animal models. PAS staining of unilateral ureteral obstruction (UUO) at (A) day 3, (B) day 5, (C) day 7, and (D) day 10 showed increased tubular damage during time-course compared with (E) healthy control. (F–H) Similar increase in kidney injury was observed in time course of adenine nephropathy. Representative picture of PAS staining showed the expected damage in (I) folic acid nephropathy (FAN), (J) ischemia reperfusion (I/R), and (K, L) 4- and 8-week-old Alport mice. (M–O) Staining of healthy murine kidney for K8 in combination with PAS confirmed expression of K8 in collecting ducts and (M) parietal epithelial cells. (P) Urothelium served as internal positive control of K8 staining. Bars ¼ 50 mm. Figure S2. Confocal laser scanning microscopy of K18 immunofluorescence staining. Confocal laser scanning microscopy confirmed up-regulation and circumferential expression of K18 in (B) unilateral ureteral obstruction (UUO) compared with (A) healthy control. Even at highest magnification, the single keratin filaments could not be resolved. A’ and B’ are higher magnifications. Bars ¼ 10 mm. Figure S3. Expression of all 4 keratins is progressively increased during the time course of the murine adenine nephropathy model. (A) Compared with K8 expression in healthy renal tissue, progressively increasing expression was found from (B–D) day 5 to day 28 of the Kidney International (2016) 89, 792–808

disease model. At day 14 and 28, albeit weak, K8 expression was found in almost all tubuli (K18, K7, and K19 showed the same staining pattern as K8 at all analyzed time points, pictures not shown). (E–H) K8, K18, K7, and K19 quantified by morphometric image analysis confirmed the significantly increased expression during the disease progression. Significant up-regulation of (I) Krt8, (J) Krt18, (K) Krt7, and (L) Krt19 mRNA expression was found during the time course of adenine nephropathy in comparison to healthy control cells. Compared with healthy kidneys, (M) Western blot analysis and (N–Q) quantification confirmed the increased expression of K8, K18, K7, and K19 during the time course of adenine nephropathy. Dashed lines represent healthy control cells. Bars ¼ 50 mm. *P < 0.05, **P < 0.01, ***P < 0.001. d, day(s). Figure S4. Keratin expression increased in a murine unilateral ischemia-reperfusion (I/R) model. Staining and quantification of (A and E) K8, (B and F) K18, (C and G) K7, and (D and H) K19 immunohistochemical tubular expression in healthy control cells in comparison to I/R showed a significant increase of all analyzed keratins. Significant up-regulation of (I–L) Krt8, Krt18, Krt7, and Krt19 mRNA expression was found in I/R in comparison to healthy control subjects. Compared with healthy kidneys, (M) Western blot analysis and (N–Q) quantification confirmed the increased expression of K8, K18, K7, and K19. Bars ¼ 50 mm. *P < 0.05, **P < 0.01. Figure S5. Keratin expression increased in murine folic acid nephropathy (FAN) model. (A–D) K8, K18, K7, and K19 immunohistochemical tubular expression in healthy control cells in comparison to (E–H) FAN and (I–L) their quantification showed a significant increase of K8, K18, K7, and K19 expression in FAN compared with healthy control subjects. Significant up-regulation of all 4 keratins was confirmed by (M–P) quantitative reverse transcriptase polymerase chain reaction and (Q–U) Western blot analysis. Bars ¼ 50 mm. *P < 0.05, **P < 0.01, ***P < 0.001. Figure S6. Slight increase in keratin expression in 4-week-old Alport mice. Immunohistochemical analyses of (A–D) K8, K18, K7, and K19 in 4-week-old Alport mice showed a (E–H) slight increase in keratin expression compared with age-matched littermates that, however, reached statistical significance only for K7. Bars ¼ 50 mm. *P < 0.05. Figure S7. Keratin expression in publicly available datasets. (A) The analyses of available microarray data from normal murine kidney tissues revealed a basal expression of the 4 renal keratins we analyzed (http://www.ebi.ac.uk). (B) The analyses of datasets comparing differential gene expression between healthy and diseased kidneys showed an up-regulation of all 4 keratins during disease. AKI, acute kidney injury; DN, diabetic nephropathy; HyperTN, hypertensive; I/R, ischemia reperfusion; NS, not significant; Tg, transgenic; UUO, unilateral ureteral obstruction. Figure S8. Negative control subjects confirm the specificity of the performed stainings. Specificity of keratin staining was confirmed using negative control subjects employing irrelevant IgG instead of a specific primary antibody. No unspecific staining was detectable for (A, E) K8, (B, F) K18, (C, G) K7, and (D, H) K19 neither in humans nor in mice. (A–D) Bars ¼ 20 mm; (E–H) bars ¼ 50 mm. Supplementary material is linked to the online version of the paper at www.kidney-international.org. REFERENCES 1. Nangaku M. Mechanisms of tubulointerstitial injury in the kidney: final common pathways to end-stage renal failure. Intern Med. 2004;43:9–17. 2. Smeets B, Boor P, Dijkman H, et al. Proximal tubular cells contain a phenotypically distinct, scattered cell population involved in tubular regeneration. J Pathol. 2013;229:645–659. 3. Boor P, Ostendorf T, Floege J. Renal fibrosis: novel insights into mechanisms and therapeutic targets. Nat Revi Nephrol. 2010;6: 643–656. 4. Quaggin SE, Kapus A. Scar wars: mapping the fate of epithelialmesenchymal-myofibroblast transition. Kidney Int. 2011;80:41–50.

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