Urinary podocyte mRNAs precede microalbuminuria as a progression risk marker in human type 2 diabetic nephropathy

Abstract

Earlier detection of progression risk in diabetic nephropathy will allow earlier intervention to reduce progression. The hypothesis that urinary pellet podocyte mRNA is a more sensitive progression risk marker than microalbuminuria was tested. A cross sectional cohort of 165 type 2 diabetics and 41 age and sex-matched controls were enrolled. Podocyte stress (Urinary pellet podocin:nephrin mRNA ratio), podocyte detachment (Urinary pellet podocin mRNA:creatinine ratio: UPPod:CR) and a tubular marker (Urinary pellet aquaporin 2:creatinine ratio) were measured in macro-albuminuric, micro-albuminuric and norm-albuminuric groups. eGFR was reassessed after 4 years in 124 available diabetic subjects. Urinary pellet podocyte and tubular mRNA markers were increased in all diabetic groups in cross-sectional analysis. After 4 years of follow-up univariable and multivariate model analysis showed that the only urinary markers significantly related to eGFR slope were UPPod:CR (P < 0.01) and albuminuria (P < 0.01). AUC analysis using K-fold cross validation to predict eGFR loss of ≥ 3 ml/min/1.73m²/year showed that UPPod:CR and albuminuria each improved the AUC similarly such that combined with clinical variables they gave an AUC = 0.70. Podocyte markers and albuminuria had overlapping AUC contributions, as expected if podocyte depletion causes albuminuria. In the norm-albuminuria cohort (n = 75) baseline UPPod:CR was associated with development of albuminuria (P = 0.007) and, in the tertile with both normal kidney function (eGFR 84 ± 11.7 ml/min/1.73m²) and norm-albuminuria at baseline, UPPod:CR was associated with eGFR loss rate (P = 0.003). In type 2 diabetics with micro- or macro-albuminuria UPPod:CR and albuminuria were equally good at predicting eGFR loss. For norm-albuminuric type 2 diabetics UPPod:CR predicted both albuminuria and eGFR loss.

Figure 1

Urinary pellet podocyte mRNA markers and albuminuria in healthy control and at various stages of diabetic kidney disease. (A) Urinary albumin:creatinine ratio. (B) Urinary pellet podocin:nephrin mRNA ratio. (C) Urinary pellet podocin mRNA:creatinine ratio. (D) Urinary pellet aquaporin 2 mRNA:creatinine ratio. (E) Urinary pellet podocin:aquaporin 2 mRNA ratio. Urinary pellet excretion of podocyte-specific markers (measured using the podocin mRNA marker) and tubular marker (measured using the aquaporin 2 mRNA marker) were both increased in diabetics. These data are compatible with injury occurring to both glomerular and tubular compartments in diabetes. The macro-albuminuric group was losing podocytes more rapidly than tubular cells.*P < 0.05 and **P < 0.01 vs control, #P < 0.05 and ##P < 0.01 vs macro-albuminuria, assessed using the Kruskal–Wallis test followed by the Dunn test.

Figure 2

AUC analysis comparing albuminuria to urinary pellet podocyte mRNA excretion to predict decrease in eGFR over a 4 year follow-up period. No statistical difference was observed between these two markers. Thus once albuminuria is present it predicts rate of eGFR decline as well as, but no better than, the UPPod:CR.

Figure 3

Relationship between baseline podocyte detachment (UPPod:CR) and the predicted UAlb:CR in normo-albuminuric patients adjusted for various clinical factors. The amount of UPPod:CR at baseline was significantly associated with increased albumin excretion rates (UAlb:CR) over the 4 year period of observation. The predictive margins were calculated using linear mixed models clustered at the patient level.

Figure 4

Relationship between baseline level of podocyte detachment (UPPod:CR) (A) and albuminuria (UAlb:Cre) (B) and GFR decline among those with normoalbuminria at baseline in the tertile with eGFR. In this group UAlb:CR within the normal range was not associated with eGFR decline (r = 0.09, P = 0.63). In contrast, UPPod:CR was significantly associated with eGFR decline (r =  − 0.56, P = 0.003) in the highest tertile of baseline eGFR (n = 25, Mean eGFR = 84 ± 11.7 ml/min/1.73 m², range 72–127 ml/min/1.73 m²).