Urine podocyte mRNAs mark progression of renal disease

Abstract

Because loss of podocytes associates with glomerulosclerosis, monitoring podocyte loss by measuring podocyte products in urine may be clinically useful. To determine whether a single episode of podocyte injury would cause persistent podocyte loss, we induced limited podocyte depletion using a diphtheria toxin receptor (hDTR) transgenic rat. We monitored podocyte loss by detecting nephrin and podocin mRNA in urine particulates with quantitative reverse transcriptase-PCR. Aquaporin 2 mRNA served as a kidney reference gene to account for variable kidney contribution to RNA amount and quality. We found that a single injection of diphtheria toxin resulted in an initial peak of proteinuria and podocyte mRNAs (podocin and nephrin) followed 8 d later by a second peak of proteinuria and podocyte mRNAs that were podocin positive but nephrin negative. Proteinuria that persisted for months correlated with podocin-positive, nephrin-negative mRNAs in urine. Animals with persistent podocyte mRNA in urine progressed to ESRD with global podocyte depletion and interstitial scarring. Podocytes in ectatic tubules expressed podocalyxin and podocin proteins but not nephrin, compatible with detached podocytes' having an altered phenotype. Parallel human studies showed that biopsy-proven glomerular injury associated with increased urinary podocin:aquaporin 2 and nephrin:aquaporin 2 molar ratios. We conclude that a single episode of podocyte injury can trigger glomerular destabilization, resulting in persistent podocyte loss and an altered phenotype of podocytes recovered from urine. Podocyte mRNAs in urine may be a useful clinical tool for the diagnosis and monitoring of glomerular diseases.

Figure 1.

Four groups based on urine protein:creatinine ratio and serum creatinine: Mean urine protein:creatinine ratio data for four groups of rats. Progressor rats had an initial biphasic peak of proteinuria followed by high level proteinuria until reaching ESKD. Limited proteinuric rats had a single peak of proteinuria that returned to close to control levels. Control and nonproteinuric groups are superimposable and had no increased proteinuria. Serum creatinine values measured at 6 mo or at ESKD were as follows: Control 0.37 ± 0.09 mg/dl; nonproteinuric 0.37 ± 0.10 mg/dl; limited proteinuric 0.30 ± 0.12 mg/dl; progressors 3.03 ± 0.46 mg/dl. Progressors had significantly higher values compared with other groups (P < 0.01, by Kruskal-Wallis test and Scheffe test).

Figure 2.

Representative histology for each group: Podocytes were identified using GLEPP1/peroxidase staining shown without counterstain (left) and with periodic acid-Schiff (PAS)/hematoxylin counterstain (middle). Masson's Trichrome staining is shown on right. From top to bottom, micrographs are from control, nonproteinuric, limited proteinuric and progressor groups. Note that progressors showed almost total loss of peroxidase product indicating loss of almost all podocytes in association with progression (glomeruli shown by arrowheads). In contrast the limited proteinuric group showed patchy loss of podocytes from some glomeruli (arrows). The nonproteinuric group showed an occasional glomerulus with patchy podocytes loss (arrow). Progressors showed extensive interstitial scarring as indicated by Masson's Trichrome staining. Limited progressors showed patchy interstitial scarring.

Figure 3.

Quantification of histology: Quantitative information related to Figure 2. (A) Percentage of glomerular tuft area containing GLEPP1 is significantly reduced in progressors and limited proteinurics in relation to controls. (B) Percentage of glomeruli with segmental or global lesions is significantly increased in progressor and limited proteinuric groups compared with control. *P < 0.05 and **P < 0.01 as assessed by Kruskal-Wallis test and then Scheffe test.

Figure 4.

Immunofluorescent photomicrographs developed for nephrin, podocin, and podocalyxin protein expression in progressor (chronic kidney disease) compared with normal kidney. The immunofluorescent color codes for each protein are shown above each set of panels. (A and B) Progressor (chronic kidney disease) rat glomeruli (delineated by white blocks) show absence of podocyte markers (podocalyxin, nephrin, or podocin) except in parts of glomeruli where intact podocytes still persisted in some glomeruli. (C and D) Podocytes in ectatic tubules in progressor renal cortex contain detached podocytes (identified by intense green podocalyxin immunofluorescent cell surface) that express podocin (D) but not nephrin (C). A higher magnification view of the detached podocytes is shown in the insets. (E and F) Normal glomerular podocytes express podocalyxin, nephrin, and podocin.

Figure 5.

RNA parameters for the progressor group: Excretion of total RNA (A), urine AQP2 mRNA (B), urine nephrin mRNA (C), and urine podocin mRNA (D) for the acute (left) and chronic (right) progressor phases of injury. *P < 0.05 and **P < 0.01 as assessed by Kruskal-Wallis test and then Scheffe test. Unless otherwise specified, units are arbitrary, based on standard curves from wild-type animals as described in the Concise Methods section.

Figure 6.

Comparison of urine nephrin and podocin mRNA excretion between groups through day 56: The progressor group excreted high-level urine nephrin mRNA (red) as an initial peak followed by low-level mRNA excretion. Podocin mRNA (blue) was excreted persistently throughout the time course. A similar pattern but reduced in quantity was seen for the limited proteinuric group. Unless otherwise specified, units are arbitrary, based on standard curves from wild-type animals as described in the Concise Methods section.

Figure 7.

Urine nephrin and podocin mRNA expressed as a ratio with AQP2 mRNA. In the acute phase (left), both nephrin:AQP2 (top) and podocin:AQP2 (bottom) mRNA ratios were highest in progressors and still significantly increased in limited proteinurics. In the chronic phase (right), only urine podocin:AQP2 (bottom) ratios remained significantly increased. *P < 0.05 and **P < 0.01 as assessed by Kruskal-Wallis test and then Scheffe test.

Figure 8.

Time course of proteinuria versus urine nephrin:AQP2 and podocin:AQP2 excretion in progressors. The urine protein:creatinine ratio profile (top) shows two peaks (A and B) during the initial injury phase as indicated by the dotted lines. The first peak (A) corresponds temporally to the nephrin:AQP2 mRNA peak, and the second peak (B) corresponds to the podocin:AQP2 peak (bottom). Persistent proteinuria during the chronic phase of progression corresponds to continued high-level podocin:AQP2 and low-level nephrin:AQP2 mRNA excretion.

Figure 9.

Relationship between proteinuria and mRNA excretion of urine nephrin and podocin: In the acute phase (left), proteinuria correlated highly with urine nephrin mRNA excretion (top; r² = 0.84) but not with urine podocin mRNA excretion (bottom; r² = 0.29). In the chronic phase (right), both nephrin and podocin mRNA correlated with proteinuria (r² = 0.80 and 0.69, respectively). Unless otherwise specified, units are arbitrary, based on standard curves from wild-type animals as described in the Concise Methods section.

Figure 10.

Human urine RNA decay rates: mRNAs from normal subjects decayed rapidly at room temperature (RT; top). The rate of decay was slower at 4°C (middle), but, by 6 h, 30 to 40% decay was observed for all mRNAs. The rate of decay was similar for all mRNAs so that expression of podocyte mRNA data divided by a reference gene (AQP2) was relatively constant even after 48 h at 4°C (bottom).