Urine podocyte mRNAs mark disease activity in IgA nephropathy

Abstract

Background: Podocyte depletion is a major mechanism driving glomerulosclerosis. We and others have previously projected from model systems that podocyte-specific mRNAs in the urine pellet might serve as glomerular disease markers. We evaluated IgA nephropathy (IgAN) to test this concept.

Methods: From 2009 to 2013, early morning voided urine samples and kidney biopsies from IgAN patients (n = 67) were evaluated in comparison with urine samples from healthy age-matched volunteers (n = 28). Urine podocyte (podocin) mRNA expressed in relation to either urine creatinine concentration or a kidney tubular marker (aquaporin 2) was tested as markers.

Results: Urine podocyte mRNAs were correlated with the severity of active glomerular lesions (segmental glomerulosclerosis and acute extracapillary proliferation), but not with non-glomerular lesions (tubular atrophy/interstitial fibrosis) or with clinical parameters of kidney injury (serum creatinine and estimated glomerular filtration rate), or with degree of accumulated podocyte loss at the time of biopsy. In contrast, proteinuria correlated with all histological and clinical markers. Glomerular tuft podocyte nuclear density (a measure of cumulative podocyte loss) correlated with tubular atrophy/interstitial fibrosis, estimated-glomerular filtration rate and proteinuria, but not with urine podocyte markers. In a subset of the IgA cohort (n = 19, median follow-up period = 37 months), urine podocyte mRNAs were significantly decreased after treatment, in contrast to proteinuria which was not significantly changed.

Conclusions: Urine podocyte mRNAs reflect active glomerular injury at a given point in time, and therefore provide both different and additional clinical information that can complement proteinuria in the IgAN decision-making paradigm.

Keywords: IgA nephropathy; glomerular disease; podocyte; proteinuria; urine podocyte mRNA.

FIGURE 1:

Comparison of urinary markers in patients with IgAN and healthy volunteers. (a) Urine protein:creatinine ratio. (b) Urine podocin:AQP2 mRNA ratio (U-PodAR). (c) Urine podocin mRNA:creatinine ratio (U-PodCR). (d) Relationship between levels of proteinuria and U-PodAR in patients with IgAN. (e) Relationship between levels of proteinuria and U-PodCR in patients with IgAN. Proteinuria and urine podocyte mRNA levels in patients with IgAN were significantly increased compared with healthy volunteer controls (P < 0.001). Urine podocyte mRNA levels were correlated with proteinuria (U-PodAR, r = 0.45, P = 0.0001, and U-PodCR, r = 0.56, P < 0.0001, [n = 67]).

FIGURE 2:

Relationship between urine podocyte mRNAs, proteinuria and the Oxford IgAN histological classification. (a) The levels of proteinuria and urine podocyte mRNAs with or without mesangial hypercellularity (M1: n = 23, M0: n = 44). (b) The levels of proteinuria and urine podocyte mRNAs with or without segmental glomerulosclerosis (S1: n = 56, S0: n = 11). (c) The levels of proteinuria and urine podocyte mRNAs with or without endocapillary hypercellularity (E1: n = 26, E0: n = 41). (d) The levels of proteinuria and urine podocyte mRNAs with or without tubular atrophy/interstitial fibrosis (T1, 2: n = 15, T0: n = 52). Both podocyte markers and proteinuria were significantly increased in association with segmental glomerulosclerosis lesions. The tubular atrophy/interstitial fibrosis lesions were associated with proteinuria, but not podocyte markers. *P < 0.05 and **P < 0.01, assessed by Mann–Whitney U test.

FIGURE 3:

Relationship between urine podocyte mRNAs, proteinuria and histological findings of acute extracapillary proliferative lesions. (a) The levels of proteinuria and urine podocyte mRNAs with (n = 35) or without (n = 32) extracapillary lesion (crescent formation). (b) Relationship between levels of proteinuria and % crescent formation. (c) Relationship between levels of urine podocin:AQP2 mRNA ratio and % crescent formation. (d) Relationship between levels of urine podocin mRNA:creatinine ratio and % crescent formation. By group analysis, urine podocyte mRNAs and proteinuria were significantly increased in association with acute extracapillary proliferative lesions (P < 0.001). Furthermore, urine podocyte mRNAs (U-PodAR and U-PodCR) and proteinuria also correlated significantly with the extent of acute extracapillary proliferative lesions (% crescent formation; U-ProtCR, r = 0.54, P < 0.0001; U-PodAR, r = 0.62, P < 0.0001; U-PodCR, r = 0.64, P < 0.0001; [n = 67]). *P < 0.05 and **P < 0.01, as assessed by Mann–Whitney U test.

FIGURE 4:

Relationship between histological findings of podocyte depletion and urine podocyte mRNAs in patients with IgAN. Representative histological findings of podocyte depletion as demonstrated by TLE4 immunostaining (a) mild podocyte depletion (<25%), (b) moderate podocyte depletion (25–50%) and (c) severe podocyte depletion (>50%). (d) Relationship between the urine protein:creatinine ratio and TLE4 podocyte nuclear density. (e) Relationship between the urine podocin:AQP2 mRNA ratio and TLE4 podocyte nuclear density. (f) Relationship between the urine podocin mRNA:creatinine ratio and TLE4 podocyte nuclear density. (g) Relationship between levels of serum creatinine and TLE4 podocyte nuclear density. (h) Relationship between estimated-glomerular filtration rate (e-GFR) and TLE4 podocyte nuclear density. (i) Relationship between levels of proteinuria and serum creatinine. (j) Relationship between the urine podocin:AQP2 mRNA ratio and serum creatinine. (k) Relationship between the urine podocin mRNA:creatinine ratio and serum creatinine. (l) Relationship between levels of proteinuria and e-GFR. (m) Relationship between the urine podocin:AQP2 mRNA ratio and e-GFR. (n) Relationship between the urine podocin mRNA:creatinine ratio and e-GFR. Podocyte nuclear density correlated with proteinuria, but not with urine podocyte mRNA levels. Furthermore, podocyte nuclear density and proteinuria correlated highly with kidney function (serum creatinine or e-GFR), in contrast urine podocyte markers did not correlate with kidney function (either serum creatinine or e-GFR).

FIGURE 5:

Relationship between degree of podocyte depletion measured in biopsies, the Oxford classification and acute extracapillary proliferative lesions of IgAN. (a) The podocyte density, with or without mesangial hypercellularity (M1: n = 23, M0: n = 44). (b) The podocyte density with or without segmental glomerulosclerosis (S1: n = 56, S0: n = 11). (c) The podocyte density with or without endocapillary hypercellulariy (E1: n = 26, E0: n = 41). (d-1) The podocyte density with or without tubular atrophy/interstitial fibrosis (T1, 2: n = 15, T0: n = 52). (d-2) The e-GFR as associated with severity of tubular atrophy/interstitial fibrosis. (e) The podocyte density with or without extracapillary lesion (crescent formation; Ex1: n = 35, Ex0: n = 32). Tubular atrophy/interstitial fibrosis lesions were significantly associated with degree of podocyte nuclear density reduction and e-GFR.

FIGURE 6:

Levels of proteinuria and urine podocyte mRNAs after treatment in patients with IgAN. (a) e-GFR after treatment. (b) Proteinuria after treatment. (c) The urine podocin:AQP2 mRNA ratio after treatment. (d) The urine podocin mRNA:creatinine ratio after treatment. Urine podocyte mRNAs were significantly decreased compared with before treatment (U-PodAR, U-PodCR, P = 0.01, P < 0.01, respectively), however, U-ProtCR was not (P = 0.35).