Kidney injury molecule-1 expression in murine polycystic kidney disease

E. Wolfgang Kuehn1, Kwon Moo Park1, Stefan Somlo2, and Joseph V. Bonventre1,3

1 Renal Unit and Department of Medicine, Massachusetts General Hospital, Charlestown 02129; 2 Section of Nephrology, Yale University School of Medicine, New Haven, Connecticut 06519; and 3 Harvard-Massachusetts Institute of Technology, Division of Health Sciences and Technology, Cambridge, Massachusetts 02139


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Kidney injury molecule-1 (Kim-1) is a type 1 membrane protein maximally upregulated in proliferating and dedifferentiated tubular cells after renal ischemia. Because epithelial dedifferentiation, proliferation, and local ischemia may play a role in the pathophysiology of autosomal dominant polycystic kidney disease, we investigated Kim-1 expression in a mouse model of this disease. In the Pkd2WS25/- mouse model for autosomal dominant polycystic kidney disease, cystic kidneys show markedly upregulated Kim-1 levels compared with noncystic control kidneys. Kim-1 is present in a subset of cysts of different sizes and segmental origins and in clusters of proximal tubules near cysts. Kim-1-expressing tubular cells show decreased complexity and quantity of basolateral staining for Na-K-ATPase. Other changes in polarity characteristic of ischemic injury are not present in Kim-1-expressing pericystic tubules. Polycystin-2 expression is preserved in Kim-1-expressing tubules. The interstitium surrounding Kim-1-expressing tubules shows high proliferative activity and staining for smooth muscle alpha -actin, characteristic of myofibroblasts. Although the functional role of the protein in cysts remains unknown, Kim-1 expression in tubules is strongly associated with partial dedifferentiation of epithelial cells and may play a role in the development of interstitial fibrosis.

Na-K-ATPase; cell polarity; fibrosis; Tim; ischemia; kidney obstruction


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

AUTOSOMAL DOMINANT POLYCYSTIC kidney disease (ADPKD) is the most commonly inherited monogenetic renal disease, affecting >1 in 1,000 live births. In the United States, it leads to end-stage renal disease in >1,500 patients/yr (24). In most patients, the disease is caused by an inherited defect in one of two polycystin genes, PKD1 or PKD2 (10a, 16). Random loss of heterozygosity in individual tubular epithelial cells is thought to be responsible for the transformation of epithelial cells through somatic inactivation of the second copy of the affected gene. The resulting dedifferentiation and proliferation causes progressive cyst formation (31). The PKD2 gene product polycystin-2 is a nonselective cation channel, and polycystin-1 may be required for its proper localization and function (8, 11, 13). The molecular mechanisms underlying the epithelial transformation are unknown. Epithelial cells in cysts have been shown to be dedifferentiated and to have increased proliferative indices (18, 21). The growth of cysts leading to massively enlarged kidneys is thought to be responsible for regional ischemia, which contributes to elevations in plasma renin and hypertension (5). Less than 1% of all nephrons are directly affected by cysts; therefore, direct nephron loss through cyst formation does not explain the decline of renal function. Progressive nephron loss is thought to involve apoptosis (30) and fibrosis (19) in areas adjacent to cysts, but this process is not very well characterized.

Kidney injury molecule-1 (Kim-1)1 is a type 1 transmembrane protein that is expressed at very low levels in normal kidneys and is maximally upregulated in the S3 segment of the proximal tubule 24-48 h after exposure of the kidney to transient ischemia (12). It is expressed in human kidneys with acute tubular necrosis and can be detected in the urine of these patients (10). In the postischemic kidney, Kim-1 is expressed in vimentin-positive cells and cells that take up 5-bromo-2'-deoxyuridine (12), suggesting a role for the protein in the dedifferentiation and proliferation of epithelial cells. Because epithelial dedifferentiation, proliferation, and ischemia may play a role in the pathophysiology of ADPKD, we examined the pattern of Kim-1 expression in cystic kidneys in the Pkd2WS25/- mouse model. We found that Kim-1 is expressed in polycystic kidneys but not in normal kidneys. Kim-1 expression is found in a small subset of cysts of varying sizes and different segmental origins. Additionally, we found a striking pattern of Kim-1 expression in clusters of noncystic tubules adjacent to cysts in regions characterized by interstitial cell proliferation and fibrosis. In tubular cells, Kim-1 expression is associated with a decrease in complexity and quantity of Na-K-ATPase expression, without other features of loss of cell polarity, as reflected by actin, villin, and E-cadherin staining patterns. The functional role of Kim-1 expression in cysts is unclear. The tubular and peritubular findings support the possibility that Kim-1 may play a role in interstitial fibrosis and nephron loss in ADPKD.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and tissues. Pkd2WS25/- mice (31) and wild-type (WT) littermates were genotyped by Southern blot analysis. Unilateral ureteral obstruction and renal ischemia with reperfusion were performed as previously described by our laboratory (20). For total protein lysates, the kidneys were harvested unfixed and shock frozen in liquid nitrogen. For immunohistochemical analysis, mice were perfused via the left ventricle with paraformaldehyde-lysine-periodate solution containing 2% paraformaldehyde, 38 mM phosphate buffer, 60 mM lysine, 10 nM sodium periodate, and 5% sucrose. Subsequently, kidneys were immersed in 2% paraformaldehyde for 4 h, washed in PBS, and cryoprotected by overnight submersion in 30% sucrose in 1× PBS. Five-micrometer frozen sections were cut in a cryostat.

Antibodies. Primary antibodies included the following. AKG7 is a mouse monoclonal antibody against the extracellular domain of human KIM-1 (1a). R9 is a rabbit polyclonal antibody, which was raised to the intracellular domain of rat Kim-1 (see Fig. 1) (12). Monoclonal anti-Na-K-ATPase 6F was obtained from the Developmental Studies Hybridoma Bank (University of Iowa). It recognizes the alpha 1-subunit and has been well characterized (1). Monoclonal anti-E-cadherin was purchased from BD Transduction Laboratories. Mouse anti-PCNA was purchased from DAKO, and mouse anti-smooth muscle alpha -actin is from Sigma. Polyclonal rabbit anti-polycystin 2 YCC is a gift from Dr. Yiqiang Cai (Yale University School of Medicine) (4). Polyclonal rabbit anti-villin and rabbit anti-aquaporin-2 are gifts from Dr. Dennis Brown (Massachusetts General Hospital) (2). Polyclonal rabbit anti-aquaporin-1 is a gift from Dr. Alfred Van Hoek (Massachusetts General Hospital) (23). Secondary antibodies were Cy3-conjugated goat anti-rabbit and FITC-conjugated goat anti-mouse (Sigma). Actin was visualized by using tetramethylrhodamine isothiocyanate-labeled phalloidin (Sigma).


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 1.   Alignment of the rat kidney injury molecule-1 (KIM-1; see footnote) and mouse Kim-1 amino acid sequences. Identical amino acids are shaded in dark grey, and similar ones are in light grey. The peptide region in the rat sequence, to which the rabbit polyclonal antibody R9 was made, is underlined. Note the high degree of homology in this region.

Apoptosis. TdT-mediated dUTP-X nick-end-labeling staining was performed with materials obtained from Boehringer according to instructions supplied by the manufacturer.

Subcloning. Expressed sequence tag (EST) AA547594 was identified by the Basic Local Alignment Search Tool (BLAST) to have significant sequence homologies to rat Kim-1 and was obtained from the IMAGE consortium (clone 960204) (14). After further sequencing, it was found to contain the full open reading frame of putative mouse Kim-1. Subcloning of the entire open reading frame was performed by PCR in pCR2.1 (Invitrogen), using primers 5'-CGCGTGGACCATGAATCAGATTC-3' and 5'-CTGCCCCTCAAGGTCTATCTTC-3'. Subsequently, the insert was subcloned into the EcoRI site of pCDNA3 (Invitrogen). The sequence was confirmed through double-stranded sequencing. Sequence analysis was performed with MacVector 6.5 software (Oxford Molecular).

Cell cultures and transient transfection. COS1 cells were maintained in DMEM supplemented by 10% fetal calf serum. Transient transfections were carried out with the superfect reagent (Qiagen) according to instructions by the manufacturer.

Proteins and Western blot. For SDS-PAGE, shock-frozen kidney tissue was homogenized in sample buffer containing 125 mM Tris, pH 6.8, 4.1% SDS, 20% glycerol, and 2% mercaptoethanol. Lysis of cells for protein analysis, electrophoresis, transfer, and Western blotting were performed as previously described (20).

Immunohistochemistry. Tissue sections were boiled in 0.1 M Na citrate buffer for 10 min, incubated with 1% SDS in PBS for 5 min, washed with 1× PBS, and blocked in 1× PBS with 2% FCS, 5% sucrose, and 0.1% IPEGAL CA-630 (Sigma). Incubation with primary antibody was done overnight at 4°C or for 1 h at room temperature. Secondary antibodies were applied for 1 h at room temperature. For immunofluorescence, slides were mounted with Vectashield (Vector Labs) in 1:1 dilution with 1.5 M Tris, pH 8.9, and examined under an epifluorescence microscope (Nikon Microphot FXA). Images were captured with a Hamamatsu Orca charge-coupled device camera and processed with IPLab Spectrum software (Scanalytics, Vienna, VA).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cloning of a mouse ortholog to rat Kim-1 and confirmation of cross-reactivity of the anti-rat Kim-1 antibody R9 with mouse Kim-1. With the purpose of studying Kim-1 expression in a mouse model of ADPKD, we searched the database for mouse genes with homologies to human KIM-1 and rat Kim-1. A putative mouse ortholog of rat Kim-1 was identified by BLAST search of the mouse EST database. EST AA547594 was found to contain the full coding region of putative mouse Kim-1 (mKim-1). Comparison of the deduced amino acid sequence with rat Kim-1 shows 58% identity. There is a high degree of homology in the protein region to which a rabbit anti-rat-Kim-1 antibody, R9, had been raised (Fig. 1). Binding of R9 to mKim-1 was demonstrated by Western blot analysis of total cell lysates of COS cells transfected with the mKim-1 expression construct (Fig. 2).


View larger version (60K):
[in this window]
[in a new window]
 
Fig. 2.   Western blot analysis of mouse Kim-1, with polyclonal anti-rat Kim-1 antibody (R9), demonstrates cross-reactivity of the R9 antibody between species. COS cells were transfected with a mouse Kim-1 expression construct or a green fluorescent protein control construct and compared with protein lysates of a postischemic mouse kidney, which were obtained 24 h after 30 min of renal ischemia and subsequent reperfusion (20). Kim-1 is highly glycosylated, resulting in three bands that represent different degrees of glycosylation (12). The predicted weight of the unglycosylated protein is 32 kDa. The bands are shifted downward in the COS cell lysates compared with the bands in the postischemic kidney. This likely represents differences in glycosylation patterns between transfected fibroblasts in culture and epithelial cells in vivo (12).

Mouse Kim-1 is expressed in the Pkd2WS25/- mouse model of ADPKD. Expression of mouse Kim-1 is markedly increased in kidneys of Pkd2WS25/- mice, compared with minimal basal expression in kidneys from WT mice (Fig. 3). Kim-1 staining is present in 2-5% of cysts and can be seen in cysts of different sizes (Fig. 4A). Two patterns of Kim-1 expression are observed in Pkd2WS25/- kidneys. One pattern of Kim-1 staining in cyst epithelia is apical (Fig. 4B), whereas the other is intracellular (Fig. 4C). In addition to Kim-1-staining cysts, occasional clusters of Kim-1-staining noncystic tubules are seen in areas adjacent to Kim-1-negative cysts (Fig. 4D). No staining for Kim-1 is present in normal kidneys from WT mice.


View larger version (47K):
[in this window]
[in a new window]
 
Fig. 3.   Kim-1 expression is increased in cystic kidneys of Pkd2WS25/- mice compared with wild-type (WT) littermates. Kidneys from 2 Pkd2WS25/- (aged 4 wk) or 2 WT mice were removed and shock frozen. The postischemic control kidney was removed and shock frozen 24 h after 40 min of unilateral ischemia in a WT mouse. The kidneys were homogenized in sample buffer and analyzed by SDS-PAGE and Western blot with the R9 anti-Kim-1 antibody. Only the 64-kDa band of Kim-1 is shown. Equal loading was demonstrated by stripping the membrane and probing with anti-P38 antibody.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 4.   Immunofluorescence staining for mouse Kim-1 with polyclonal anti-Kim-1 (R9) antibody and anti-rabbit Cy3 secondary antibody. Pkd2WS25/- mouse (aged 10 wk) kidneys were perfused with paraformaldehyde-lysine-periodate fixative solution. Cryostat sections (5 µm) were analyzed by indirect immunofluorescence staining. A: intermediate-sized cyst with a diameter of 550 µm shows nearly circumferential staining for Kim-1 in cyst-lining cells. Staining for Kim-1 is found in cysts of various sizes. Most cysts are negative for Kim-1 staining (not shown) B and C: pattern of Kim-1 expression differs among cysts. B: cyst epithelium with apical expression of Kim-1. C: cyst epithelium with intracellular staining for Kim-1. D: Kim-1-expressing tubules are clustered around cysts (arrows). Counterstaining was with monoclonal anti-E-cadherin antibody and anti-mouse FITC antibody. Bars = A, 100 µm; B and C, 10 µm; D, 50 µm.

Kim-1-expressing cysts are derived from different segmental origins. Kim-1 expression after ischemia-reperfusion injury is localized to the proximal tubule (12). To evaluate whether Kim-1 expression occurs only in cysts of proximal tubular origin, we examined Kim-1-expressing cysts for staining with the proximal tubular marker aquaporin-1 and the collecting duct marker aquaporin-2. Some Kim-1-positive cysts express aquaporin-1 (Fig. 5B) but not aquaporin-2 (Fig. 5C), whereas others stain for aquaporin-2 (Fig. 5F) but not aquaporin-1 (Fig. 5E). Some Kim-1-positive cysts do not express either aquaporin-1 or aquaporin-2 (data not shown). These findings indicate that Kim-1 expression in cysts is not related to their segmental origin.


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 5.   Expression of a proximal and a distal segmental marker in Kim-1-positive cysts. Kim-1-staining cysts were examined by immunofluorescence for aquaporin-1, which is expressed in the proximal tubule and thin descending limb, and aquaporin-2, which is expressed in the collecting duct. Secondary staining was with anti-rabbit Cy3 antibody. A-C: Kim-1-expressing cyst is derived from the proximal tubule. A: Kim-1-expressing cyst. B: cyst epithelium stains positive for aquaporin-1. Nearby proximal tubules also express aquaporin-1. C: no staining for aquaporin-2 is visible in the cyst-lining epithelium. Two adjacent collecting ducts stain positive for aquaporin-2. D-F: Kim-1-positive cyst is derived from collecting duct. D: Kim-1-staining cyst. E: staining for aquaporin-1 is negative in the cyst epithelium. The surrounding proximal tubules stain positive for aquaporin-1. F: staining for aquaporin-2 in the cyst-lining epithelium is positive. No surrounding collecting ducts are seen in the visualized portion of the cortex. G: Kim-1 expression in pericystic tubules is apical and sometimes lateral. H and I: Kim-1 staining in kidneys from Pkd2WS25/- mice in proximal tubules occurs in subsets of tubular cells and differs from the staining pattern seen in postobstructed kidneys. H: staining pattern for Kim-1 in individual tubules is discontinuous, frequently involving less than one-half of the cells in an individual tubule (arrows). I: WT mouse was subjected to unilateral ureteral obstruction for the duration of 24 h. Kidneys were perfused with paraformaldehyde-lysine-periodate fixative solution 48 h after the release of obstruction. R9 staining involves all cells of Kim-1-expressing tubules. Bars: A-F = 50 µm; G-I = 10 µm.

The staining pattern for Kim-1 differs between pericystic tubules and obstructed WT kidneys. In tubules adjacent to cysts, the staining pattern is apical and occasionally lateral (Fig. 5G). Staining for Kim-1 is often discontinuous, with only some of the epithelial cells in a given tubule expressing detectable amounts (Fig. 5H). Because it has been proposed that tubular obstruction plays a role in the pathophysiology of ADPKD (25), we compared the Kim-1 expression pattern in ADPKD to that observed in obstructed kidneys. We found Kim-1 uniformly expressed in all cells of a tubular segment 48 h after transient ureteral obstruction in WT mice (Fig. 5I), a pattern markedly different from the pattern of sporadic cell staining within a given tubule in cystic kidneys.

Kim-1-expressing tubular epithelial cells have decreased complexity and quantity of Na-K-ATPase expression but maintain normal expression of actin, villin, and E-cadherin. Because Kim-1 is expressed in dedifferentiated epithelial cells after ischemic injury (12), we analyzed Kim-1-expressing tubular cells for the distribution of Na-K-ATPase. Basolateral expression of Na-K-ATPase is a marker of terminal differentiation of epithelial cells (28) and is decreased after ischemic injury (33). In tubules adjacent to cysts, single Kim-1-positive cells (Fig. 6, A and D) exhibit a loss of complexity (Fig. 6E) and decreased quantity (Fig. 6, B, C, E, and F) of Na-K-ATPase expression, whereas Kim-1-negative cells in the same tubule have a normal staining pattern for Na-K-ATPase. We additionally examined the staining pattern of actin and villin, which have been shown to partially redistribute from the apical brush border of epithelial cells to the basolateral membrane after renal ischemia (2). A normal distribution of apical brush-border staining for actin (not shown) and villin is present in Kim-1-expressing pericystic tubules (Fig. 6, G and H). Because ischemia has been shown to result in breakdown of the adherens junction and loss of lateral E-cadherin expression in epithelial cells (3), we also examined E-cadherin staining in Kim-1-expressing tubules. E-cadherin staining is preserved in the lateral membrane of Kim-1-expressing cells (Fig. 6, I-L). These results indicate that Kim-1 expression in epithelial cells is associated with a partial loss of polarity. This is not likely to be due to ischemia.


View larger version (46K):
[in this window]
[in a new window]
 
Fig. 6.   Kim-1, Na-K-ATPase, E-cadherin, and villin expression in tubules of cystic kidneys from Pkd2WS25/- mice. Sections were stained with polyclonal anti-Kim-1 (R9), monoclonal anti-Na-K-ATPase (6F), monoclonal anti-E-cadherin, and polyclonal anti-villin antibodies. Secondary antibodies were Cy3-coupled anti-rabbit and FITC-coupled anti-mouse antibodies. A-C: basal membrane expression of Na-K-ATPase is greatly diminished in individual Kim-1-expressing proximal tubular cells. A: Kim-1 is expressed in individual epithelial cells of proximal tubules (red). B: staining for Na-K-ATPase (green) demonstrates individual cells that have strongly reduced staining of Na-K-ATPase at the basal and lateral membrane (arrows) C: combined image of A and B shows that the cells that express Kim-1 have greatly diminished Na-K-ATPase expression at the basal and lateral membrane (arrows). D-F: another example of reduced amounts and complexity of Na-K-ATPase in Kim-1-expressing tubule cells. D: one-half of the cells of this tubule stain for Kim-1. E: cells in the upper one-half of the tubule show a strong reduction in the complexity and quantity of staining for Na-K-ATPase. F: in the combined image of D and E, reduced expression of Na-K-ATPase in individual cells coincides with apical Kim-1 expression. G and H: apical villin staining is preserved in Kim-1-expressing proximal tubules. G: pair of Kim-1-expressing tubules (arrows) show apical staining for R9 in red. H: in a neighboring section, apical villin expression in the Kim-1-expressing tubules (arrows) is present only in the apical brush border but not in the basolateral membrane, as has been described after ischemia-reperfusion (2). I-L: lateral E-cadherin staining is preserved in Kim-1-expressing proximal tubular cells in sections adjacent to G and H. I: Kim-1-expressing proximal tubule cells show apical staining (red). K: E-cadherin (in green) localizes to the lateral membranes. L: in the combined image of I and K, staining for anti-E-cadherin in the lateral membrane is preserved in the Kim-1-expressing tubule. Bars = 10 µm.

Different distribution patterns of Na-K-ATPase are found in Kim-1-expressing cysts. Because Kim-1-expressing tubular cells show alterations of Na-K- ATPase expression that are consistent with a state of partial dedifferentiation and dedifferentiation of epithelial cells has been implicated to play a role in cyst formation, we investigated the staining pattern for Na-K-ATPase in Kim-1-expressing cysts. Cysts with apical Kim-1 expression have preserved basolateral staining for Na-K-ATPase (Fig. 7, A-C), whereas cystic epithelia with cytoplasmic Kim-1 expression show diffuse staining for Na-K-ATPase (Fig. 7, D-F). Diffuse, as well as basolateral, staining for Na-K-ATPase in cysts is also found in the absence of Kim-1 expression. Lateral staining for E-cadherin is preserved in all Kim-1-expressing cysts, as reflected in an example of a Kim-1-positive cyst with diffuse expression of Na-K-ATPase (Fig. 7, G-I). These findings of apical Kim-1 expression with basolateral staining for Na-K-ATPase in cysts are different from the findings in Kim-1-positive tubular cells, wherein the expression of Kim-1 is associated with loss of quantity and complexity of basolateral Na-K-ATPase staining.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 7.   Expression of Kim-1, Na-K-ATPase, and E-cadherin in Pkd2WS25/- kidney cysts. Sections were stained by indirect immunofluorescence with polyclonal anti-Kim-1 (R9), monoclonal anti-Na-K-ATPase (6F), and monoclonal anti-E-cadherin antibodies. Secondary antibodies were Cy3-coupled anti-rabbit and FITC-coupled anti-mouse antibodies. The pattern of Na-K-ATPase differs between cysts with apical vs. intracellular expression of Kim-1. A-C: cyst epithelia with apical expression of Kim-1 have preserved basolateral staining for Na-K-ATPase. A: cyst epithelium expressing Kim-1 at the apical membrane (red). B: Na-K-ATPase staining (green) is mostly lateral. C: in the combined image of A and B, Na-K-ATPase staining is localized to the lateral membrane of Kim-1-expressing cells in a cyst epithelium. D-F: cyst epithelia with cytoplasmic Kim-1 expression lack an organized pattern of Na-K-ATPase staining. D: cyst with cytoplasmic staining for Kim-1. E: staining for Na-K-ATPase (green) is diffuse. F: combined image of D and E; no organized pattern of Na-K-ATPase expression is seen in this cyst with cytoplasmic Kim-1 expression. G-I: Kim-1-expressing epithelia in cysts with a cytoplasmic pattern of Kim-1 expression have preserved lateral staining for E-cadherin. G: Kim-1-expressing cyst epithelium (red). H: same section using a FITC filter shows E-cadherin staining (green) in the lateral membranes. I: combined image of G and H shows preserved expression of E-cadherin in the lateral membranes of cells in a cyst epithelium with cytoplasmic Kim-1 expression. Bars = 10 µm.

Kim-1-expressing tubular cells are surrounded by proliferating cells and interstitial myofibroblasts. Kim-1 is expressed in proliferating tubular epithelial cells after ischemic injury (12). Because increased cell proliferation has been observed in ADPKD (18, 21), we examined cellular proliferation in Kim-1-expressing epithelial cells by PCNA staining. We observed PCNA staining in only a few positive cells that express Kim-1, either in cysts or in pericystic tubules (data not shown), but found a greater number of PCNA-positive cells in the peritubular interstitium surrounding Kim-1-expressing tubules (Fig. 8, A, C, and E). Increased numbers of PCNA-positive cells were also found in some non-Kim-1-expressing tubules in these same areas, consistent with a "field effect," as previously described (18). In contrast, very few proliferating cells are found in the interstitial areas adjacent to Kim-1-negative tubules (Fig. 8, B, D, and F).


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 8.   Kim-1 and PCNA staining in Pkd2WS25/- kidneys. Sections were stained with polyclonal anti-Kim-1 (R9) and monoclonal anti-PCNA antibodies. Secondary antibodies were Cy3-coupled anti-rabbit and FITC-coupled anti-mouse antibodies. Clusters of Kim-1-expressing proximal tubules adjacent to cysts coincide with a high degree of cell proliferation in the surrounding interstitium (A, C, and E) relative to areas of absent Kim-1 staining (B, D, and F). A: clusters of tubules near a cyst (*) stain positive (red) for apical Kim-1 expression. B: in a different area adjacent to a cyst, no tubular staining for Kim-1 is observed. C: large number of PCNA-positive nuclei (green) are present in the interstitium, as well as in some tubules. D: PCNA staining shows markedly fewer proliferating nuclei (arrows) compared with C. E: in the combined image of A and C, the proliferating cells in the interstitium and the tubules are near Kim-1-expressing tubules. F: combined image of B and D shows that the absence of Kim-1 expression in tubules coincides with a markedly reduced proliferative activity in the interstitium. Bars = 50 µm.

Because cellular proliferation in the renal interstitium is observed during the development of interstitial fibrosis in ADPKD (21), we evaluated these areas for the presence of myofibroblasts by staining for smooth muscle alpha -actin. Smooth muscle alpha -actin is found in vascular smooth muscle cells and in fibrogenic myofibroblasts but not in resident interstitial fibroblasts (7). Its expression has been associated with the progression of interstitial fibrosis (22). We found that smooth muscle alpha -actin staining is enhanced in regions of the kidney surrounding Kim-1-expressing clusters of pericystic tubules (Fig. 9, A, C, and E). Smooth muscle alpha -actin expression is not observed in the interstitium adjacent to Kim-1-negative tubules (Fig. 9, B, D, and F). Because nephron loss in ADPKD may be due to apoptosis, we checked TdT-mediated dUTP-X nick-end-labeling staining. There is no increase in the numbers of apoptotic cells in either Kim-1-positive cysts or tubules or in the surrounding interstitium, compared with Kim-1-negative cysts or areas without Kim-1-expressing tubules (not shown). Thus Kim-1 is expressed in tubules that are surrounded by early fibrogenic activity.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 9.   Kim-1 and smooth muscle alpha -actin staining in Pkd2WS25/- kidneys. Sections were stained with polyclonal anti-Kim-1 (R9) and monoclonal anti-smooth muscle alpha -actin antibodies, respectively. Secondary antibodies were Cy3-coupled anti-rabbit and FITC-coupled anti-mouse antibodies. Clusters of Kim-1-expressing proximal tubules adjacent to cysts are seen in areas of interstitial myofibroblast activity, as indicated by the presence of smooth muscle alpha -actin (A, C, and E). No interstitial smooth muscle alpha -actin expression is seen in Kim-1-negative areas (B, D, and F). A: clusters of tubules near a cyst (*) stain positive (red) for apical Kim-1. B: different area adjacent to the same cyst does not show Kim-1 expression in surrounding tubules. C: smooth muscle alpha -actin is present (green) in the interstitium. D: staining for smooth muscle alpha -actin is strong in arteries and present only in minimal amounts in the interstitium. E: combined image of A and C demonstrates that smooth muscle alpha -actin is present in the interstitium surrounding Kim-1-expressing tubules. F: combined image of B and D demonstrates the absence of Kim-1 expression and interstitial smooth muscle alpha -actin. Bars = 50 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Despite important insights into the molecular genetics of ADPKD (24) and recent data about the function of the polycystins (8, 11, 13), the precise mechanism of cyst formation and the pathophysiology leading to interstitial fibrosis and nephron loss in ADPKD remain unknown. In this report, we demonstrate that Kim-1 is expressed in a subset of cysts, without a clear association with cyst size, segment of origin, or proliferative activity. Kim-1 expression in tubules is associated with a partial loss of polarity, as indicated by a loss of complexity and quantity of Na-K-ATPase expression, but preserved staining for actin, villin, and E-cadherin. These findings raise the possibility that Kim-1 plays a role in the dedifferentiation of epithelial cells. Hypothetically, this could explain Kim-1 expression in cysts. The close relationship between apical Kim-1 staining in tubules and disordered Na-K-ATPase expression is not found in cysts as might be expected if Kim-1 expression was tightly coupled to Na-K-ATPase expression. Our findings do not rule out a more complex relationship between Kim-1 and Na-K-ATPase expression in cysts or an effect of Kim-1 expression on the differentiation status of epithelial cells in cysts.

In addition to Kim-1 expression in cysts, we found a striking pattern of Kim-1-expressing noncystic tubules clustered near cysts. These express polycystin-2 by immunostaining (data not shown); hence, Kim-1-positive tubular cells are not characterized by loss of heterozygosity, which is associated with cyst formation in this model. Because Kim-1 expression in proximal tubules is most pronounced after ischemia (12), and ischemia has been proposed to play a role in the pathophysiology of ADPKD (5), we looked for evidence of ischemic injury in Kim-1-expressing pericystic tubular cells. Ischemia is associated with breakdown of the adherens junction and disruption of the apical brush-border actin and villin localization pattern (2, 3). Our findings of normal distribution patterns for E-cadherin, actin, and villin make it very unlikely that tubular Kim-1 expression near cysts is a consequence of ischemic injury.

Tubular obstruction has also been proposed to play a role in progressive nephron loss in ADPKD (25). We found that obstructed kidneys express Kim-1. However, the expression pattern of Kim-1 in postobstructed kidneys is markedly different from the pattern in ADPKD. In postobstructed kidneys, Kim-1 is expressed in entire tubular segments, involving all cells of a given tubule, as opposed to the intermittent epithelial cell staining pattern in cystic kidneys. It is therefore unlikely that Kim-1 expression in pericystic tubules in ADPKD is a consequence of tubular obstruction.

The large number of PCNA-positive cells in the interstitium surrounding Kim-1-expressing tubules together with the presence of smooth muscle alpha -actin-positive cells suggests that Kim-1 expression in proximal tubules adjacent to cysts is associated with interstitial proliferation and fibrosis. This is particularly important because the loss of renal function in patients with ADPKD is not believed to be due solely to the cysts themselves. Interstitial fibrosis and inflammation have been implicated in nephron loss (32). It is possible that pericystic Kim-1 expression in ADPKD kidneys is a consequence of epithelial injury caused by a proliferative and fibrogenic interstitial process. Alternatively, Kim-1 expression in tubular epithelial cells could be a primary event leading to interstitial fibrosis. Proximal tubular cells have been previously invoked in the pathogenesis of tubulointerstitial damage and fibrosis in various animal models of chronic renal disease, such as systemic lupus erythematosus (26), diabetic nephropathy (29), and ureteral obstruction (27), and in studies of human tissues from patients with chronic renal disease (9). The mechanisms resulting in interstitial fibrosis are not understood, but chemokine expression, such as monocyte chemoattractant protein-1, by tubular epithelial cells has been implicated in the recruitment of interstitial macrophages and the development of interstitial fibrosis (26, 27). Recent data from our laboratory that the extracellular portion of Kim-1 is shed from the cell (1a) raise the possibility that shed Kim-1 reaches the interstitium and plays a role in the appearance of myofibroblasts and the proliferative and fibrogenic response. Further support for the concept that Kim-1 is an immunologically active molecule comes from recently published evidence that suggests that molecules of the Kim family are involved in T cell differentiation and macrophage activation (15, 17).

In summary, we demonstrate that Kim-1 is expressed in murine ADPKD but not in normal kidneys. It is expressed in a small number of cysts and in a pattern of proximal tubule clusters adjacent to cysts. Kim-1 expression in single tubule cells is associated with a partial loss of polarity, which is likely unrelated to ischemic injury. Kim-1-expressing tubules are surrounded by a highly proliferative and fibrogenic interstitial response. In conclusion, we propose that Kim-1 expression may play a pathogenic role in the development of interstitial fibrosis and subsequent nephron loss in ADPKD.


    ACKNOWLEDGEMENTS

The authors thank Drs. Sayoko Nishimura and Xin Tian for assistance with the Pkd2WS25/- mice. We thank Dr. Dennis Brown for helpful discussions.


    FOOTNOTES

E. W. Kuehn was supported by Deutsche Forschungsgemeinschaft Grant DFG-Ku 1322/1-1 and the National Kidney Foundation. S. Somlo was supported by National Institutes of Health (NIH) Grants DK-54053 and DK-57328. J. V. Bonventre was supported by NIH Grants DK-39773, DK-38452, DK-46267, and NS-10828. The core facility used for the immunohistochemistry was partially supported by the Center for the Study of Inflammatory Bowell Disease Grant DK-43351.

Address for reprint requests and other correspondence: J. V. Bonventre, Massachusetts General Hospital East, Renal Unit 4th floor, Rm. 4002, 149 13th St., Charlestown, MA 02129 (E-mail: joseph_bonventre{at}hms.harvard.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1  In Ref. 12, rat KIM-1 was capitalized. We have modified the nomenclature and reserved the capitalized form for human KIM-1. We refer to the rodent rat or mouse form as Kim-1 throughout this manuscript.

July 24, 2002;10.1152/ajprenal.00166.2002

Received 30 April 2002; accepted in final form 12 July 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Arystarkhova, E, and Sweadner KJ. Isoform-specific monoclonal antibodies to Na,K-ATPase alpha subunits. Evidence for a tissue-specific posttranslational modification of the alpha subunit. J Biol Chem 271: 23407-23417, 1996[Abstract/Free Full Text].

1a.  Bailly V, Zhang Z, Meier W, Cate R, Sanicola M, and Bonventre JV. Shedding of kidney injury molecule-1, a putative adhesion protein involved in renal regeneration. J Biol Chem In press.

2.   Brown, D, Lee R, and Bonventre JV. Redistribution of villin to proximal tubule basolateral membranes after ischemia and reperfusion. Am J Physiol Renal Physiol 273: F1003-F1012, 1997[Abstract/Free Full Text].

3.   Bush, KT, Tsukamoto T, and Nigam SK. Selective degradation of E-cadherin and dissolution of E-cadherin-catenin complexes in epithelial ischemia. Am J Physiol Renal Physiol 278: F847-F852, 2000[Abstract/Free Full Text].

4.   Cai, Y, Maeda Y, Cedzich A, Torres VE, Wu G, Hayashi T, Mochizuki T, Park JH, Witzgall R, and Somlo S. Identification and characterization of polycystin-2, the PKD2 gene product. J Biol Chem 274: 28557-28565, 1999[Abstract/Free Full Text].

5.   Chapman, AB, Johnson A, Gabow PA, and Schrier RW. The renin-angiotensin-aldosterone system and autosomal dominant polycystic kidney disease. N Engl J Med 323: 1091-1096, 1990[Abstract].

7.   Eddy, AA. Molecular basis of renal fibrosis. Pediatr Nephrol 15: 290-301, 2000[ISI][Medline].

8.   Gonzalez-Perret, S, Kim K, Ibarra C, Damiano AE, Zotta E, Batelli M, Harris PC, Reisin IL, Arnaout MA, and Cantiello HF. Polycystin-2, the protein mutated in autosomal dominant polycystic kidney disease (ADPKD), is a Ca2+-permeable nonselective cation channel. Proc Natl Acad Sci USA 98: 1182-1187, 2001[Abstract/Free Full Text].

9.   Grandaliano, G, Gesualdo L, Ranieri E, Monno R, Montinaro V, Marra F, and Schena FP. Monocyte chemotactic peptide-1 expression in acute and chronic human nephritides: a pathogenetic role in interstitial monocytes recruitment. J Am Soc Nephrol 7: 906-913, 1996[Abstract].

10.   Han, WK, Bailly V, Abichandani R, Thadhani R, and Bonventre JV. Kidney injury molecule-1 (KIM-1): a novel biomarker for human renal proximal tubule injury. Kidney Int 62: 237-244, 2002[ISI][Medline].

10a.   The International Polycystic Kidney Disease Consortium. Polycystic kidney disease: the complete structure of the PKD1 gene and its protein. Cell 81: 289-298, 1995[ISI][Medline].

11.   Hanaoka, K, Qian F, Boletta A, Bhunia AK, Piontek K, Tsiokas L, Sukhatme VP, Guggino WB, and Germino GG. Co-assembly of polycystin-1 and -2 produces unique cation-permeable currents. Nature 408: 990-994, 2000[ISI][Medline].

12.   Ichimura, T, Bonventre JV, Bailly V, Wei H, Hession CA, Cate RL, and Sanicola M. Kidney injury molecule-1 (KIM-1), a putative epithelial cell adhesion molecule containing a novel immunoglobulin domain, is up-regulated in renal cells after injury. J Biol Chem 273: 4135-4142, 1998[Abstract/Free Full Text].

13.   Koulen, P, Cai Y, Geng L, Maeda Y, Nishimura S, Witzgall R, Ehrlich BE, and Somlo S. Polycystin-2 is an intracellular calcium release channel. Nat Cell Biol 4: 191-197, 2002[ISI][Medline].

14.   Lennon, G, Auffray C, Polymeropoulos M, and Soares MB. The I.M.A.G.E. Consortium: an integrated molecular analysis of genomes and their expression. Genomics 33: 151-152, 1996[ISI][Medline].

15.   McIntire, JJ, Umetsu SE, Akbari O, Potter M, Kuchroo VK, Barsh GS, Freeman GJ, Umetsu DT, and DeKruyff RH. Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family. Nat Immun 2: 1109-1116, 2001[ISI].

16.   Mochizuki, T, Wu G, Hayashi T, Xenophontos SL, Veldhuisen B, Saris JJ, Reynolds DM, Cai Y, Gabow PA, Pierides A, Kimberling WJ, Breuning MH, Deltas CC, Peters DJ, and Somlo S. PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein. Science 272: 1339-1342, 1996[Abstract].

17.   Monney, L, Sabatos CA, Gaglia JL, Ryu A, Waldner H, Chernova T, Manning S, Greenfield EA, Coyle AJ, Sobel RA, Freeman GJ, and Kuchroo VK. Th1-specific cell surface protein Tim-3 regulates macrophage activation and severity of an autoimmune disease. Nature 415: 536-541, 2002[ISI][Medline].

18.   Nadasdy, T, Laszik Z, Lajoie G, Blick KE, Wheeler DE, and Silva FG. Proliferative activity of cyst epithelium in human renal cystic diseases. J Am Soc Nephrol 5: 1462-1468, 1995[Abstract].

19.   Okada, H, Ban S, Nagao S, Takahashi H, Suzuki H, and Neilson EG. Progressive renal fibrosis in murine polycystic kidney disease: an immunohistochemical observation. Kidney Int 58: 587-597, 2000[ISI][Medline].

20.   Park, KM, Kramers C, Vayssier-Taussat M, Chen A, and Bonventre JV. Prevention of kidney ischemia/reperfusion-induced functional injury, MAPK and MAPK kinase activation, and inflammation by remote transient ureteral obstruction. J Biol Chem 277: 2040-2049, 2002[Abstract/Free Full Text].

21.   Ramasubbu, K, Gretz N, and Bachmann S. Increased epithelial cell proliferation and abnormal extracellular matrix in rat polycystic kidney disease. J Am Soc Nephrol 9: 937-945, 1998[Abstract].

22.   Roberts, IS, Burrows C, Shanks JH, Venning M, and McWilliam LJ. Interstitial myofibroblasts: predictors of progression in membranous nephropathy. J Clin Pathol 50: 123-127, 1997[Abstract].

23.   Sabolic, I, Valenti G, Verbavatz JM, Van Hoek AN, Verkman AS, Ausiello DA, and Brown D. Localization of the CHIP28 water channel in rat kidney. Am J Physiol Cell Physiol 263: C1225-C1233, 1992[Abstract/Free Full Text].

24.   Somlo, S, and Markowitz GS. The pathogenesis of autosomal dominant polycystic kidney disease: an update. Curr Opin Nephrol Hypertens 9: 385-394, 2000[ISI][Medline].

25.   Tanner, GA, Gretz N, Connors BA, Evan AP, and Steinhausen M. Role of obstruction in autosomal dominant polycystic kidney disease in rats. Kidney Int 50: 873-886, 1996[ISI][Medline].

26.   Tesch, GH, Maifert S, Schwarting A, Rollins BJ, and Kelley VR. Monocyte chemoattractant protein 1-dependent leukocytic infiltrates are responsible for autoimmune disease in MRL-Fas(lpr) mice. J Exp Med 190: 1813-1824, 1999[Abstract/Free Full Text].

27.   Vielhauer, V, Anders HJ, Mack M, Cihak J, Strutz F, Stangassinger M, Luckow B, Grone HJ, and Schlondorff D. Obstructive nephropathy in the mouse: progressive fibrosis correlates with tubulointerstitial chemokine expression and accumulation of CC chemokine receptor 2- and 5-positive leukocytes. J Am Soc Nephrol 12: 1173-1187, 2001[Abstract/Free Full Text].

28.   Wagner, MC, and Molitoris BA. Renal epithelial polarity in health and disease. Pediatr Nephrol 13: 163-170, 1999[ISI][Medline].

29.   Wang, SN, and Hirschberg R. Growth factor ultrafiltration in experimental diabetic nephropathy contributes to interstitial fibrosis. Am J Physiol Renal Physiol 278: F554-F560, 2000[Abstract/Free Full Text].

30.   Woo, D. Apoptosis and loss of renal tissue in polycystic kidney diseases. N Engl J Med 333: 18-25, 1995[Abstract/Free Full Text].

31.   Wu, G, D'Agati V, Cai Y, Markowitz G, Park JH, Reynolds DM, Maeda Y, Le TC, Hou H, Jr, Kucherlapati R, Edelmann W, and Somlo S. Somatic inactivation of Pkd2 results in polycystic kidney disease. Cell 93: 177-188, 1998[ISI][Medline].

32.   Zeier, M, Fehrenbach P, Geberth S, Mohring K, Waldherr R, and Ritz E. Renal histology in polycystic kidney disease with incipient and advanced renal failure. Kidney Int 42: 1259-1265, 1992[ISI][Medline].

33.   Zuk, A, Bonventre JV, Brown D, and Matlin KS. Polarity, integrin, and extracellular matrix dynamics in the postischemic rat kidney. Am J Physiol Cell Physiol 275: C711-C731, 1998[Abstract].


Am J Physiol Renal Fluid Electrolyte Physiol 283(6):F1326-F1336
0363-6127/02 $5.00 Copyright © 2002 the American Physiological Society