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Urinary and Kidney Podocalyxin and Podocin Levels in Diabetic Kidney Disease: A Kidney Biopsy Study

  • Lingfeng Zeng
    Affiliations
    Carol & Richard Yu Peritoneal Dialysis Research Centre, Department of Medicine & Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong, China
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  • Winston Wing-Shing Fung
    Affiliations
    Carol & Richard Yu Peritoneal Dialysis Research Centre, Department of Medicine & Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong, China
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  • Gordon Chun-Kau Chan
    Affiliations
    Carol & Richard Yu Peritoneal Dialysis Research Centre, Department of Medicine & Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong, China
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  • Jack Kit-Chung Ng
    Affiliations
    Carol & Richard Yu Peritoneal Dialysis Research Centre, Department of Medicine & Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong, China
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  • Kai-Ming Chow
    Affiliations
    Carol & Richard Yu Peritoneal Dialysis Research Centre, Department of Medicine & Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong, China
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  • Cheuk-Chun Szeto
    Correspondence
    Address for Correspondence: Cheuk-Chun Szeto, MD, Department of Medicine & Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, China.
    Affiliations
    Carol & Richard Yu Peritoneal Dialysis Research Centre, Department of Medicine & Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong, China

    Li Ka Shing Institute of Health Sciences (LiHS), Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong, China
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Open AccessPublished:November 13, 2022DOI:https://doi.org/10.1016/j.xkme.2022.100569

      Rationale & Objective

      Diabetic kidney diseases (DKDs) are the most common cause of dialysis-dependent kidney disease around the world. Previous studies have suggested that urinary level of podocyte-associated molecules may predict the prognosis of DKD.

      Study Design

      Observational cohort.

      Setting & Participants

      118 consecutive patients with biopsy-proven DKD; 13 nondiabetic patients with hypertensive nephrosclerosis as controls.

      Predictors

      Urinary podocalyxin and podocin levels were obtained by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) and the corresponding intrarenal levels by western blotting.

      Outcomes

      Dialysis-free survival; kidney event-free survival; rate of kidney function decline in 12 months.

      Analytic Approach

      Correlation and time to event analysis.

      Results

      Urinary podocalyxin level was closely correlated with its messenger RNA (mRNA) level (r = 0.562, P < 0.001), but this did not predict the progression of DKD. Intrarenal podocalyxin level had only modest correlation with its urinary mRNA and ELISA levels, was an independent predictor of dialysis-free survival (adjusted HR, 1.85; 95% CI, 1.21-2.82; P = 0.005), and showed an insignificant trend of predicting kidney event-free survival (adjusted HR, 1.36; 95% CI, 0.94-1.95; P = 0.10). Urinary podocin level by ELISA had a modest correlation with the rate of kidney function decline (r = 0.238, P = 0.01) but did not predict dialysis-free survival.

      Limitations

      Small sample size; lack of serial measurement.

      Conclusions

      Intrarenal podocalyxin level, but not its urinary level, was an independent predictor of dialysis-free survival, whereas urinary podocin level by ELISA correlated with the rate of kidney function decline. Although intrarenal podocalyxin level has prognostic value, it may not be suitable for routine clinical use.

      Index Words

      Diabetic kidney diseases (DKDs) are the most common cause of dialysis-dependent kidney disease around the world. Previous studies suggested that the urinary level of podocyte-associated molecules may predict the prognosis of DKD. We studied 118 patients with biopsy-proven DKD. Their urinary and kidney levels of podocalyxin and podocin were measured. We found that podocalyxin level in the kidney, but not the urine, independently predicted dialysis-free survival. However, it is an invasive test and may not be suitable for routine clinical use.
      Diabetic kidney diseases (DKDs) are the most common cause of dialysis-dependent kidney disease around the world.
      • Flyvbjerg A.
      The role of the complement system in diabetic nephropathy.
      Recent studies have shown that 25%-40% of patients with type 1 diabetes and 5%-40% of patients with type 2 diabetes eventually develop DKD.
      • Shimizu M.
      • Furuichi K.
      • Wada T.
      Epidemiology and pathogenesis of diabetic nephropathy.
      Although DKD is generally diagnosed by albuminuria and kidney function tests, novel biomarkers are needed for the prognosis, prediction of treatment response, and monitoring of DKD.
      • Schutte E.
      • Gansevoort R.T.
      • Benner J.
      • Lutgers H.L.
      • Lambers Heerspink H.J.
      Will the future lie in multitude? A critical appraisal of biomarker panel studies on prediction of diabetic kidney disease progression.
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      • Mayer G.
      BEAt-DKD Consortium
      Multimarker panels in diabetic kidney disease: the way to improved clinical trial design and clinical practice?.
      Podocytes play a key role in the maintenance of normal kidney function and are the primary focus of many kidney diseases.
      • Ishii H.
      • Kaneko S.
      • Yanai K.
      • et al.
      MicroRNAs in podocyte injury in diabetic nephropathy.
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      • Tufro A.
      • Veron D.
      VEGF and podocytes in diabetic nephropathy.
      Various disease processes lead to podocyte damage and detachment from the glomerular basement membrane, and viable podocytes and their cellular fragments are detectable in the urine.
      • Mundel P.
      Urinary podocytes: lost and found alive.
      ,
      • Sun D.
      • Zhao X.
      • Meng L.
      Relationship between urinary podocytes and kidney diseases.
      There is a wealth of literature showing that the urinary levels of many podocyte-associated molecules may serve as biomarkers for DKD and other kidney diseases.
      • Zeng L.
      • Szeto C.C.
      Urinary podocyte markers in kidney diseases.
      • Sullivan K.M.
      • Scholey J.
      • Moineddin R.
      • et al.
      Urinary podocyte-derived microparticles in youth with type 1 and type 2 diabetes.
      • Zhang L.H.
      • Zhu X.Y.
      • Eirin A.
      • et al.
      Early podocyte injury and elevated levels of urinary podocyte-derived extracellular vesicles in swine with metabolic syndrome: role of podocyte mitochondria.
      However, podocytes have many separate cellular compartments, and most of the previous studies focused on slit-diaphragm and foot-process proteins in the urine as biomarkers of kidney diseases.
      • Jim B.
      • Ghanta M.
      • Qipo A.
      • et al.
      Dysregulated nephrin in diabetic nephropathy of type 2 diabetes: a cross sectional study.
      • Kostovska I.
      • Tosheska-Trajkovska K.
      • Topuzovska S.
      • et al.
      Urinary nephrin is earlier, more sensitive and specific marker of diabetic nephropathy than microalbuminuria.
      • Petrica L.
      • Ursoniu S.
      • Gadalean F.
      • et al.
      Urinary podocyte-associated mRNA levels correlate with proximal tubule dysfunction in early diabetic nephropathy of type 2 diabetes mellitus.
      • Fukuda A.
      • Minakawa A.
      • Kikuchi M.
      • et al.
      Urinary podocyte mRNAs precede microalbuminuria as a progression risk marker in human type 2 diabetic nephropathy.
      • Wang G.
      • Lai F.M.
      • Lai K.B.
      • et al.
      Urinary messenger RNA expression of podocyte-associated molecules in patients with diabetic nephropathy treated by angiotensin-converting enzyme inhibitor and angiotensin receptor blocker.
      For example, urinary levels of nephrin, a podocyte slit-diaphragm protein, correlated with the level of albuminuria and inversely with kidney function and is an earlier, more sensitive, and specific marker of diabetic nephropathy than microalbuminuria.
      • Jim B.
      • Ghanta M.
      • Qipo A.
      • et al.
      Dysregulated nephrin in diabetic nephropathy of type 2 diabetes: a cross sectional study.
      ,
      • Kostovska I.
      • Tosheska-Trajkovska K.
      • Topuzovska S.
      • et al.
      Urinary nephrin is earlier, more sensitive and specific marker of diabetic nephropathy than microalbuminuria.
      Urinary messenger (mRNA) level of podocin, another slit-diaphragm protein, helps evaluate podocyte loss and monitor treatment response of DKD.
      • Petrica L.
      • Ursoniu S.
      • Gadalean F.
      • et al.
      Urinary podocyte-associated mRNA levels correlate with proximal tubule dysfunction in early diabetic nephropathy of type 2 diabetes mellitus.
      • Fukuda A.
      • Minakawa A.
      • Kikuchi M.
      • et al.
      Urinary podocyte mRNAs precede microalbuminuria as a progression risk marker in human type 2 diabetic nephropathy.
      • Wang G.
      • Lai F.M.
      • Lai K.B.
      • et al.
      Urinary messenger RNA expression of podocyte-associated molecules in patients with diabetic nephropathy treated by angiotensin-converting enzyme inhibitor and angiotensin receptor blocker.
      Podocalyxin is the major transmembrane protein specifically expressed on the apical side of podocyte (the so-called “top membrane”) and is responsible for the regulation of podocyte morphology and glomerular permeability.
      • Nielsen J.S.
      • McNagny K.M.
      The role of podocalyxin in health and disease.
      ,
      • Economou C.G.
      • Kitsiou P.V.
      • Tzinia A.K.
      • et al.
      Enhanced podocalyxin expression alters the structure of podocyte basal surface.
      Preliminary studies showed that urinary podocalyxin level of DKD patients was higher than that of the control group, and the level positively correlated with glycemic control and urinary albumin excretion, indicating that urinary podocalyxin level might serve as a biomarker of DKD.
      • Hara M.
      • Yamagata K.
      • Tomino Y.
      • et al.
      Urinary podocalyxin is an early marker for podocyte injury in patients with diabetes: establishment of a highly sensitive ELISA to detect urinary podocalyxin.
      ,
      • Shoji M.
      • Kobayashi K.
      • Takemoto M.
      • Sato Y.
      • Yokote K.
      Urinary podocalyxin levels were associated with urinary albumin levels among patients with diabetes.
      In the present study, we compared the role of urinary and intrarenal levels of podocalyxin and podocin (a prototype slit-diaphragm protein) biomarker in patients with biopsy-proven DKD.

      Methods

      The study was approved by the Clinical Research Ethical Committee of the Chinese University of Hong Kong (approval number CRE-2019.283). All participants provided written inform consent for the study. All study procedures were in compliance with the Declaration of Helsinki.

      Participants

      We recruited 118 consecutive patients who had type 2 diabetes and kidney biopsy-proven diabetic nephropathy in our center. We also studied 13 nondiabetic patients with biopsy-proved hypertensive nephrosclerosis and 3 healthy kidney donors as controls. All kidney biopsy specimens were assessed by a single experienced pathologist and validated by an independent expert. Whole-stream early-morning timed (8 hours) urine was collected on the day of kidney biopsy. We also reviewed the participants’ demographic and clinical data including serum creatinine and proteinuria. The estimated glomerular filtration rate (eGFR) was calculated by the Chronic Kidney Disease Epidemiology Collaboration equation.
      • Levey A.S.
      • Stevens L.A.
      • Schmid C.H.
      • et al.
      A new equation to estimate glomerular filtration rate.

      RNA Extraction

      The method of mRNA extraction and quantification in urinary sediment has been described previously.
      • Kostovska I.
      • Tosheska-Trajkovska K.
      • Topuzovska S.
      • et al.
      Urinary nephrin is earlier, more sensitive and specific marker of diabetic nephropathy than microalbuminuria.
      Briefly, urine samples were centrifuged immediately after collection at 4°C for 15 min, 3,200g. The supernatant was removed, the pellet suspended in 1.5 mL diethyl pyrocarbonate-treated phosphate-buffered saline, and then centrifuged at 12,000g for 5 min at 4°C. The washed pellet was resuspended in lysis buffer (RNeasy; Qiagen), and then kept frozen at −80°C until RNA extraction. The urinary pellet was then purified using an RNeasy mini kit (Qiagen), and cDNA was prepared with the SuperScript IV First-Strand Synthesis System (ThermoFisher).

      RNA Preparation and Real-Time Quantitative Polymerase Chain Reaction (PCR)

      Quantitation of podocin mRNA was performed with the StepOnePlus real-time PCR system (Applied Biosystems) using TaqMan Fast Advanced Master Mix (ThermoFisher) in a final volume of 10 μL per reaction. Commercially available TaqMan primers and probes, including 2 unlabeled PCR primers and one fluorescein amidite dye-labeled TaqMan minor groove binder probe, were used for both target genes (all from ThermoFisher). Each sample was run in triplicate. Results were analyzed with the use of Sequence Detection software, version 1.9 (Applied Biosystems). Gene expression for each signal was calculated by using the difference-in-threshold-cycle procedure. For the quantification of the target mRNA abundance, differences of threshold cycles between target genes were calculated. The cDNA standard curves generated from known concentrations of synthetic DNA oligonucleotides (all from ThermoFisher) that were identical in sequence to the corresponding target were constructed using these serially diluted standards. Assays were accepted only if R2 was 0.97 for the standard curve. cDNAs of known sequence and concentration were used as standards for each assay. Quantitation of podocalyxin mRNA was performed using the Quantstudio 3D Digital PCR system (Applied Biosystems) including the chip loader, ProFlex thermal cycler, and chip reader. Reactions were prepared in 15 μL volumes with Quantstudio 3D Digital PCR Mastermix v2 (Applied Biosystems), TaqMan gene expression assays (podocalyxin: Hs00210532_m1). The concentration of podocalyxin was calculated using Quantasoft software (Applied Biosystems).

      Urine Podocin and Podocalyxin Levels

      Urinary supernatant podocin and podocalyxin levels were detected by commercially available enzyme-linked immunosorbent assay (ELISA) kits (Bioassay Technology Laboratory) following the manufacturer’s protocol. The enzymatic reaction was detected at 450 nm in an automatic microplate reader (Spectrafluorplus; Tecan) and adjusted to urinary creatinine level as measured by a creatinine colorimetric assay kit (MilliporeSigma). Each sample was measured in duplicate, and all measured creatinine concentrations were within range of the standard curve.

      Intrarenal Podocin and Podocalyxin Levels

      Intrarenal podocin and podocalyxin levels were determined by western blotting with β-actin level used as the reference. The protein electrophoresis, transfer apparatus, and acrylamide gel were obtained from Bio-Rad. The polyvinylidene fluoride membrane, protein detection reagent, and radiograph films were obtained from Bio-Rad. Total protein from the kidney biopsy specimen was lysed with radioimmunoprecipitation assay buffer containing protease inhibitors. The protein concentrations were quantified with a BCA Protein Assay Kit (Beyotime Institute of Biotechnology). Individual proteins were then separated from 20 μg of total protein extract by acrylamide gel electrophoresis in Mini-PROTEAN Cell and transferred to Hybond-P polyvinylidene fluoride membrane. The membranes were then probed with primary antibodies against podocin (1:1,000, Abcam), podocalyxin (1:1,000, Abcam), and β-actin (1:1,000, Abcam). The corresponding secondary antibody was obtained from Abcam. The membrane was exposed to Amersham Hyperfilm Blue. The areas of the bands were estimated with the Image J software. The protein expression level of a sample was calculated by dividing the area of the protein of interest by the area of β-actin of the same sample.

      Morphometric Study of Kidney Biopsy

      The method of morphometry study of kidney scarring has been described in previous studies.
      • Wang G.
      • Szeto C.C.
      Methods of microRNA quantification in urinary sediment.
      ,
      • Wang G.
      • Lai F.M.
      • Chow K.M.
      • et al.
      Urinary mRNA levels of ELR-negative CXC chemokine ligand and extracellular matrix in diabetic nephropathy.
      Briefly, Jones’ silver staining was performed on 5 μm thick sections of kidney biopsy specimen. Semiquantitative computerized image analysis was performed with the Leica Twin Pro image analysis system (Leica Microsystems), which was connected to a Leica DC500 digital camera on a Leica DMRXA2 microscope with a ×40 objective (final calibration: 0.258 mm/pixel). Image analysis was performed with MetaMorph 4.0 image-analyzing software (Universal Imaging Corporation). Ten glomeruli and 10 randomly selected areas were assessed in each patient’s sample and the average percentage of scarred glomerular and tubulointerstitial areas, as represented by the area with positive silver staining, were computed.

      Outcome Measures

      All patients were followed for at least 12 months. All patients were followed by nephrologists and their treatment was not affected by the study and was only adjusted according to clinical need of the individual patients. Kidney function was monitored every 3 months. The primary outcome measures were dialysis-free survival and kidney event-free survival. Kidney event was defined as death of any cause, need for dialysis, or ≥40% decline in eGFR as compared to baseline. Secondary outcome measure includes the rate of eGFR decline, which was calculated by the least-square regression method.

      Statistical Analysis

      Statistical analysis was performed using SPSS for Windows software version 17.0. All the results are presented in mean ± standard deviation for normally distributed data and median (lower and upper quartiles) for the others. Because the data of gene expression levels were highly skewed, we used the Mann-Whitney U test to compare gene expression levels between groups and Spearman’s rank-order correlations to test associations between gene expression levels and other parameters. Data were further analyzed with univariate and multivariable Cox regression analysis for dialysis-free survival and kidney event-free survival. In addition to podocin and podocalyxin level quartiles, the multivariable Cox regression model was constructed by age, baseline eGFR, proteinuria, severity of glomerulosclerosis, and tubulointerstitial fibrosis. A P value of below 0.05 was considered statistically significant. All probabilities were 2-tailed.

      Results

      A total of 118 patients with biopsy-proven DKD were recruited. We also studied 13 nondiabetic patients with biopsy-proven hypertensive nephrosclerosis and 3 healthy kidney donors as controls. Their baseline demographic and clinical characteristics are summarized and compared in Table 1. For the DKD group, the average duration of diabetes was 6.0 ± 2.0 years. Their average hemoglobin A1c was 6.7 ± 0.8%; 47 patients (39.8%) were receiving insulin therapy.
      Table 1Baseline Demographic and Clinical Data
      DKDHTNCTLP
      No. of patients118133
      Sex (M:F)80:386:73:0< 0.001a
      Age (y)61.3 ± 12.261.3 ± 13.856.6±0.90.85b
      Serum creatinine (μmol/L)217.9 ± 162.2282.3 ± 162.663.0±15.60.14b
      eGFR (mL/min/1.73 m2)41.4 ± 31.328.1 ± 21.8110.3 ± 15.8< 0.001b
      Proteinuria (g/d)2.5 (1.7-4.5)0.6 (0.4-2.4)0.01c
      Histological damage (%)
       glomerulosclerosis32.7 ± 21.725.5 ± 19.30.04b
       tubulointerstitial fibrosis30.2 ± 17.432.5 ± 20.40.58b
      Abbreviations: CTL, healthy control group; DKD, diabetic kidney disease; eGFR, estimated glomerular filtration rate; F, female; HTN, hypertensive nephrosclerosis; M, male.
      Data are presented as mean ± standard deviation or median (interquartile range) and compared between the DKD and HTN groups by. aχ2 test bt test cMann-Whitney U test.

      Urinary and Intrarenal Podocyte Marker Levels

      The urinary and intrarenal levels of podocin and podocalyxin are summarized in Table 2. Representative western blot images are shown in Fig 1. Urinary and intrarenal levels of podocin and podocalyxin were further compared according to baseline chronic kidney disease stage, proteinuria level, and severity of glomerulosclerosis (Fig 2). In essence, urinary podocalyxin mRNA level, but not its urinary level measured by ELISA or intrarenal level, correlated with the severity of proteinuria. Urinary podocalyxin mRNA level was also marginally higher in patients with 25%-50% glomerulosclerosis than those with >25% or >50% glomerulosclerosis. The internal correlations between urinary and intrarenal podocyte marker levels in the DKD group are summarized in Table 3.
      Table 2Urinary and Intrarenal Podocyte Marker Levels
      DKDHTNCTLP
      Podocin
       Urinary level by ELISA (ng per mmol-Cr)47.12 (11.05-178.64)5.83 (1.90-19.48)36.46 (21.44-52.50)0.005
       Urinary mRNA level (copy number/μL)
      copy number per μL of resuspended sediment
      911.48 (225.9-3125.25)110.17 (51.97-339.94)436.60 (301.05-622.28)0.71
       Intrarenal level
      copy number per μL of resuspended sediment
      39,603.5 (37,049.0-45,179.3)41418.5 (37,957.5-43,740.5)0.82
      Podocalyxin
       Urinary level by ELISA (ng per mmol-Cr)1.44 (1.23-1.93)1.38 (1.05-2.10)1.24 (1.19-2.93)0.12
       Urinary mRNA level (copy number/μL)
      copy number per μL of resuspended sediment
      251.15 (57.79-697.31)90.42 (30.91-506.05)46.89 (35.44-73.06)0.49
       Intrarenal level
      relative band density in western blotting.
      24,813.0 (19,823.0-29,810.0)20953.5 (19,231.0-22,153.0)0.52
      Abbreviations: Cr, creatinine; CTL, healthy control group; DKD, diabetic kidney disease; ELISA, enzyme-linked immunosorbent assay; HTN, hypertensive nephrosclerosis; mRNA, messenger RNA.
      Data are presented as median (interquartile range) and compared between the DKD and HTN groups by Mann-Whitney U test.
      a copy number per μL of resuspended sediment
      b relative band density in western blotting.
      Figure thumbnail gr1
      Figure 1Representative images of western blots for intrarenal levels of podocin and podocalyxin. There was no significant difference in intrarenal podocin or podocalyxin levels between diabetic kidney disease (DKD) and hypertensive nephrosclerosis (HTN).
      Figure thumbnail gr2ab
      Figure 2Urinary and intrarenal levels of podocin and podocalyxin grouped according to: (A) baseline chronic kidney disease (CKD) stage; (B) proteinuria level; and (C) severity of glomerulosclerosis. Data were compared by Kruskal-Wallis test. Abbreviations: Cr, creatinine; ELISA, enzyme-linked immunosorbent assay; mRNA, messenger RNA. ∗Arbitrary unit for the relative band density in western blotting.
      Figure thumbnail gr2c
      Figure 2Urinary and intrarenal levels of podocin and podocalyxin grouped according to: (A) baseline chronic kidney disease (CKD) stage; (B) proteinuria level; and (C) severity of glomerulosclerosis. Data were compared by Kruskal-Wallis test. Abbreviations: Cr, creatinine; ELISA, enzyme-linked immunosorbent assay; mRNA, messenger RNA. ∗Arbitrary unit for the relative band density in western blotting.
      Table 3Internal Correlations Between Urinary and Intrarenal Podocyte Marker Levels in Diabetic Kidney Disease
      Urinary mRNA levelIntrarenal level
      Podocin
       Urinary level by ELISAr = 0.182, P = 0.04r = -0.237, P = 0.03
       Urinary mRNA levelr = -0.214, P = 0.04
      Podocalyxin
       Urinary level by ELISAr = 0.562, P < 0.001r = -0.228, P = 0.04
       Urinary mRNA levelr = -0.220, P = 0.04
      Note: All data were compared by Spearman’s rank correlation coefficient.
      Abbreviations: ELISA, enzyme-linked immunosorbent assay; mRNA, messenger RNA.

      Relationship With Clinical and Histological Parameters

      The relationship between podocyte marker levels and clinical and pathological parameters of the DKD group are summarized in Tables 4 and 5. In essence, there were modest but statistically significant correlations between the severity of tubulointerstitial fibrosis and urinary sediment mRNA levels of podocin and podocalyxin (but not the urinary levels measured by ELISA), whereas the amount of proteinuria had a modest correlation with urinary mRNA level of podocalyxin but not podocin. The severity of glomerulosclerosis had a modest correlation with intrarenal podocalyxin, but not podocin, level.
      Table 4Relationship Between Podocin and Podocalyxin Levels
      Podocin and Podocalyxin
      Urinary level by ELISAr = 0.521, P < 0.001
      Urinary mRNA levelr = -0.029, P = 0.74
      Intrarenal levelr = 0.048, P = 0.65
      Abbreviations: ELISA, enzyme-linked immunosorbent assay; mRNA, messenger RNA.
      Table 5Relationship Between Podocyte Marker Levels and Clinical and Pathological Parameters
      eGFRProteinuriaGlomerulosclerosisTubulointerstitial Fibrosis
      Podocin
       Urinary level by ELISAr = -0.056, P = 0.55r = 0.176, P = 0.07r = -0.090, P = 0.33r = -0.081, P = 0.39
       Urinary mRNA levelr = 0.022, P = 0.82r = 0.073, P =0.42r = -0.114, P = 0.22r = 0.196, P = 0.02
       Intrarenal levelr = -0.093, P = 0.41r = 0.016, P = 0.89r = 0.025, P = 0.82r = -0.040, P = 0.72
      Podocalyxin
       Urinary level by ELISAr = -0.049, P = 0.60r = -0.010, P = 0.92r = -0.049, P = 0.60r = -0.078, P = 0.41
       Urinary mRNA levelr = -0.124, P = 0.18r = 0.198, P = 0.04r = -0.059, P = 0.53r = 0.230, P = 0.01
       Intrarenal levelr =-0.128, P = 0.25r = -0.015, P = 0.90r = 0.211, P = 0.05r = 0.202, P = 0.07
      Note: Data were compared by Spearman’s rank correlation coefficient.
      Abbreviations: eGFR, estimated glomerular filtration rate; ELISA, enzyme-linked immunosorbent assay; mRNA, messenger RNA.

      Relationship With Clinical Outcome

      The DKD group was followed for an average of 19.0 ± 16.9 months. During this period, none of the patients died; 66 patients progressed to dialysis-dependent kidney failure, and another 21 patients had ≥40% decline in eGFR. The eGFR was measured 12.4 ± 5.8 times per patient, and the average rate of eGFR decline was -14.6 ± 19.9 mL/min/1.73 m2 per year. Patients with low intrarenal podocalyxin level had significantly higher risk for developing the kidney end point as compared with those with high level (Figure 3). The relationship between urinary and intrarenal podocin and podocalyxin levels and clinical outcome by univariate analysis is summarized in Table 6. With univariate analysis, only intrarenal podocalyxin level, but neither its urinary level nor any podocin marker, was associated with dialysis-free survival and kidney event-free survival. In contrast, urinary podocin level measured by ELISA had a modest but significant correlation with the rate of eGFR decline. After adjusting the clinical parameters by multivariable Cox regression analysis, intrarenal podocalyxin level remained an independent predictor of dialysis-free survival (adjusted hazard ratio, 1.85; 95% confidence interval, 1.21-2.82; P = 0.005) (Table 7). In this model, baseline eGFR was the other independent predictor of dialysis-free survival. By a similar analysis, intrarenal podocalyxin level also had a trend of predicting kidney event-free survival (adjusted hazard ratio, 1.36; 95% confidence interval, 0.94-1.95; P = 0.10), but the result did not reach statistical significance (Table 7). By multiple linear regression model analysis, urinary podocin level was still independently correlated with the rate of eGFR decline (Table 7). In this series of analyses, proteinuria did not predict dialysis-free survival, kidney event-free survival, or the rate of eGFR decline after adjusting for urinary and intrarenal podocin and podocalyxin level, baseline eGFR, and histological parameters.
      Figure thumbnail gr3
      Figure 3Kaplan-Meier plot of (A) dialysis-free survival; and (B) kidney event-free survival of the diabetic kidney diseases group. Patients were divided according to the quartiles of intrarenal podocalyxin level, with quartile I indicating the lowest level. Relative band intensity of podocalyxin western blotting for quartile I, II, III, and IV were <20,000, 20,000 to <25,000, 25,000 to <30,000, and ≥30,000, respectively. Data were compared with the log rank test.
      Table 6Relationship Between Podocyte Marker Levels and Clinical Outcome
      Dialysis-free SurvivalKidney Event-free SurvivalSlope of eGFR Decline
      Spearman’s rank correlation coefficient.
      Podocin
       Urinary level by ELISA0.97 (0.85-1.09), P = 0.580.95 (0.84-1.08), P = 0.46r = 0.238, P = 0.01
       Urinary mRNA level1.24 (0.99-1.55), P = 0.061.17 (0.94-1.45), P = 0.17r = -0.093, P = 0.37
       Intrarenal level1.25 (0.70-2.21), P = 0.450.86 (0.45-1.48), P = 0.50r = 0.002, P = 0.99
      Podocalyxin
       Urinary level by ELISA2.76 (0.25-30.50), P = 0.416.84 (0.50-93.01), P = 0.15r = 0.026, P = 0.79
       Urinary mRNA level1.32 (0.69-1.08), P = 0.200.92 (0.73-1.15), P = 0.46r = 0.193, P = 0.06
       Intrarenal level1.81 (1.01-3.23), P = 0.052.01 (1.11-3.65), P = 0.02r = -0.170, P = 0.15
      aUnadjusted hazard ratio (95% confidence interval) by univariate Cox analysis.
      b Spearman’s rank correlation coefficient.
      Table 7Summary of Multivariable Analysis Models
      Multivariable Cox Regression Model for Dialysis-free SurvivalMultivariable Cox Regression Model for Kidney Event-free SurvivalMultiple Linear Regression Model for the Rate of eGFR Decline
      AHR95%CIPAHR95% CIPUnstandardized B95% CIP
      Age0.980.95-1.000.100.970.95-1.000.080.344-0.069 to 0.7570.10
      Baseline eGFR0.950.93-0.97< 0.0011.000.98-1.010.73-0.196-0.034 to -0.3580.02
      Proteinuria1.000.90-1.120.961.050.94-1.170.400.142-1.565 to 1.8490.87
      Glomerulosclerosis1.000.99-1.010.881.021.00-1.040.08-0.112-0.346 to 0.1210.34
      Tubulointerstitial fibrosis1.031.01-1.040.011.000.98-1.030.97-0.128-0.465 to 0.2090.45
      Intrarenal podocalyxin1.041.00-1.090.031.001.00-1.010.75
      Urinary podocin level1.8460.166-3.5270.03
      Abbreviations: AHR, adjusted hazard ratio; CI, confidence interval; eGFR, estimated glomerular filtration rate.

      Discussion

      In our study, we found that urinary podocalyxin level closely correlated with its mRNA level, but they did not appear to predict the progression of DKD. Intrarenal podocalyxin level, but not its urinary level, was an independent predictor of dialysis-free survival, whereas urinary podocin level determined by ELISA correlated with the rate of eGFR decline.
      Our results are in line with but slightly different from that of previous studies, which showed that urinary podocin and podocalyxin are early-stage biomarkers of DKD. Similar to our present result, 2 previous studies reported that urinary podocin mRNA level was higher in patients with DKD than normal controls, and the level correlated with the severity of albuminuria and urinary podocyte count.
      • Wei P.Z.
      • Kwan B.C.
      • Chow K.M.
      • et al.
      Urinary mitochondrial DNA level in non-diabetic chronic kidney diseases.
      ,
      • Wang G.
      • Lai F.M.
      • Lai K.B.
      • Chow K.M.
      • Li K.T.
      • Szeto C.C.
      Messenger RNA expression of podocyte-associated molecules in the urinary sediment of patients with diabetic nephropathy.
      Urinary podocin mRNA-to-creatinine ratio had been considered as a marker of podocyte detachment and predicted the rate of subsequent kidney function decline.
      • Petrica L.
      • Ursoniu S.
      • Gadalean F.
      • et al.
      Urinary podocyte-associated mRNA levels correlate with proximal tubule dysfunction in early diabetic nephropathy of type 2 diabetes mellitus.
      As for podocalyxin, Kostavska et al
      • Petrica L.
      • Ursoniu S.
      • Gadalean F.
      • et al.
      Urinary podocyte-associated mRNA levels correlate with proximal tubule dysfunction in early diabetic nephropathy of type 2 diabetes mellitus.
      reported that urinary podocalyxin cutoff level of 43.8 ng/mL had 73.3% sensitivity and 93.3% specificity to detect early-stage DKD, and Hara et al
      • Kostovska I.
      • Trajkovska K.T.
      • Cekovska S.
      • et al.
      Role of urinary podocalyxin in early diagnosis of diabetic nephropathy.
      reported that urinary podocalyxin level was higher than the cutoff value in 53.8% of patients with diabetes at the normoalbuminuric stage. Similar to previous studies, we found that urinary podocalyxin level correlated with the degree of proteinuria in DKD.
      • Fukuda A.
      • Minakawa A.
      • Kikuchi M.
      • et al.
      Urinary podocyte mRNAs precede microalbuminuria as a progression risk marker in human type 2 diabetic nephropathy.
      ,
      • Hara M.
      • Yamagata K.
      • Tomino Y.
      • et al.
      Urinary podocalyxin is an early marker for podocyte injury in patients with diabetes: establishment of a highly sensitive ELISA to detect urinary podocalyxin.
      However, although intrarenal podocalyxin level inversely correlated with the urinary podocalyxin mRNA and protein levels, only the intrarenal podocalyxin level correlated with the severity of glomerulosclerosis and dialysis-free survival. In contrast, urinary podocin level measured by ELISA, but not urinary podocin mRNA level as reported previously, correlated with the rate of eGFR decline.
      • Shoji M.
      • Kobayashi K.
      • Takemoto M.
      • Sato Y.
      • Yokote K.
      Urinary podocalyxin levels were associated with urinary albumin levels among patients with diabetes.
      In this study, urinary podocin and podocalyxin mRNA levels had little correlation with the corresponding levels determined by ELISA. It is important to note that we measured podocin and podocalyxin levels in urinary supernatant by ELISA, whereas the mRNA levels were quantified from the cellular sediment. The 2 methods probably measure targets from different origins and may reflect different disease status. Specifically, mRNA in urinary sediment likely comes from denuded podocytes or their cellular fragments, whereas soluble podocin and podocalyxin in urinary supernatant probably originates from cellular leak following sublethal podocyte injury, but this hypothesis has not been proven.
      Our results suggest that although podocin and podocalyxin are both podocyte-specific markers, they have different biological and clinical implications. From a biological point of view, podocalyxin is the major negatively charged protein synthesized by podocytes, and the expression is restricted to the apical membrane during the maturation of podocytes.
      • Akankwasa G.
      • Jianhua L.
      • Guixue C.
      • Changjuan A.
      • Xiaosong Q.
      Urine markers of podocyte dysfunction: a review of podocalyxin and nephrin in selected glomerular diseases.
      Podocin, on the other hand, is a hairpin-like protein and is part of the slit-diaphragm protein complex responsible for connecting nephrin to the actin cytoskeleton under the cell membrane.
      • Kestilä M.
      • Lenkkeri U.
      • Männikkö M.
      • et al.
      Positionally cloned gene for a novel glomerular protein--nephrin--is mutated in congenital nephrotic syndrome.
      As to the clinical implication, intrarenal podocalyxin level significantly predicted dialysis-free survival and was less reliable in predicting kidney event-free survival, whereas urinary podocin level predicted the rate of eGFR decline. As an outcome measure, the recent consensus is that dialysis-free survival focuses on short-term progression and is biased toward rapid progressors, whereas the rate of eGFR decline put emphasis on the slow progressors in the long term.
      • Krolewski A.S.
      • Skupien J.
      • Rossing P.
      • Warram J.H.
      Fast renal decline to end-stage renal disease: an unrecognized feature of nephropathy in diabetes.
      Our result seems to indicate that intrarenal podocalyxin level is a marker of rapid progression, suggesting that this protein is induced in DKD, possibly as a result of podocyte stimulation or overactivation. However, the exact mechanism is unknown and deserves further study. In contrast, urinary podocin level represents the rate of progression for indolent cases. Although urinary podocalyxin level had a modest correlation with its intrarenal expression, it was not a predictor of dialysis-free survival, and our results would lead to the conclusion that urinary podocalyxin level may not be a suitable noninvasive biomarker for DKD.
      Another interesting observation of our study was that intrarenal podocalyxin level inversely correlated with the urinary podocalyxin level, which is consistent with the notion that podocyte fragments are detached from diseased glomeruli into the urine. There is a wealth of literature to support that podocyte depletion and glomerulosclerosis have a direct relationship in the puromycin aminonucleoside-treated rat, and podocyte loss correlated with the progression of glomerular injury in type 2 diabetes.
      • Kim Y.H.
      • Goyal M.
      • Kurnit D.
      • et al.
      Podocyte depletion and glomerulosclerosis have a direct relationship in the PAN-treated rat.
      ,
      • Pagtalunan M.E.
      • Miller P.L.
      • Jumping-Eagle S.
      • et al.
      Podocyte loss and progressive glomerular injury in type II diabetes.
      Because the correlation between intrarenal and urinary podocalyxin levels was substantially higher than that of podocin, our result seems to suggest that cellular fragments, presumably from the apical membrane of diseased podocytes, rather than intact podocytes are the predominant component shed into the urine. In the present study, we did not measure the rate of intact podocyte loss in urine, and our hypothesis would need further studies to confirm. It is also important to note that the traditional method of detecting “intact” podocyte in urine uses cytospin techniques with immunofluorescence study by specific anti-podocalyxin antibody, which also detects podocyte apical membrane fragments and is prone to false-positive results.
      • Hara M.
      • Yamamoto T.
      • Yanagihara T.
      • et al.
      Urinary excretion of podocalyxin indicates glomerular epithelial cell injuries in glomerulonephritis.
      In addition, there are several other limitations of this study. First, this was a retrospective study on patients with DKD who underwent kidney biopsy. There may be referral (ie, patients with typical DKD might not be referred for biopsy) and selection bias (ie, patients with substantial kidney scarring and insufficient kidney tissue in the biopsy specimen would not be recruited). Second, this was a single-center study, and the sample size was small. The external validity of our findings is unknown, and the limited sample size precluded an extensive multivariable analysis. Similarly, additional analysis to determine the subgroup of patients for which podocyte markers provide valuable prognostic information was limited by the small sample size and number of events. In theory, urinary podocin and podocalyxin levels could also be measured by western blotting, but absolute quantification is less robust as compared to the simple ELISA assay. Unfortunately, the size of the available kidney biopsy tissue was small, and the ELISA test was not feasible. Because we only measured the urinary podocin and podocalyxin levels at 1 time point (ie, around the time of kidney biopsy), the intraindividual variability of the measurement is unknown, and it remains to be determined whether serial monitoring of urinary podocin or podocalyxin, either at mRNA or protein level, would provide additional prognostic information. Notably, introduction of effective treatment that reduces proteinuria or improves kidney function may lead to a change in the urinary podocyte marker levels, and further studies in this area are needed.
      Nonetheless, our results show that the urinary podocin level may be a prognostic marker of DKD. Although the intrarenal podocalyxin level also has prognostic value, it does not appear to be a promising clinical marker in view of the invasive nature of the test, and urinary podocalyxin, either at mRNA or protein level, does not provide additional prognostic information. Our results also suggest that qualitative changes in various cellular compartments of the podocyte, rather than the change in the absolute podocyte number, may be an important pathological alteration in DKD.

      Article Information

      Authors’ Full Names and Academic Degrees

      Lingfeng Zeng, MB, Winston Wing-Shing Fung, MBBCh (Cantab), MRCP (UK), Gordon Chun-Kau Chan, MBChB, MRCP (UK), Jack Kit-Chung Ng, MBChB, MRCP (UK), Kai-Ming Chow, MBChB, FRCP, and Cheuk-Chun Szeto, MD, FRCP.

      Authors’ Contributions

      Research idea and study design: LZ, CCS; data acquisition: LZ, WWSF, GCKC, JKCN; data analysis/interpretation: LZ, CCS; statistical analysis: LZ, CCS; supervision or mentorship: KMC, CCS. Each author contributed important intellectual content during manuscript drafting or revision and accepts accountability for the overall work by ensuring that questions pertaining to the accuracy or integrity of any portion of the work are appropriately investigated and resolved.

      Support

      This study was supported by the Research Grant Council Research Impact Fund (project reference R4012-18), Chinese University of Hong Kong research accounts 6905134, 2410026, and 3133200. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

      Financial Disclosure

      The authors declare that they have no relevant financial interests.

      Acknowledgements

      The authors would like to thank Professor Fernand MacMoune Lai, Department of Anatomical & Cellular Pathology, and Dr Ka-Bik Lai, Li Ka Shing Institute of Health Sciences (LiHS), Faculty of Medicine, the Chinese University of Hong Kong, for their assistance in the pathological assessment of the kidney biopsy samples.

      Peer Review

      Received February 18, 2022. Evaluated by 2 external peer reviewers, with direct editorial input from the Statistical Editor, an Associate Editor, and the Editor-in-Chief. Accepted in revised form September 11, 2022.

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