Title: Expression of Notch pathway proteins correlates with albuminuria, glomerulosclerosis, and renal function
Abstract: Recent studies indicate that the Notch signaling pathway plays an important role in the development of diabetic kidney disease and focal segmental glomerulosclerosis (FSGS). Here we analyzed the degree of expression and localization of Notch ligands (Jagged1 and Delta1) and activated (cleaved) receptors (Notch1 and Notch2) in healthy human kidneys and in renal biopsies from a wide variety of kidney diseases. These included patients with minimal change disease, membranous nephropathy, lupus nephritis ISN/RPS classes III/IV/V, hypertensive nephrosclerosis, crescentic glomerulonephritis, tubulointerstitial fibrosis, IgA nephropathy, diabetic kidney disease, and FSGS. We found that cleaved Notch1, Notch2, and Jagged1 are expressed on podocytes in proteinuric nephropathies and their level of expression correlated 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 cleaved Notch1 in the tubulointerstitium. Hence, our results raise the possibility that Notch pathway activation is a common mechanism in the pathophysiology of a wide range of acquired renal diseases. Recent studies indicate that the Notch signaling pathway plays an important role in the development of diabetic kidney disease and focal segmental glomerulosclerosis (FSGS). Here we analyzed the degree of expression and localization of Notch ligands (Jagged1 and Delta1) and activated (cleaved) receptors (Notch1 and Notch2) in healthy human kidneys and in renal biopsies from a wide variety of kidney diseases. These included patients with minimal change disease, membranous nephropathy, lupus nephritis ISN/RPS classes III/IV/V, hypertensive nephrosclerosis, crescentic glomerulonephritis, tubulointerstitial fibrosis, IgA nephropathy, diabetic kidney disease, and FSGS. We found that cleaved Notch1, Notch2, and Jagged1 are expressed on podocytes in proteinuric nephropathies and their level of expression correlated 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 cleaved Notch1 in the tubulointerstitium. Hence, our results raise the possibility that Notch pathway activation is a common mechanism in the pathophysiology of a wide range of acquired renal diseases. Diseases of the glomerulus, diabetic (DKD) and hypertensive kidney disease and focal segmental glomerulosclerosis (FSGS), are responsible for >75% of chronic kidney disease cases in the United States.1.Centers for Disease Control and Prevention (CDC) State-specific trends in chronic kidney failure—United States, 1990–2001.MMWR Morb Mortal Wkly Rep. 2004; 53: 918-920PubMed Google Scholar Genetic studies identified mutations in a handful of genes (NPHS1, NPHS2, CD2AP, ACN4, TRPC6, PLCE1, MYH9 and so on) responsible for glomerulosclerosis.2.Mundel P. Schwarz K. Reiser J. Podocyte biology: a footstep further.Adv Nephrol Necker Hosp. 2001; 31: 235-241PubMed Google Scholar The majority of end-stage renal disease 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 the development of albuminuria and glomerulosclerosis. Interestingly, the progression of glomerulosclerosis and chronic renal disease on a phenotypic level seems to follow a similar pattern, potentially indicating that a common pathway has a role in disease progression.3.Kriz W. Elger M. Hosser H. et al.How does podocyte damage result in tubular damage?.Kidney Blood Press Res. 1999; 22: 26-36Crossref PubMed Scopus (25) Google Scholar, 4.Pavenstadt H. Kriz W. Kretzler M. Cell biology of the glomerular podocyte.Physiol Rev. 2003; 83: 253-307Crossref PubMed Scopus (1117) Google Scholar The Notch signaling pathway is a basic cell–cell communication mechanism. Its main components are: the ligands Jagged (Jag1 and 2) and Delta (Dll1, 3 and 4); and the Notch transmembrane receptor proteins (Notch1–4).5.Ilagan M.X. Kopan R. SnapShot: notch signaling pathway.Cell. 2007; 128: 1246Abstract Full Text PDF PubMed Scopus (116) Google Scholar 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 intracellular domain of Notch. The intracellular domain of Notch then travels to the nucleus and binds to other transcriptional regulators (mainly of the CBF1/RBP-JK, SU(H), Lag1 family) to trigger the transcription of target genes (classically Hes and Hey genes).6.Kopan R. Cagan R. Notch on the cutting edge.Trends Genet. 1997; 13: 465-467Abstract Full Text PDF PubMed Scopus (38) Google Scholar The spatially and temporally orchestrated expression of different Notch pathway proteins has a key role in kidney development.7.Cheng H.T. Kim M. Valerius M.T. et al.Notch2, but not Notch1, is required for proximal fate acquisition in the mammalian nephron.Development (Cambridge, England). 2007; 134: 801-811Crossref PubMed Scopus (261) Google Scholar, 8.Cheng H.T. Kopan R. The role of Notch signaling in specification of podocyte and proximal tubules within the developing mouse kidney.Kidney Int. 2005; 68: 1951-1952Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 9.Cheng H.T. Miner J.H. Lin M. et al.Gamma-secretase activity is dispensable for mesenchyme-to-epithelium transition but required for podocyte and proximal tubule formation in developing mouse kidney.Development (Cambridge, England). 2003; 130: 5031-5042Crossref PubMed Scopus (161) Google Scholar, 10.Kopan R. Cheng H.T. Surendran K. Molecular insights into segmentation along the proximal-distal axis of the nephron.J Am Soc Nephrol. 2007; 18: 2014-2020Crossref PubMed Scopus (56) Google Scholar 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.Chen L. Al-Awqati Q. Segmental expression of Notch and Hairy genes in nephrogenesis.Am J Physiol. 2005; 288: F939-F952PubMed Google Scholar, 12.Piscione T.D. Wu M.Y. Quaggin S.E. Expression of Hairy/Enhancer of Split genes, Hes1 and Hes5, during murine nephron morphogenesis.Gene Expr Patterns. 2004; 4: 707-711Crossref PubMed Scopus (70) Google Scholar Both Notch1 and Notch2 are expressed in the S-shaped body.10.Kopan R. Cheng H.T. Surendran K. Molecular insights into segmentation along the proximal-distal axis of the nephron.J Am Soc Nephrol. 2007; 18: 2014-2020Crossref PubMed Scopus (56) Google Scholar Elegant studies carried out by Cheng et al.9.Cheng H.T. Miner J.H. Lin M. et al.Gamma-secretase activity is dispensable for mesenchyme-to-epithelium transition but required for podocyte and proximal tubule formation in developing mouse kidney.Development (Cambridge, England). 2003; 130: 5031-5042Crossref PubMed Scopus (161) Google Scholar showed that although 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 an important subtype specificity for the two Notch receptors. Recent studies from our and other laboratories indicated that activation of the Notch pathway in podocytes has a critical functional role in the development of proteinuria and glomerulosclerosis.13.Niranjan T. Bielesz B. Gruenwald A. et al.The Notch pathway in podocytes plays a role in the development of glomerular disease.Nat Med. 2008; 14: 290-298Crossref PubMed Scopus (294) Google Scholar, 14.Waters A.M. Wu M.Y. Onay T. et al.Ectopic notch activation in developing podocytes causes glomerulosclerosis.J Am Soc Nephrol. 2008; 19: 1139-1157Crossref PubMed Scopus (92) Google Scholar Immunohistological experiments showed that cleaved Notch1 was expressed in podocytes of human and murine diabetic kidneys and in patients with FSGS.13.Niranjan T. Bielesz B. Gruenwald A. et al.The Notch pathway in podocytes plays a role in the development of glomerular disease.Nat Med. 2008; 14: 290-298Crossref PubMed Scopus (294) Google Scholar 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.13.Niranjan T. Bielesz B. Gruenwald A. et al.The Notch pathway in podocytes plays a role in the development of glomerular disease.Nat Med. 2008; 14: 290-298Crossref PubMed Scopus (294) Google Scholar, 15.Niranjan T. Murea M. Susztak K. The pathogenic role of notch activation in podocytes.Nephron. 2009; 111: e73-e79PubMed Google Scholar 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.Walsh D.W. Roxburgh S.A. McGettigan P. et al.Co-regulation of Gremlin and Notch signalling in diabetic nephropathy.Biochim Biophys acta. 2008; 1782: 10-21Crossref PubMed Scopus (86) Google Scholar Jagged1 was also identified as one of the top differentially expressed transcripts by microarray studies carried out on murine model of tubulointerstitial fibrosis (unilateral ureteral obstruction).17.Morrissey J. Guo G. Moridaira K. et al.Transforming growth factor-beta induces renal epithelial jagged-1 expression in fibrotic disease.J Am Soc Nephrol. 2002; 13: 1499-1508Crossref PubMed Scopus (82) Google Scholar, 18.Zavadil J. Cermak L. Soto-Nieves N. et al.Integration of TGF-beta/Smad and Jagged1/Notch signalling in epithelial-to-mesenchymal transition.EMBO J. 2004; 23: 1155-1165Crossref PubMed Scopus (583) Google Scholar, 19.Niimi H. Pardali K. Vanlandewijck M. et al.Notch signaling is necessary for epithelial growth arrest by TGF-beta.J Cell Biol. 2007; 176: 695-707Crossref PubMed Scopus (112) Google Scholar The aim of the this 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. A total of 86 biopsy samples from subjects with acquired kidney diseases were examined and grouped on the basis of the histopathological diagnosis: minimal change disease (n=3), FSGS (n=9), DKD (n=9), idiopathic membranous nephropathy (n=9), lupus nephritis International Society of Nephrology/Renal Pathology Society class III focal- or IV diffuse-proliferative (n=9), lupus nephritis International Society of Nephrology/Renal Pathology Society class V (membranous, n=9), immunoglobulin (Ig)A nephropathy (n=8), hypertensive nephrosclerosis (n=8), tubulointerstitial nephritis (n=8), and crescentic vasculitis (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 1. The average age of our cohort was 43 years, with overrepresentation of females (female=59%). The highest degree of proteinuria was observed in patients with DKD (7.9 g/day) and with idiopathic membranous nephropathy (7.8 g/day). Patients with crescentic glomerulonephritis had the worst kidney function (estimated glomerular filtration rate (GFR)=26 cm3/min per 1.73 m2) at the time of the kidney biopsy.Table 1Demographics and clinical characteristics of the research participantsDiagnosisNo. of casesGender M/FAge (years)HematuriaProteinuria (g/day)eGFR (ml/min)Control74/347±9.960.8±0.990 (0, 0.2)92.87±11.29DKD94/552.5±6.70±06 (5, 10)45.45±21.39FSGS93/626.8±18.50.2±0.412 (1, 2)77.24±25.23MCD31/244±22.60.3±0.41.5 (0.5, 4)75.19±17.56IMN93/552±19.70.3±0.667.5 (4, 9)65.42±30.64LN class V93/540.5±13.80.6±0.663.5 (3, 5)67.18±37.96LN class III/IV91/833±14.061.2±0.782 (1.5, 6.5)77.17±29.23IgAN85/341.8±18.71.7±0.821 (0.8, 1.65)77.23±28.97HN84/457.3±160.2±0.330.95 (0.65, 3.5)40.92±25.5TIN85/334.6±15.90.6±0.991.25 (0.6, 2.25)40.84±16.41Crescentic72/547.5±16.62±0.922 (1.5, 4)26.52±21.18Abbreviations: DKD, diabetic kidney disease; eGFR, estimated glomerular filtration rate; FSGS, focal segmental glomerulosclerosis; HN, hypertensive nephrosclerosis; IgAN, immunoglobulin A nephropathy; IMN, idiopathic membranous nephropathy; LN class III/IV, lupus proliferative glomerulonephritis classes III and IV; LN class V, lupus membranous nephropathy; MCD, minimal change disease; TIN, tubulointerstitial nephritis.Values are expressed as means±s.d. for age, hematuria, and eGFR as median and interquartile range for proteinuria.eGFR was calculated using the Modification of Diet in Renal Disease (MDRD) formula, hematuria is quantified as described under methods. Open table in a new tab Abbreviations: DKD, diabetic kidney disease; eGFR, estimated glomerular filtration rate; FSGS, focal segmental glomerulosclerosis; HN, hypertensive nephrosclerosis; IgAN, immunoglobulin A nephropathy; IMN, idiopathic membranous nephropathy; LN class III/IV, lupus proliferative glomerulonephritis classes III and IV; LN class V, lupus membranous nephropathy; MCD, minimal change disease; TIN, tubulointerstitial nephritis. Values are expressed as means±s.d. for age, hematuria, and eGFR as median and interquartile range for proteinuria. eGFR was calculated using the Modification of Diet in Renal Disease (MDRD) formula, hematuria is quantified as described under methods. Table 2 summarizes the baseline histopathological changes observed in the kidney biopsy samples. We used the diagnosis and the results provided by the clinical pathologist and used during clinical patient care. We analyzed biopsy samples from a 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. Previous histopathological studies indicate that the degree of glomerulosclerosis and tubulointerstitial fibrosis correlates with renal function across the different disease entities.20.Nath K.A. Tubulointerstitial changes as a major determinant in the progression of renal damage.Am J Kidney Dis. 1992; 20: 1-17Abstract Full Text PDF PubMed Scopus (805) Google Scholar, 21.Agarwal A. Nath K.A. Effect of proteinuria on renal interstitium: effect of products of nitrogen metabolism.Am J Nephrol. 1993; 13: 376-384Crossref PubMed Scopus (19) Google Scholar Similar to previous reports, in our cohort we found correlation between the degree of tubulointerstitial fibrosis across all disease groups and the estimated GFR (Supplementary Table SI). From the different disease groups, we observed the highest degree of glomerulosclerosis (69%) and tubulointerstitial fibrosis (73%) in the DKD biopsy samples, followed by samples obtained from tubulointerstitial nephritis and hypertensive nephrosclerosis (Table 2). The degree of glomerulosclerosis and tubulointerstitial fibrosis was low in the control samples (3 and 6%, respectively).Table 2Histopathological characteristics of the kidney biopsy samplesDisease groupNo. of glomeruliGlomerulosclerosis (%)Tubulointerstitial fibrosis (%)Control10.50 (0, 7)0 (0, 10)DKD19.875 (42, 100)80 (60, 85)FSGS15.440 (25, 55)10 (10, 30)MCD833 (5, 100)20 (20, 20)IMN15.516 (8.5, 23.5)22.5 (15, 62.5)LN class V2115 (0, 25)20 (17.5, 35)LN class III/IV16.516.5 (0, 57.5)20 (10, 32.5)IgAN13.822.5 (12.5, 54)20 (15, 25)HN12.857.5 (29, 82.5)35 (15, 75)TIN11.124 (0, 45)50 (45, 65)Crescentic10.233 (0, 77)15 (10, 50)Abbreviations: DKD, diabetic kidney disease; FSGS, focal segmental glomerulosclerosis; HN, hypertensive nephrosclerosis; IgAN, immunoglobulin A nephropathy; IMN, idiopathic membranous nephropathy; LN class III/IV, lupus proliferative glomerulonephritis classes III and IV; LN class V, lupus membranous nephropathy; MCD, minimal change disease; TIN, tubulointerstitial nephritis.Values are expressed as median (interquartile range) for glomerulosclerosis and tubulointerstitial fibrosis. Open table in a new tab Download .ppt (5.05 MB) Help with ppt files Supplementary Information Abbreviations: DKD, diabetic kidney disease; FSGS, focal segmental glomerulosclerosis; HN, hypertensive nephrosclerosis; IgAN, immunoglobulin A nephropathy; IMN, idiopathic membranous nephropathy; LN class III/IV, lupus proliferative glomerulonephritis classes III and IV; LN class V, lupus membranous nephropathy; MCD, minimal change disease; TIN, tubulointerstitial nephritis. Values are expressed as median (interquartile range) for glomerulosclerosis and tubulointerstitial fibrosis. 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 Notch1 and Notch2.13.Niranjan T. Bielesz B. Gruenwald A. et al.The Notch pathway in podocytes plays a role in the development of glomerular disease.Nat Med. 2008; 14: 290-298Crossref PubMed Scopus (294) Google Scholar, 22.Hozumi K. Negishi N. Suzuki D. et al.Delta-like 1 is necessary for the generation of marginal zone B cells but not T cells in vivo.Nat Immunol. 2004; 5: 638-644Crossref PubMed Scopus (264) Google Scholar, 23.Sharma M. Fopma A. Brantley J.G. et al.Coexpression of Cux-1 and Notch signaling pathway components during kidney development.Dev Dyn. 2004; 231: 828-838Crossref PubMed Scopus (28) Google Scholar, 24.Vooijs M. Ong C.T. Hadland B. et al.Mapping the consequence of Notch1 proteolysis in vivo with NIP-CRE.Development (Cambridge, England). 2007; 134: 535-544Crossref PubMed Scopus (110) Google Scholar Podocyte-specific expression was confirmed by double immunostaining with podocyte marker Wilms tumor (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 Supplementary Figures S3 and S4). Similar to previous reports, we could not observe expression of activated Notch1, Notch2, and Delta1 in the glomeruli of control ‘healthy’ human kidney biopsy samples (Figure 1).13.Niranjan T. Bielesz B. Gruenwald A. et al.The Notch pathway in podocytes plays a role in the development of glomerular disease.Nat Med. 2008; 14: 290-298Crossref PubMed Scopus (294) Google Scholar, 24.Vooijs M. Ong C.T. Hadland B. et al.Mapping the consequence of Notch1 proteolysis in vivo with NIP-CRE.Development (Cambridge, England). 2007; 134: 535-544Crossref PubMed Scopus (110) Google Scholar Our antibody detected a faint signal for Jagged1 in healthy glomeruli, and double immunostaining for Jagged1 and WT-1 indicated a possible podocyte-specific localization (Figure 1). In the tubules of healthy adult kidneys there was no significant expression of Delta1,25.Kobayashi T. Terada Y. Kuwana H. et al.Expression and function of the Delta-1/Notch-2/Hes-1 pathway during experimental acute kidney injury.Kidney Int. 2008; 73: 1240-1250Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar but we occasionally observed positive immunostaining for cleaved Notch1 and Notch2 in tubule segments (Figure 1).24.Vooijs M. Ong C.T. Hadland B. et al.Mapping the consequence of Notch1 proteolysis in vivo with NIP-CRE.Development (Cambridge, England). 2007; 134: 535-544Crossref PubMed Scopus (110) Google Scholar In summary, there was no significant expression of cleaved Notch receptors in healthy adult human glomerulus, whereas there was minimal expression of cleaved Notch1 and Notch2 in the tubulointerstitium. Next we analyzed the level of expression and localization of cleaved Notch1 and Notch2 proteins in diseased kidney samples. The amplitude of nuclear expression (examined by 4′,6-diamidino-2-phenylindole (DAPI) costaining) was scored on a 0–4 scale (see Materials and Methods). We validated this method by using computerized image analysis (Image J), which showed excellent correlation (P=0.01) with the dual observer semiquantitative assessment. In addition, this method has been used successfully recently and in the past by other investigators.26.Giannico G. Yang H. Neilson E.G. et al.Dystroglycan in the diagnosis of FSGS.Clin J Am Soc Nephrol. 2009; 4: 1747-1753Crossref PubMed Scopus (32) Google Scholar Representative staining images from a single biopsy are shown in Figure 2. In addition, images from representative scoring values are presented in Supplementary Figure S1. First, the presence of positive immunostaining for activated Notch1 and Notch2 in human DKD and FSGS samples was confirmed. Although 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 that are generally not associated with heavy proteinuria, such as hypertensive nephrosclerosis and tubulointerstitial nephritis samples (Figures 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 (Figures 2 and 3). Tubulointerstitial expression of cleaved Notch2 was increased in almost all disease groups, and we could not observe a clear disease-specific distribution (Figures 2 and 4). Cleaved Notch1 expression in the tubulointerstitium was significantly increased in the DKD samples (Figure 4). There was significantly increased expression of Jagged1 and Delta1 in many different diseases, without a clear disease-specific distribution (Figures 2 and 4). Next, we examined whether there is a correlation between the expression of different Notch proteins in the glomerular and tubulointerstitial compartment (Supplementary Table SI). Such analysis revealed that Notch2 expression on podocytes correlated with Notch1 and Jagged1 expression on podocytes. Jagged1 expression in podocytes directly correlated with Jagged1 and Notch2 expressions in the tubulointerstitial compartment (Supplementary Table SI). Expression of Delta1 in the tubulointerstitial compartment correlated with tubular and podocyte expression of Notch2 and podocyte expression of Jagged1 (Supplementary Table SI). In summary, our results indicate that Notch ligands and receptors are expressed in a wide range of renal diseases. 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 and 0.004, respectively) (Table 3a and Figure 5a). 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) (Table 3 and Figure 5b).Table 3Result of the linear regression analysis of Notch expression and histological and functional renal parametersB co-efficient95% CIP-value(a) Proteinuria (log)Podocyte ICN1 (n=79)0.17-0.004, 0.350.056Podocyte ICN2 (n=90)0.220.007, 0.430.04Podocyte Jag1 (n=71)0.330.05, 0.610.02Tubular ICN1 (n=72)0.21-0.01, 0.430.07Tubular ICN2 (n=72)-0.03-0.27, 0.210.80Tubular Jag1 (n=75)-0.10-0.44, 0.230.54Tubular Delta1 (n=80)0.13-0.07, 0.320.22(b) eGFRPodocyte ICN1 (n=85)1.24-4.29, 6.780.66Podocyte ICN2 (n=86)-1.68-8.42, 5.070.62Podocyte Jag1 (n=75)8.660.29, 17.00.04Tubular ICN1 (n=78)-12.2-18.4, -5.93<0.001Tubular ICN2 (n=84)10.573.36, 17.80.005Tubular Jag1 (n=75)7.59-2.25, 17.40.13Tubular Delta1 (n=80)1.16-4.86, 7.190.70(c) GlomerulosclerosisPodocyte ICN1 (n=83)8.683.31, 14.00.002Podocyte ICN2 (n=83)2.22-4.78, 9.230.53Podocyte Jag1 (n=75)-6.9-15.7, 1.890.12Tubular ICN1 (n=83)9.873.20, 16.50.004Tubular ICN2 (n=83)-4.02-11.8, 3.740.31Tubular Jag1 (n=75)-4.95-15.3, 5.370.34Tubular Delta1 (n=79)1.71-4.37, 7.790.58(d) Tubulointerstitial fibrosisPodocyte ICN1 (n=83)4.940.32, 9.560.04Podocyte ICN2 (n=83)4.02-1.75, 9.800.17Podocyte Jag1 (n=80)-1.83-9.28, 5.620.63Tubular ICN1 (n=78)9.143.63, 14.70.001Tubular ICN2 (n=78)-4.50-10.9, 1.920.17Tubular Jag1 (n=75)-1.38-10.0, 7.290.75Tubular Delta1 (n=79)-0.36-5.48, 4.770.89Abbreviation: ICN, Notch intracellular domain.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 0.002 or the unadjusted P-value 0.05 for this analysis were considered statistically significant. Open table in a new tab Abbreviation: ICN, Notch intracellular domain. 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 0.002 or the unadjusted P-value 0.05 for this analysis were considered statistically significant. 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. 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 podocyte-specific expression level of active Notch1, Notch2, and Jagged1 (Table 3c and Figure 5c). Expression of Notch proteins in the tubulointerstitium on the other hand did not show significant correlation with the amount of proteinuria (Table 3c). This finding might further support the central role of podocytes in the development of proteinuria. Estimated GFR, at the time of biopsy, showed a 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, that is, 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 (Table 3d). Our results indicate that Notch expression across multiple renal disease groups is correlated with renal functional parameters. 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 has a crucial role in the kidney development.11.Chen L. Al-Awqati Q. Segmental expression of Notch and Hairy genes in nephrogenesis.Am J Physiol. 2005; 288: F939-F952PubMed Google Scholar Our study indicates that the Notch pathway—like most developmental pathways 27.van Es J.H. van Gijn M.E. Riccio O. et al.Notch/gamma-secretase inhibition turns proliferative cells in intestinal crypts and adenomas into goblet cells.Nature. 2005; 435: 959-963Crossref PubMed Scopus (1182) Google Scholar—is mostly silenced in the glomeruli of normal mature human k