Conceived and designed the experiments: BJ MG JCH. Performed the experiments: BJ MG AQ YF PC. Analyzed the data: BJ MG AQ HWC JCH. Contributed reagents/materials/analysis tools: HWC DBT MA. Wrote the paper: BJ MG AQ HWC JCH.
DBT is employed by Nephrocor. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.
Podocyte specific proteins are dysregulated in diabetic nephropathy, though the extent of their expression loss is not identical and may be subject to different regulatory factors. Quantifying the degree of loss may help identify the most useful protein to use as an early biomarker of diabetic nephropathy.
Protein expression of synaptopodin, podocin and nephrin were quantified in 15 Type 2 diabetic renal biopsies and 12 control patients. We found statistically significant downregulation of synaptopodin (P<0.0001), podocin (P = 0.0002), and nephrin (P<0.0001) in kidney biopsies of diabetic nephropathy as compared with controls. Urinary nephrin levels (nephrinuria) were then measured in 66 patients with Type 2 diabetes and 10 healthy controls by an enzyme-linked immunosorbent assay (Exocell, Philadelphia, PA). When divided into groups according to normo-, micro-, and macroalbuminuria, nephrinuria was found to be present in 100% of diabetic patients with micro- and macroalbuminuria, as well as 54% of patients with normoalbuminuria. Nephrinuria also correlated significantly with albuminuria (
These data suggest that key podocyte-specific protein expressions are significantly and differentially downregulated in diabetic nephropathy. The finding that nephrinuria is observed in a majority of these normoalbuminuric patients demonstrates that it may precede microalbuminuria. If further research confirms nephrinuria to be a biomarker of pre-clinical diabetic nephropathy, it would shed light on podocyte metabolism in disease, and raise the possibility of new and earlier therapeutic targets.
Diabetes affecting the kidney, or diabetic nephropathy (DN), affects approximately one third of patients with either Type 1 or Type 2 diabetes mellitus
Small amounts of albumin in the urine, or microalbuminuria is the current early biomarker. However, its association with progression to renal failure is unclear, as microalbuminuria does not always lead to progressive renal failure
We now know that much of the early inciting events stem from podocyte pathology. The podocyte is a specialized visceral epithelial cell that helps to establish the glomerular filtration barrier and prevents protein loss, along with the glomerular basement membrane and the endothelial cell layer. Occurrence of podocytopenia (decreased number) and podocyturia (podocytes in urine) in DN are well established
Synaptopodin, a proline rich protein, directly interacts with the α-actinin-induced actin filaments. Downregulation of synaptopodin expression leads to structural and functional changes such as loss of stress fibers, aberrant formation of filopodia, and impaired cell migration
In the current study, we investigated the morphologic alterations of podocyte-specific proteins in DN biopsies from patients with Type 2 diabetes and found significant downregulation of synaptopodin, podocin and nephrin expression in the diabetic group as compared to controls. Given the availability of a reliable method of quantifying nephrin in the urine (nephrinuria), we measured its levels in diabetic patients and found that it was detected in 54% of normoalbuminuric patients, suggesting its potential role as an early biomarker.
Our goal is to study the expression of podocyte specific proteins in renal biopsies of Type 2 diabetic patients, and then to select the most downregulated protein as a marker for early detection of renal disease.
Study samples consisted of kidney biopsies of 15 DN patients that were obtained at Jacobi Medical Center in Bronx, New York, USA and archived at Columbia-Presbyterian Hospital in New York, New York, USA. Indications for biopsy included: presence of nephrotic syndrome without retinopathy, unexplained acute kidney injury, or presence of hematuria. All clinical data at time of biopsy were retrieved from electronic medical records at Jacobi Medical Center. Twelve control biopsies consisted of normal kidney tissue from tumor nephrectomies. This study has been approved by the Internal Review Board of the Albert Einstein College of Medicine.
Patients were selected from the outpatient nephrology clinic at Jacobi Medical Center. A one-time random urine sample was collected by the clean catch method on all study and control subjects. Inclusion criteria for study patients were: history of type 2 diabetes, absence of another renal or urinary tract disease, and absence of pregnancy. There were no exclusion criteria based on race, age, or gender. Diabetic patients were categorized as having normoalbuminuria when urine albumin-to-creatinine ratio (UACR) <30 mg/g, microalbuminuria when UACR was between 30 and 300 mg/g, and macroalbuminuria when UACR was >300 mg/g. All control subjects were healthy volunteers from the medical staff with no history of diabetes, hypertension, or renal disease. Demographic and clinical data were recorded, including age, sex, weight, height, medications, blood pressure, blood urea nitrogen (BUN), serum creatinine, HbA1C, random urine albumin, urine creatinine, and urine protein. All clinical laboratories values were measured at the Department of Clinical Laboratories at Jacobi Medical Center. Estimated glomerular filtration rate (eGFR) was calculated by the 4-variable Modification of Diet in Renal Disease study equation
Paraffin-embedded tissues sections were deparaffinized in microwave oven for 3 minutes. The sections were rehydrated in graded series of xylene and alcohols. Endogenous peroxidase activity was blocked by 3% hydrogen peroxide in distilled water for 15 minutes. Antigen retrieval was achieved by steam heating in a solution of citrate buffer, pH 6.0 for 30 minutes. Sections were blocked with 10% normal horse serum for monoclonal antibodies and 10% normal goat serum for polyclonal antibodies. Sections were then incubated overnight with primary antibodies monoclonal mouse anti-human synaptopodin antibody (1∶20) (gift of Dr. Peter Mundel), polyclonal rabbit anti-human podocin antibody (1∶1000) (gift of Dr. Peter Mundel), monoclonal mouse anti-human nephrin antibody (1∶50) (gift of Dr. Karl Tryggvason), and polyclonal rabbit anti-human nephrin antibody (1∶200) (Enzo Life Sciences Inc., Farmingdale, NY) at 4°C. Horse anti-mouse IgG and goat anti-rabbit IgG (Dako Inc. Carpinteria, CA) were used as secondary antibodies (1∶1000) for 30 minutes. The sections were then incubated in avidin-biotin complex at 1∶25 dilution (Vector Labs, Burlingame, CA) and developed using diaminobenzidine (DAB) as chromogen. After washing, the sections were counter-stained with hematoxylin and coverslipped. Negative controls were carried out by incubation in the absence of the primary antibody.
Areas of positive DAB staining as a percentage of the entire glomerulus were calculated using the ImageJ software (US National Institutes of Health) for each glomerulus in the biopsy sample. Subsequently, a mean value of positive DAB staining was calculated based on the number of glomeruli for each biopsy sample.
Urinary nephrin was determined by a competitive enzyme-linked immunosorbent assay, using polyclonal antibodies against the extracellular domain (amino acids 23–322) of human nephrin (Exocell Inc., Philadelphia, PA). The assay was performed by Exocell Inc. according to manufacturer's instructions. Briefly, urine samples were diluted in the range of 1∶10 to 1∶500 (depending on degree of proteinuria in urines sample). A 50-µl diluted sample was added to each well already coated with rat nephrin followed by the addition of a 50-µl of rabbit anti-nephrin antibody for an incubation of 60 minutes at room temperature. Plates were washed followed by the incubation with 100-µl of anti-rabbit HRP conjugate to each well for 60 minutes. After plates were washed and color developed, absorbance was read at 450 nm. Elevated levels of urinary nephrin or nephrinuria was defined as urine nephrin-creatinine ratio (UNCR) (mg/g) ≥0.1 mg/g. This value was based on 10 healthy controls who consistently exhibited UNCR <0.1 mg/g.
Archival human kidney biopsies and random urine samples were collected at Jacobi Medical Center, Bronx, New York as part of exempted protocols “Differential Protein Expression in Nephrotic Diseases” and “Analysis of Urinary Proteins in Nephrotic Syndrome”, both of which were approved by the Institutional Review Board of the Albert Einstein College of Medicine of Yeshiva University. Since all clinical data were de-identified, no consent was required.
Analyses were performed comparing study and control biopsies and also between 4 groups defined as normal controls (no diabetes), normo-, micro-, and macroalbuminuria. Categorical variables were presented as percentage while continuous variables were presented as median (interquartile range) or mean ± standard deviation (SD) if normality assumptions were not substantially violated. Differences between mean rank expression of proteins between study and control biopsies were calculated using Mann-Whitney test. Difference in UNCR between groups was determined Kruskal-Wallis. Spearman correlations were calculated to assess trend for continuous variables and Chi-square was used to assess associations of categorical variables. Both UACR and UNCR were log transformed for graphical depiction. Analyses were performed with STATA Version 8.2 and GraphPad Prism Version 5.0 for Windows software and results were considered statistically significant if P<0.05.
Clinical characteristics of diabetic and control patients are summarized in
Representative staining of synaptopodin in Con (A), DN (B), podocin in Con (E) and DN (F), and nephrin in Con (I) and DN (J). Negative controls without administration of primary antibody are represented for synaptopodin (C), podocin (G), and nephrin (K). Quantification of synaptopodin (D), podocin (H), and nephrin (L) positive area per glomerular tuft in Con (12 patients) and DN (15 patients). Horizontal lines represent the median value. *Number of DN biopsies for nephrin was 11 due to lack of remaining tissue.
Clinical Characteristic | Control (n = 12) | Diabetic Nephropathy (n = 15) | P value |
Age (years) | 56 (52;60) | 56 (51.9;63.5) | 0.83 |
Male sex (%) | 75% | 66% | 0.67 |
Urine protein-to-creatinine ratio (g/g) | – |
5.3 (3.9;7.8) | – |
Blood urea nitrogen (mg/dL) | 15 (12.8;17.3) | 51 (39.5;63.3) | <0.0001 |
Serum creatinine (mg/dl) | 1.0 (0.8;1.1) | 4.1 (2.7;4.8) | 0.0005 |
estimated GFR (ml/min/1.73 m2) | 86.8 (61.8;103.7) | 13.35 (12.5;40.7) | 0.0008 |
Serum albumin (g/dl) | 4.2 (3.7;4.4) | 2.5 (2.3;3.6) | 0.0002 |
Systolic blood pressure mm/Hg | 127 (120;130) | 150 (140;166) | 0.0002 |
Diastolic blood pressure (mm/Hg) | 80.5 (73.4;86.2) | 85 (76;91) | 0.49 |
HbA1C% | 6.5 (4.5;10.9) | 9.3 (7.8;11.8) | 0.10 |
ACEI |
25% | 73% | 0.026 |
ARB |
8.3% | 26% | 0.25 |
Data are presented as median (interquartile range) for continuous variables and % for categorical variables.
Urine protein-creatinine ratio not available for control patients as their urinalyses did not show proteinuria.
ACEI: angiotensin converting enzyme inhibitors.
ARB: angiotensin receptor blockers.
We subsequently measured urinary nephrin in 66 diabetic patients of all chronic kidney disease (CKD) stages and levels of albuminuria and compared them to 10 healthy control subjects. Clinical characteristics are summarized in
A) Log transformed UNCR correlates significantly with macroalbuminuria (
Control n = 10 | Normo-albuminuria n = 26 | Micro-albuminuria n = 11 | Macro-albuminuria n = 29 | P value | |
Men (%) | 40% | 44% | 45% | 53% | 0.85 |
Age | 61±12.2 | 62±14 | 68±7.3 | 59±13 | <0.0001 |
HbA1C | – | 7.6 (6.5,8.1) | 7.2 (6.3,8.0) | 7.5 (6.8,9.0) | 0.6 |
UACR |
7.32 (3.7,14.5) | 7.9 (5.6,12.8) | 93.8 (79.1,185.4) | 1644.3 (750.3,3013.3) | <0.0001 |
Serum creatinine (mg/dL) | 0.9 (0.8,1.0) | 1.4 (0.9,1.6) | 1.8 (1.5,2.1) | 2.1 (1.6,2.9) | <0.0001 |
estimated GFR (ml/min/1.73m2) | 89 (65,120) | 47 (36.2,72.2) | 37 (29,48) | 30 (20,44.5) | <0.0001 |
Systolic blood pressure (mmHg) | 125 (102,149) | 132 (120,145) | 139 (129,159) | 144 (136,154) | 0.0001 |
Diastolic blood pressure (mmHg) | 67 (54,85) | 68 (54,76) | 70 (64,72) | 76 (66,88) | 0.21 |
% Diabetic Retinopathy | – | 43% | 20% | 55% | 0.15 |
UNCR |
0.07 (.03,.098) | 0.11(.08,.17) |
1.16 (.35,1.60) | 9.15 (5.68,12.08) | <0.0001 |
% ACEI/ARB treatment | 0% (0/10) | 73% (19/26) | 54% (6/11) | 62% (18/29) | 0.0004 |
% UNCR >0.1 (mg/g) | 0% (0/10) | 54% (14/26) | 100% (11/11) | 100% (29/29) | 0.001 |
Data are means +/− SD or median (interquartile range).
UACR: urine albumin-to-creatinine ratio.
UNCR: urine nephrin-to-creatinine ratio.
P for trend across the 4 categories.
P <0.05 vs. Control.
Parameter | n value |
|
P value |
UACR |
61 | 0.89 | <0.001 |
Serum creatinine (mg/dL) | 65 | 0.43 | 0.0002 |
Blood urea nitrogen (mg/dL) | 65 | 0.37 | 0.001 |
estimated GFR (ml/min/1.73m2) | 65 | −0.33 | 0.005 |
HbA1C | 63 | 0.20 | 0.10 |
Serum albumin (g/dL) | 55 | −0.48 | 0.0001 |
Systolic blood pressure (mmHg) | 64 | 0.32 | 0.007 |
Diastolic blood pressure (mmHg) | 64 | 0.21 | 0.07 |
Presence of diabetic retinopathy | 58 | 0.14 | 0.27 |
Presence of ACEI |
66 | −0.04 | 0.75 |
Correlations were determined by calculations of Spearman
UACR: urine albumin-to-creatinine ratio.
ACEI: angiotensin converting enzyme inhibitor.
ARB: angiotensin receptor blocker.
Our results substantiated the dysregulated podocyte phenotype in human DN and quantified expressions of synaptopodin, podocin and nephrin in all Type 2 diabetic biopsies. Decreased expression of protein and mRNA levels has been described in podocyte-associated molecules in both animal models
Furthermore, nephrin may be unique in that it modulates the podocyte's response to insulin. Coward et al. has shown that podocytes that are nephrin deficient results in at state of insulin resistance
That nephrinuria is seen in early disease (in 54% of patients with normoalbuminuria) and increases in overt disease (macroalbuminuria) supports a previous finding by Patari et al. who had described nephrinuria in one-third of diabetic patients with normoalbuminuria
There are a few limitations to this study. As renal biopsies are not routinely performed to diagnose DN, these patients may represent a separate category of patients which may skew podocyte expression. For example, they may demonstrate a more severe subset with rapidly progressive renal failure or exhibit concomitant diagnoses with the presence of hematuria. Our biopsies indicate a more advanced group of patients, given that the mean eGFR was 27.2 ml/min/1.73 m2. Unfortunately, it is difficult to avoid this limitation as it may be unethical to biopsy all classic presentations of DN, though we used only biopsies that had pathognomonic findings. Also, since the comparison of nephrinuria with clinical data was a cross-sectional study, we do not know if nephrinuria is part of a causal mechanism, or if early nephrinuria will consistently predict subsequent DN. Measuring nephrinuria prospectively in diabetic patients with normoalbuminuria will help to answer these questions, though this type of study will require a prospective study for at least 10–20 years after onset of diabetes.
In conclusion, we have shown significantly reduced expressions of synaptopodin and podocin, and nephrin in DN. As a corollary, we have shown that a high percentage of these patients exhibit nephrinuria without albuminuria, suggesting its potential utility as an early biomarker. If further research confirms nephrinuria to be a biomarker of pre-clinical DN, it would shed light on podocyte metabolism in disease, and raise the possibility of new and earlier therapeutic targets.
We thank Ms. Ann-Sofie Nilsson of the Karolinska Institutet, Stockholm, Sweden, for sharing the nephrin antibody from Dr. Karl Tryggvason.