Abstract
Recent studies indicate that the Notch signaling pathway plays an important role in diabetic kidney disease (DKD) and focal segmental glomerulosclerosis (FSGS) development, but the specificity and the clinical significance of Notch activation have not been studied in a broader set of diseases.
Here we analyzed the degree of expression and localization of Notch ligands (Jagged1 and Delta1) and Notch receptors (Notch1 and Notch2) in healthy human kidneys and in biopsy samples obtained from patients with minimal change disease, membranous nephropathy, lupus nephritis ISN/RPS classes III/IV/V, hypertensive nephrosclerosis, crescentic glomerulonephritis, tubulointerstitial fibrosis, IgA nephropathy, DKD and FSGS.
We found that cleaved Notch1, Notch2 and Jagged1 are expressed on podocytes in proteinuric nephropathies and their level of expression correlates with the amount of proteinuria (across all disease groups). The degree of glomerulosclerosis correlated with podocyte expression of cleaved Notch1, while the severity of tubulointerstitial fibrosis and the estimated glomerular filtration rate correlated with expression of cleavedNotch1 in the tubulointerstitium.
In summary, here we show that the expression of Notch pathway proteins correlates with proteinuria and kidney dysfunction in a wide range of acquired renal diseases. Our results raise the possibility that Notch pathway activation is a common mechanism in the development of albuminuria, glomerulosclerosis and kidney dysfunction.
INTRODUCTION
Diseases of the glomerulus; diabetic-(DKD)and hypertensive-kidney disease and focal segmental glomerulosclerosis (FSGS), are responsible for >75% of chronic kidney disease (CKD) cases in the US (1). Genetic studies identified mutations in a handful of genes (NPHS1, NPHS2, CD2AP, ACN4, TRPC6, PLCE1, MYH9 etc.) responsible for glomerulosclerosis (2). The majority of ESRD cases, however, are not caused by a single genetic mutation. The mechanism of glomerulosclerosis development is unclear even when the disease mutation is identified. Recent studies highlighted the critical role of podocytes in development of albuminuria and glomerulosclerosis. Interestingly, the progression of glomerulosclerosis and chronic renal disease on a phenotypic level appears to follow a similar pattern, potentially indicating that a common pathway plays role in disease progression(3, 4).
The Notch signaling pathway is a basic cell-cell communication mechanism. The main components of its are: the ligands Jagged (Jag1 and 2) and Delta (Dll1, 3 and 4); and the Notch transmembrane receptor proteins (Notch1–4) (5). Activation of this signaling pathway requires cell-cell contact. Binding of the ligand leads to a series of proteolytic cleavages of the Notch receptor and finally to the release of the active Notch intracellular domain (ICN). ICN then travels to the nucleus and binds to other transcriptional regulators (mainly of the CSL family) to trigger the transcription of target genes (classically Hes and Hey genes)(6).
The spatially and temporally orchestrated expression of different Notch pathway proteins plays a key role in kidney development(7–10). Notch1, Notch2, Delta1 and Jagged1 mRNA can be detected in the renal vesicle and its derivatives; Notch2 and Jagged1 are also expressed in the collecting duct; Notch4 expression is mainly restricted to endothelial cells, and Notch3 to the distal portion of the S-shaped body(11, 12). Both Notch1 and Notch2 are expressed in the S-shaped body (10). Elegant studies performed by Cheng et al. (9) showed that while both Notch1 and Notch2 expression were detected in the early renal vesicle. Genetic deletion of Notch1 did not alter kidney development, but in the absence of Notch2 proximal kidney (glomerular and proximal tubule) development was significantly impaired. These observations indicate important subtype specificity for the two Notch receptors.
Recent studies from our and other laboratories indicated that activation of the Notch pathway in podocytes plays critical functional role in the development of proteinuria and glomerulosclerosis (13, 14). Immunohistological experiments showed that cleaved Notch1 was expressed in podocytes of human and murine diabetic kidneys and in patients with FSGS (13). Transgenic expression of Notch1 in podocytes caused nephrotic syndrome and glomerulosclerosis. Genetic deletion or pharmacological inhibition of the Notch pathway significantly reduced albuminuria in rodent glomerular disease models (15)(13).
In addition, Notch expression might not be limited to the glomerulus and DKD, but could be observed in tubular epithelial cells and in a variety of renal disorders as well. Gene expression arrays performed on control and DKD kidneys showed increased Jagged1 mRNA levels in the tubulointerstitial compartment of DKD patients (16). Jagged1 was also identified as one of the top differentially expressed transcripts by microarray studies performed on murine model of tubulointerstitial fibrosis (unilateral ureteral obstruction) (17),(18, 19).
The aim of the current study was to: 1) describe the degree of expression and the localization of Notch receptors and ligands in control and diseased human kidneys;2) examine whether the expression of Notch pathway molecules is specific for DKD and FSGS or can be observed in other acquired renal diseases as well;3) determine whether there are differences in the expression of different Notch ligands and receptors. Thus we analyzed Notch pathway protein expressions in control and kidney biopsy samples obtained from 10 acquired kidney disease groups and analyzed their correlations with renal functional and histological changes.
Results
Patient characteristics
A total of 86 biopsy samples from subjects with acquired kidney diseases were examined and grouped based on the histopathologic diagnosis: minimal change disease (MCD, n=3), focal segmental glomerulosclerosis (FSGS, n=9), DKD (n=9), idiopathic membranous nephropathy (IMN, n=9), lupus nephritis ISN/RPS class III focal-or IV diffuse-proliferative (LN class III/IV, n=9), lupus nephritis ISN/RPS class V (membranous, LN class V, n=9), IgA nephropathy (IgAN, n=8), hypertensive nephrosclerosis (HN, n=8), tubulointerstitial nephritis (TIN, n=8), crescentic vasculitis (crescentic, n=7). Seven biopsy samples obtained from the unaffected portion of tumor nephrectomies (n=3) or from protocol living donor biopsies (n=4) were used as controls. The demographic and clinical characteristics of the research participants at the time of the kidney biopsy are summarized in Table I. The average age of our cohort was 43, with overrepresentation of females (female=59%). The highest degree of proteinuria was observed in patients with diabetic kidney disease (7.9 g/day) and with idiopathic membranous nephropathy (7.8 g/day). Patients with crescentic glomerulonephritis had the worst kidney function (eGFR=26cc/min/1.73m2)at the time of the kidney biopsy.
TABLE 1.
Demographics and Clinical Characteristics of the Research Participants
| Diagnosis | No. of cases | Gender M/F | Age (years) | Hematuria | Proteinuria (g/day) | eGFR (ml/min) |
|---|---|---|---|---|---|---|
| Control | 7 | 4/3 | 47±9.96 | 0.8±0.99 | 0 (0, 0.2) | 92.87±11.29 |
| DKD | 9 | 4/5 | 52.5±6.7 | 0±0 | 6 (5, 10) | 45.45±21.39 |
| FSGS | 9 | 3/6 | 26.8±18.5 | 0.2±0.41 | 2 (1, 2) | 77.24±25.23 |
| MCD | 3 | 1/2 | 44±22.6 | 0.3±0.4 | 1.5 (0.5, 4) | 75.19±17.56 |
| IMN | 9 | 3/5 | 52±19.7 | 0.3±0.66 | 7.5 (4, 9) | 65.42±30.64 |
| LN class V | 9 | 3/5 | 40.5±13.8 | 0.6±0.66 | 3.5 (3, 5) | 67.18±37.96 |
| LN class III/IV | 9 | 1/8 | 33±14.06 | 1.2±0.78 | 2 (1.5, 6.5) | 77.17±29.23 |
| IgAn | 8 | 5/3 | 41.8±18.7 | 1.7±0.82 | 1 (0.8, 1.65) | 77.23±28.97 |
| HN | 8 | 4/4 | 57.3±16 | 0.2±0.33 | 0.95 (0.65, 3.5) | 40.92±25.5 |
| TIN | 8 | 5/3 | 34.6±15.9 | 0.6±0.99 | 1.25 (0.6, 2.25) | 40.84±16.41 |
| Crescentic | 7 | 2/5 | 47.5±16.6 | 2±0.92 | 2 (1.5, 4) | 26.52±21.18 |
Values are expressed as means ± SD for age, hematuria and eGFR; and as median and (IQR) for proteinuria.
Abbreviations: MCD minimal change disease; IMN idiopathic membranous nephropathy, LN lupus proliferative glomerulonephritis classes III and IV; LN class V lupus membranous nephropathy; IgAN immunoglobulin A nephropathy; DKD; diabetic kidney disease; HN hypertensive nephrosclerosis; TIN tubulointerstitial nephritis; eGFR was calculated using the MDRD formula, Hematuria is quantified as described under methods
Renal Histology
Table II summarizes the baseline histopathological changes observed in the kidney biopsies. We used the diagnosis and the results provided by the clinical pathologist and used during clinical patient care. We analyzed biopsy samples from wide range of renal disease groups, making it difficult to compare disease specific histological abnormalities. We focused on the degree of glomerulosclerosis and tubulointerstitial fibrosis. Prior histopathological studies indicate that the degree of glomerulosclerosis and tubulointerstitial fibrosis strongly correlates with renal function across the different disease entities (20, 21). Similar to prior reports, in our cohort we found correlation between the degree of tubulointerstitial fibrosis across all disease groups and the estimated GFR (Supplemental Table I). From the different disease groups we observed the highest degree of glomerulosclerosis (69%) and tubulointerstitial fibrosis (73%) in the DKD biopsies, followed by samples obtained from tubulointerstitial nephritis and hypertensive nephrosclerosis (Table II). The degree of glomerulosclerosis and tubulointerstitial fibrosis was low in the control samples (3 and 6%, respectively).
TABLE 2.
Histopathological Characteristic of the Kidney Biopsy Samples
| Disease group | No. of glomeruli | Glomerulosclerosis (%) | Tubulointerstitial fibrosis (%) |
|---|---|---|---|
| Control | 10.5 | 0 (0, 7) | 0 (0, 10) |
| DKD | 19.8 | 75 (42, 100) | 80 (60, 85) |
| FSGS | 15.4 | 40 (25, 55) | 10 (10, 30) |
| MCD | 8 | 33 (5, 100) | 20 (20, 20) |
| IMN | 15.5 | 16. (8.5, 23.5) | 22.5 (15, 62.5) |
| LN class V | 21 | 15 (0, 25) | 20 (17.5, 35) |
| LN class III/IV | 16.5 | 16.5 (0, 57.5) | 20 (10, 32.5) |
| IgAN | 13.8 | 22.5 (12.5, 54) | 20 (15, 25) |
| HN | 12.8 | 57.5 (29, 82.5) | 35 (15, 75) |
| TIN | 11.1 | 24 (0, 45) | 50 (45, 65) |
| Crescentic | 10.2 | 33 (0, 77) | 15 (10, 50) |
Values are expressed as median (IQR) for glomerulosclerosis and tubulointerstitial fibrosis
Expression of Notch receptors and ligands in healthy adult human kidney samples
First we characterized the expression of Notch ligands and receptors in control “healthy ” human kidney samples. We used the immunoflourescence method with established antibodies to determine the expression of different Notch ligands and for the receptors we used antibodies specific for the cleaved Notch 1 and Notch2 (13, 22–24). Podocyte specific expression was confirmed by double immunostaining with podocyte marker WT-1. We used the mouse monoclonal WT-1 antibody that gave a cytoplasmic stain of the human glomeruli. The quality and the specificity of the antibodies were tested on Western blots(shown in the supplemental figures 3 and 4).
Similar to prior reports, we could not observe expression of activated Notch 1, Notch 2 and Delta1 in glomeruli of control “healthy ” human kidney biopsy samples (Fig 1)(13, 24). Our antibody detected a faint signal for Jagged 1 in healthy glomeruli, and double immunostaining for Jagged 1 and WT-1 indicated a possible podocyte specific localization (Fig 1). In the tubules of healthy adult kidneys there was no significant expression of Delta1 (25), but we occasionally observed positive immunostaining for cleaved Notch1 and Notch2 in tubule segments (Fig 1)(24).
Figure 1. Expression of Notch molecules in control human kidneys.
Double immunostaining of a single control (healthy) human kidney sample with WT-1 (A, B, C, D) (FITC;green) and cleaved Notch1 (A), and Notch2 (B), Jagged1 (C) or Delta1 (D) (Cy3;red), nuclear stain with DAPI(blue) or their combination shown as indicated on the figure.
In summary, there was no significant expression of cleaved Notch receptors in healthy adult human glomerulus, while there was minimal expression of cleaved Notch1 and Notch2 in the tubulointerstitium.
Expression of Notch receptors in diseased kidney tissue
Next we analyzed the level of expression and localization of cleavedNotch1 and Notch2 proteins in diseased kidney samples. The amplitude of nuclear expression (examined by DAPI co-staining) was scored on a 0 to 4 scale (see methods). We validated this method by using computerized image analysis (Image J) which showed excellent correlation (P=0.01) with the dual observer semi-quantitative assessment. In addition, this method has been used successfully recently and in the past by other investigators (26). Representative staining images from a single biopsy are shown in Figure 2. In addition, images from representative scoring values are presented in Supplemental Figure 1.
Figure 2. Expression of Notch molecules in diseased human kidney biopsies.
Double immunostaining from a single diseased human kidney samples with WT-1 (FITC; green) and cleaved Notch1 (A), and Notch2 (B), Jagged1 (C) or Delta1 (D) (Cy3;red) nuclear stain with DAPI (blue) as indicated on the figure. The pictures were taken from a single case of membranous nephropathy.
First, the presence of positive immunostaining for activated Notch1 and Notch2 in human DKD and FSGS samples was confirmed. While these antibodies also show some background staining we only considered it as a positive label when the nuclei were stained. In addition, we observed positive glomerular Notch1 and Notch2 immunostaining in multiple other renal diseases as well. Interestingly, when we examined the pattern of expression of active Notch1 and Notch2 across all disease groups, we found significantly higher expression of these proteins in diseases that usually manifest with nephrotic-range proteinuria, such as DKD, FSGS, minimal change disease, membranous nephropathy, lupus nephritis classes III/IV/V, or can manifest with nephrotic-range proteinuria, such as IgA nephropathy. The expression was not significantly different from controls in diseases generally not associated with heavy proteinuria such as hypertensive nephrosclerosis and tubulointerstitial nephritis samples (Fig 2 and 3). Delta1 was not expressed in the glomerulus. The expression of Jagged1 on podocytes generally followed and correlated with the expression of cleaved Notch2(Fig 2 and 3).
Figure 3. Glomerular specific expression of cleaved Notch1, 2 and Jagged1 in different renal disease groups.
The relative expression of active Notch1 (blue bar) Notch2 (red bar) and Jagged1 (yellow bar) in the glomerulus was scored in each biopsy sample. The graph shows the mean and SD of the expression of each molecules. * denotes statistically significant differences (p<0.05) when compared to controls.
Tubulointerstitial expression of cleaved Notch2 was increased in almost all disease groups, and we could not observe a clear disease specific distribution (Fig 2 and 4). Cleaved Notch1 expression in the tubulointerstitium was significantly increased in the DKD samples (Fig 4). There was significantly increased expression of Jagged1 and Delta1 in many different diseases, without a clear disease specific distribution (Fig 2 and 4).
Figure 4. Tubulointerstitial expression of cleaved Notch1, 2, Jagged1 and Delta1 in different renal disease groups.
The relative expression of activeNotch1 (blue bar), Notch2 (red bar), Jagged1 (yellow bar) and Delta1 (light blue bar) in the tubulointerstitium was scored in each biopsy sample. The graph shows the mean and SD of the expression of each molecules. *denotes statistically significant differences (p<0.05) when compared to controls.
Next, we examined whether there is a correlation between the expression of different Notch proteins in the glomerular and tubulointerstitial compartment (Supplemental Table I). Such analysis revealed that Notch2, expression on podocytes correlated with Notch1 and Jagged1 expression on podocytes. Jagged1 expression in podocytes directly correlated Jagged1 and Notch2 expressions in the tubulointerstitial compartment (Supplemental Table I). Expression of Delta1 in the tubulointerstitial compartment correlated with tubular and podocyte expression of Notch2 and podocyte expression of Jagged1 (Supplemental Table I).
In summary, our results indicate that Notch ligands and receptors are expressed in a wide range of renal diseases.
Correlation of Notch ligand and receptor expression and renal histology
We detected an increased Notch expression in different renal disorders without clear disease specificity, therefore we examined whether the expression of Notch ligands and receptors shows correlation with histological parameters including glomerular and tubulointerstitial fibrosis across all disease samples. Using linear regression, we found a positive linear correlation between podocyte and tubulointerstitial expression of active Notch1 and the percent of sclerosed glomeruli in the biopsy samples (p=0.002, p=0.004, respectively) (Fig 5A and 6A). These results were significant even after adjusting for the multiple comparisons using the Bonferroni method. The expression of active Notch1 in the tubulointerstitium showed a strong positive linear correlation with the percent of tubulointerstitial fibrosis, when examined across all 10 different renal disease conditions (p=0.001, β=9.14) (Fig 5B and 6B).
Figure 5. Result of the linear regression analysis of Notch expression and histological and functional renal parameters.
Results of linear regression analysis (significance and correlation coefficient) of Notch pathway protein expression (Podocyte and tubulointerstitial active Notch1, Notch2, Jagged1 and Delta1) and proteinuria (log transformed) (A) estimated GFR (B) glomerulosclerosis (log transformed) (C), tubulointerstitial fibrosis (D). The Bonferroni adjusted p-value for this analysis is 0.002 or the unadjusted p-value 0.05 were considered statistically significant.
Figure 6. Correlation plots of PodocyteNotch1 expression and glomerulosclerosis and tubulointerstitial fibrosis and tubulointerstitial expression of active Notch1.
Scatter-plot with fitted values (red line) and 95% confidence (green line) intervals for (A) percent glomerulosclerosis and podocyte expression of Notch1 and (B) and tubular expression of Notch1 and tubulointerstitial fibrosis.
In summary, these results indicate that the expression of active Notch1 correlates with the degree of glomerular and tubulointerstitial fibrosis virtually in all renal disease categories.
Renal function parameters correlate with Notch expression
Next we studied whether there is a correlation between the expression of Notch proteins and renal functional parameters. In our dataset, across all spectrums of acquired renal diseases, the degree of proteinuria (analyzed as grams/day at the time of biopsy and normalized by log transformation) correlated with podocytes-specific expression level of active Notch1, Notch2, and Jagged1 (Fig 5C and 6C). Expression of Notch proteins in the tubulointerstitium on the other hand did not show significant correlation with the amount of proteinuria(Fig 5C). This finding might further support the central role of podocytes in the development of proteinuria.
Estimated GFR, at the time of biopsy, showed statistically significant association with tubulointerstitial expression of active Notch1 and Notch2 and podocyte expression of Jagged1. Interestingly, tubular Notch1 expression and GFR were in an inverse correlation, i.e. samples with increased Notch1 had a lower GFR, whereas tubular Notch2 expression level was in positive correlation with GFR, as samples with increased Notch2 expression had a better GFR (Fig 5D). Our results indicate that Notch expression across multiple renal disease groups correlates with renal functional parameters.
Discussion
This study represents the initial step to characterize the expression of the Notch pathway molecules in control and diseased human kidney tissue samples. The carefully choreographed expression of different Notch pathway proteins play a crucial role in kidney development (11). Our study indicates that the Notch pathway –like most developmental pathways (27)- is mostly silenced in the glomeruli of normal mature human kidney. We found that few tubular epithelial cells remain positive for active Notch protein expression in the adult human kidney. Similarly, the Kopan group using Notch1Cre mediated mouse genetic model also found some Notch activity in tubular epithelial cells of adult mice (24). It is intriguing to speculate the role these Notch positive tubular cells could play in the kidney. In other organs (best characterized in skin and intestinal epithelial cells) Notch positive cells are regarded as progenitor-like cells and are often involved in organ regeneration, maintenance and renewal(28, 29). It would be interesting to know whether Notch positive cells in the kidney play such role.
The availability of samples from a wide variety of kidney disorders enabled us to extend our initial observations in DKD and FSGS (13). We found that the expression of Notch pathway molecules is not restricted to DKD and FSGS, but can be observed in many acquired kidney disorders without following a disease specific expression. Therefore, we studied whether the expression of Notch proteins correlate with histological and functional parameters. Using this approach, when we analyzed samples across disease groups (regardless of the baseline histopathological diagnosis), we found that the expression of the cleaved Notch1 fragment in the podocytes/glomerulus correlated with albuminuria and glomerulosclerosis. The strength of this approach is that it does not depend on the clinical diagnosis, which is often less rigorous than when diagnosis is made to research studies. Glomerular Jagged1 and Notch2 expression only showed correlation with proteinuria. This observation makes us speculate that podocyte Notch expression might represent a final common (downstream) pathway for albuminuria and glomerulosclerosis development. To our knowledge, this is one of the first molecules whose expression shows correlation with renal function across multiple human disease samples.
We also studied the relationship between Notch expression and renal function parameters. Most previous immunohistological studies examined expression of a number of other molecules (podocyte nephrin, podocin, CD2AP, α-actinin, WT-1, tubular kidney injury molecule-1, tissue transglutaminase-2) and their associations with morphologic damage and baseline clinical data, but not with renal function(30, 31). A very nicely performed study by Eardley et al. showed correlation between albuminuria and MCP-1 expression in the kidney (32). Our functional analysis across multiple disease groups revealed some interesting observations. We found that while podocyte expression of Notch molecules showed the best correlation with albuminuria, Notch1 expression in the tubules showed the strongest association with eGFR and with tubulointerstitial fibrosis. These observations are consistent with some prior pathological and clinical studies indicating that the degree of tubulointerstitial fibrosis shows the strongest correlation with renal function (20, 21). Based on our results, we can further hypothesize that the Notch pathway could also play role in the development of tubulointerstitial fibrosis. Prior studies indicated the expression of Notch1/Jagged1 in a mouse model of tubulointerstitial fibrosis (17)and their role in the TGFβ induced epithelial to mesenchymal transformation in vitro (18).
It is also worth to mention that we could not detect a clear correlation between podocyte expression of Notch molecules and renal function or the degree of tubulointerstitial fibrosis at the time of biopsy. These results make us speculate on the differential role for the Notch pathway in podocytes and in albuminuria development and the expression of Notch in tubular cells and in tubulointerstitial fibrosis development.
In our study we found differences in expression of different ligands and receptors. Most importantly, increased Notch1 levels were associated with increased TIF and decreased eGFR, while tubulointerstitial expression of Notch2 was observed in samples with less TIF and better renal function. These results would suggest that similar to renal development where Notch1 and Notch2 play non-redundant role in kidney development, Notch1 and Notch2 might play different roles in tubulointestitial fibrogenesis as well (7). A single study that looked at the function of Notch2 proteins in experimental acute kidney injury showed that tubular Delta 1 and Notch2 expression is increased during ischemic injury in vivo. In vitro co-culturing experiments also showed that Delta1 stimulated proliferation of cultured tubular epithelial cells, suggesting that Delta 1 may play role in the tubular regenerative process (25). What balances the differential expression of these molecules is unknown at the present time, but one could speculate that growth factors (for example VEGF and TGFβ) could be important. While our study is purely observational in nature, nevertheless might support the concept from previous experiments indicating the different roles for the various Notch isoforms.
We used fluorescent immunohistochemistry-based analysis of renal biopsy samples to detect Notch receptors and ligands. The approach has several advantages, most importantly that it can detect active signaling unit for Notch1 and Notch2 proteins, as opposed to in situ hybridization that can only detect transcript levels. It is well recognized that transcript levels might not directly correlate with cleaved protein expression. These cleavage-site specific antibodies have been used on multiple occasions previously and appear to be highly specific (13, 24). In addition, the immunohistochemistry approach can be performed on small amounts of frozen or paraffin-embedded tissues. Potential limitations of our study are that it is from a single center and analyzed different disease groups together. For example we only had a limited number (n=3) of biopsies with minimal change disease, as it is not very common in adults. Another limitation of the study is that the results have not been adjusted for multiple comparisons and therefore they should be interpreted carefully, and need to be confirmed in larger studies with greater statistical power. It is not clear whether such adjustment should be made because this is the first and exploratory analysis defining the expression of the Notch pathway in human kidney biopsy samples and second because some of the most recent results suggest that the Bonferroni correction might be too stringent for multiple comparisons(33). However, even in case we use the very stringent p-values suggested by the Bonferroni adjustment the association between glomerulosclerosis and podocyte Notch1 expression remains statistically significant. Similarly, the association between tubule expression of Notch1 and tubulointerstitial fibrosis and glomerular filtration rate remained statistically significant, further supporting our findings.
In conclusion, we found that in the human kidney Notch ligands and receptors are expressed in a wide range of renal diseases, with podocyte specific Notch expression showing correlation with albuminuria and glomerulosclerosis. Tubulointerstitial abundance of specific Notch-signaling molecules showed association with renal function and histologic predictors of renal outcome. Our findings could indicate that Notch activation might represent a common downstream pathway for albuminuria, glomerulosclerosis and tubulointerstitial fibrosis. These results may help to understand the development of chronic renal disease and generate novel candidate molecular markers and therapeutic targets.
MATERIALS AND METHODS
Research Subjects
Renal biopsies diagnosed at Montefiore Medical Center from 2004–2007 were reviewed and cases with sufficient material (at least 5 glomeruli) for further study were selected. Renal biopsies were processed by standard techniques for light, immunofluorescent and electron microscopy and clinical diagnosis was made based on these stainings. Corresponding histopathological and clinical information was collected by chart review. The percent glomerulosclerosis and tubulointerstitial fibrosis was obtained from the standard pathology report and represent the percentage of glomeruli or tubulointerstitium with fibrosis. The study was approved by the Institutional Review Board.
Immunostaining
OCT-embedded frozen kidney tissue was cryotome cut at 5 μm. Kidney sections were fixed in ice-cold acetone for 10 min then washed with PBS for 5 min. The tissue was blocked with 1% Fish skin gelatin (FSG) for 30 min at room temperature. Primary antibodies diluted in 1% FSG (anti-notch1 1:200, anti-notch2 1:200, anti-Jagged 1:200, anti-Delta1 1:100, anti-WT1 1:100) were incubated at 4°C overnight. Thereafter, slides were washed in PBS and secondary antibodies (goat anti-rabbit Cy3 1:500, donkey anti-mouse FITC 1:150) were incubated for 30 min at room temperature. Slides then washed with PBS were counterstained with DAPI and mounted with Fluoromount. The following primary antibodies were used; cleaved Notch1 (Abcam, ab8925) rabbit polyclonal IgG, cleaved Notch2 (Abcam, ab8926) rabbit polyclonal IgG, Jagged 1 (Santa Cruz, sc-8303) rabbit polyclonal IgG, Delta 1 (Santa Cruz, sc-9102) rabbit polyclonal IgG, WT1 (Santa Cruz, sc-81617)mouse monoclonal IgG,. Specificity of the primary antibodies used in this study was tested on Western Blots(Supplemental Figure 3). Control staining with secondary antibodies only was also performed, to evaluate if there was presence of nonspecific background antibody binding (pictures shown in Supplementary figure 2).
Image analysis
Slides were examined with Nikon Eclipse TE300 fluorescence microscope (City, state) and pictures were taken with SPOT Diagnostic CCD camera. Immunostaining was evaluated in a blinded fashion. A zero to four (0–4) relative scale was used to grade the amount of immunostaining; 0 <5% staining; 1+, 5–10% staining; 2+, 10–25% immunostaining; 3+, 25–50% immunostaining; 4+>50% immunostaining. In case of linear staining, such as Jagged-1, the percentage of positive staining in relation to the total glomerular cross sectional area defines the scale. In case of discrete nuclear staining of cleaved Notch-1 and Notch-2, the percentage of positively stained nuclei per all glomerular nuclei has been used. Co-immunostaining for podocyte marker WT1 and DAPI and the use of overlapping microscope image aided in determination of glomerular podocyte or tubulointerstitial localization of Notch-complex proteins. Slides were analyzed by 2 independent observers blinded to the diagnosis. There was a very good congruence observed in the dual blinded scoring (Pearson correlation 0.97). For each stained molecule and each case, average glomerular and tubulointerstitial immunofluorescence intensity and amount was determined.
Statistical analysis
Results are presented as the mean and standard deviation, or median and interquartile range (IQR) when necessary for our descriptive statistical data. Continuous variables were assessed by ANOVA followed by Tukey test. Nonparametric data were assessed by Kruskal-Wallis test followed by Dunn’s test. Pearson’s correlations were used to evaluate the association between two continuous variables. Non-linearly distributed outcome variable for proteinuria was logarithmically transformed. Because of the hypothesis-generating exploratory analysis of this data, there is some argument in the medical literature about whether or not correction needs to be made for multiple comparisons (33). The Bonferroni adjusted p-value for this analysis is 0.002.
Linear regression was performed to evaluate associations between stained molecules and baseline parameters. A two-sided p-value < 0.05 indicated statistical significance. Error bars represent standard deviation. All statistical analyses were performed using STATA 10.0 (College Station, TX).
Supplementary Material
Supplemental Figure 1. Expression of Notch molecules in diseased human kidney samples
Double immunostaining of diseased human kidney samples with WT-1 (green) and cleaved Notch1 (A), and Notch2 (B), Jagged1 (C) or Delta1 (D) (red) showing representatives images with different scores in different diseased kidney samples.
Supplemental Figure 2. Secondary antibody labeling for the immunostaining experiments
The figure shows the labeling with secondary antibodies only (in the presence of non-immune serum). (A) Anti-rabbit Cy3 secondary antibody only; (B) Anti-rabbit Cy3 + Anti-mouse FITC (showing anti-rabbit Cy3); and (C) Anti-rabbit Cy3 + Anti-mouse FITC (showing anti-mouse FITC)
Supplemental Figure 3. Evaluation of the primary antibodies using Western blots
Western blot analysis using (A) activeNotch-1; (B) cleaved Notch-2; (C) Jagged-1; and (D) Delta-1 antibodies on kidney cell lysates. The antibodies labeled a single band of the correct molecular weight as shown on the blots.
Supplemental Table 1. Expression of Notch molecules in diseased human kidney samples
The correlation matrix between renal expression of Notch pathway molecules, renal histological and functional parameters. The p values for the statistically significant associations are shown under the correlation coefficients in bold. P-N1; podocyte Notch1 expression, P-N2 podocyte Notch2 expression, P-J1 podocyte Jagged1 expression, T-N1 tubule Notch1 expression, T-N2 tubule Notch2 expression, T-J1 tubule Jagged1 expression, T-D1 tubule delta1 expression, Alb; proteinuria at the time of biopsy (log transformed), GS; glomerulosclerosis, GFR glomerular filtration rate, TIF, tubulointerstitial fibrosis.
Acknowledgments
Source of Support: NIH R01DK076077 and JDRF RRG to K.S and NIH K23DK078774 to M.L.M.
We would like to thank Ari Benson for his help and the members of the clinical pathology lab for their help collecting the kidney tissue samples. This work was supported by the National Institute of Health (R01DK076077 to K.S) and (K23DK078774 to M.L.M) and by a Regular Research Grant from the Juvenile Diabetes Foundation (to K.S).
Footnotes
Disclosure: The authors have nothing to disclose.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Supplemental Figure 1. Expression of Notch molecules in diseased human kidney samples
Double immunostaining of diseased human kidney samples with WT-1 (green) and cleaved Notch1 (A), and Notch2 (B), Jagged1 (C) or Delta1 (D) (red) showing representatives images with different scores in different diseased kidney samples.
Supplemental Figure 2. Secondary antibody labeling for the immunostaining experiments
The figure shows the labeling with secondary antibodies only (in the presence of non-immune serum). (A) Anti-rabbit Cy3 secondary antibody only; (B) Anti-rabbit Cy3 + Anti-mouse FITC (showing anti-rabbit Cy3); and (C) Anti-rabbit Cy3 + Anti-mouse FITC (showing anti-mouse FITC)
Supplemental Figure 3. Evaluation of the primary antibodies using Western blots
Western blot analysis using (A) activeNotch-1; (B) cleaved Notch-2; (C) Jagged-1; and (D) Delta-1 antibodies on kidney cell lysates. The antibodies labeled a single band of the correct molecular weight as shown on the blots.
Supplemental Table 1. Expression of Notch molecules in diseased human kidney samples
The correlation matrix between renal expression of Notch pathway molecules, renal histological and functional parameters. The p values for the statistically significant associations are shown under the correlation coefficients in bold. P-N1; podocyte Notch1 expression, P-N2 podocyte Notch2 expression, P-J1 podocyte Jagged1 expression, T-N1 tubule Notch1 expression, T-N2 tubule Notch2 expression, T-J1 tubule Jagged1 expression, T-D1 tubule delta1 expression, Alb; proteinuria at the time of biopsy (log transformed), GS; glomerulosclerosis, GFR glomerular filtration rate, TIF, tubulointerstitial fibrosis.