Study of the natural history of diabetic kidney disease (DKD)

Introduction: Diabetes mellitus is a global public health problem affecting an increasing number of chronic kidney disease patients. The progression of diabetic kidney disease (DKD) is variable, and the natural course of the disease may be evolving. We aimed here to revisit the natural history of DKD and to evaluate the validity of a number of urinary biomarkers in predicting the progression of DKD by determination of their independent predictive values compared to more conventional risk factors. Methods: To address this, the effects of potential risk factors on the progression of DKD was studied retrospectively in a cohort of diabetic CKD patients followed at Sheffield Kidney Institute (n = 385). Another observational prospective study was performed on 102 T2DM with DKD, 21 diabetic patients without kidney disease and 21 healthy volunteers. Each provided fresh midstream urine sample with demographic and clinical parameters collected from the unit's electronic database. Commercial ELISA kits were optimised and used to measure different urinary proteins. After cytospin, urine cells were stained using anti- fibroblast specific protein-l (FSP-l) antibody and standard cytochemistry techniques to identify potential myofibroblasts. Finally, the urine matrix metalloproteinase activity was measured by using fluoresce in conjugate substrates. Results: In the retrospective DKD cohort study, baseline proteinuria, HbA 1 c and MAP as well as the presence of vascular co-morbidities were associated with a faster rate of annual eGFR decline. Whereas, in the prospective study, albuminuria was the weakest predictor of functional outcome (ROC = 64%) and transferrinuria was the strongest (ROC = 81.2%). Furthermore, the combined score of all measured proteins did not increase the predictive value considerably (86.3%) above that of urinary transferrin. Urine cytochemistry showed that FSP-I positive cells were detected in the urine of 61 % of DKD progressors group compared to 27% of non-progressors (P = 0.004). In contrast, all slides from DM patients and healthy volunteers were negatively stained for FSP-l. The predictive value of urinary FSP I positive cells (83.6%) was superior to albuminuria as well as podocyturia (66.2%). Finally. urine MMP activity was elevated in DKD. However, there was a significantly lower MMP activity in the progressors DKD patients. Conclusions: This thesis identified a number of key observations relating to the progression of DKD. Firstly, it showed that beside conventional clinical predictors of progression, vascular comorbidities were associated with a faster rate of progression of DKD in a largely older population suffering from T2DM. Urinary biomarkers analysis showed that peptiduria and in particular transferrinuria was highly predictive of DKD progression. For the first time, we show in this thesis the potential use of urine FSP-I positive cells (presumably kidney derived myofibroblasts) to predict DKD outcomes. Also, the lower urinary MMP activity predicted a faster rate of progression. In summary, this thesis successfully identifies a number of biomarkers that may prove highly valuable to predict DKD progression in T2DM affected by diffuse vascular pathology and co-morbidities.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Study of the natural history of diabetic kidney disease (DKD)

Introduction: Diabetes mellitus is a global public health problem affecting an increasing number of chronic kidney disease patients. The progression of diabetic kidney disease (DKD) is variable, and the natural course of the disease may be evolving. We aimed here to revisit the natural history of DKD and to evaluate the validity of a number of urinary biomarkers in predicting the progression of DKD by determination of their independent predictive values compared to more conventional risk factors. Methods: To address this, the effects of potential risk factors on the progression of DKD was studied retrospectively in a cohort of diabetic CKD patients followed at Sheffield Kidney Institute (n = 385). Another observational prospective study was performed on 102 T2DM with DKD, 21 diabetic patients without kidney disease and 21 healthy volunteers. Each provided fresh midstream urine sample with demographic and clinical parameters collected from the unit's electronic database. Commercial ELISA kits were optimised and used to measure different urinary proteins. After cytospin, urine cells were stained using anti- fibroblast specific protein-l (FSP-l) antibody and standard cytochemistry techniques to identify potential myofibroblasts. Finally, the urine matrix metalloproteinase activity was measured by using fluoresce in conjugate substrates. Results: In the retrospective DKD cohort study, baseline proteinuria, HbA 1 c and MAP as well as the presence of vascular co-morbidities were associated with a faster rate of annual eGFR decline. Whereas, in the prospective study, albuminuria was the weakest predictor of functional outcome (ROC = 64%) and transferrinuria was the strongest (ROC = 81.2%). Furthermore, the combined score of all measured proteins did not increase the predictive value considerably (86.3%) above that of urinary transferrin. Urine cytochemistry showed that FSP-I positive cells were detected in the urine of 61 % of DKD progressors group compared to 27% of non-progressors (P = 0.004). In contrast, all slides from DM patients and healthy volunteers were negatively stained for FSP-l. The predictive value of urinary FSP I positive cells (83.6%) was superior to albuminuria as well as podocyturia (66.2%). Finally. urine MMP activity was elevated in DKD. However, there was a significantly lower MMP activity in the progressors DKD patients. Conclusions: This thesis identified a number of key observations relating to the progression of DKD. Firstly, it showed that beside conventional clinical predictors of progression, vascular comorbidities were associated with a faster rate of progression of DKD in a largely older population suffering from T2DM. Urinary biomarkers analysis showed that peptiduria and in particular transferrinuria was highly predictive of DKD progression. For the first time, we show in this thesis the potential use of urine FSP-I positive cells (presumably kidney derived myofibroblasts) to predict DKD outcomes. Also, the lower urinary MMP activity predicted a faster rate of progression. In summary, this thesis successfully identifies a number of biomarkers that may prove highly valuable to predict DKD progression in T2DM affected by diffuse vascular pathology and co-morbidities.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Urinary Matrix Metalloproteinase Activity in Diabetic Kidney Disease: A Potential Marker of Disease Progression

Background: Progressive kidney fibrosis, associated with chronic kidney disease (CKD), results from an imbalance in extracellular matrix (ECM) homeostasis. Reduced matrix metalloproteinases (MMP) activity causing lower clearance of ECM proteins has been implicated mainly through an overproduction of tissue inhibitors of metalloproteinases (TIMP), but also by reduced MMP synthesis. We tested the hypothesis that MMP activity can be measured in human urine and can be used as a potential biomarker of the progression of diabetic kidney disease (DKD). Methods: An observational prospective study was performed on 102 DKD patients using 21 diabetic patients without kidney disease and 21 healthy volunteers as controls. The Molecular Probes EnzChek Gelatinase/Collagenase Assay Kit were used to determine urinary MMP activity using DQ™ Gelatin (total MMPs), DQ™ Collagen I (interstitial collagenases) and DQ™ Collagen IV (gelatinises) substrates. A broad-spectrum synthetic inhibitor of all MMP, 1,10-phenanthroline, was used to confirm that the proteolytic activity is due to MMP activity. All MMP values were expressed per unit of urine creatinine. Results: Overall urinary MMP activity (DQ Gelatin substrate) was significantly elevated in DKD patients (14.76 ± 3.65 Δ fl/h/mmol creatinine) compared to diabetes mellitus controls (7.09 ± 2.12 Δ fl/h/mmol creatinine) and healthy volunteers (1.87 ± 0.74 Δ fl/h/mmol creatinine) (ANOVA p = 0.01). Within the DKD cohort, there was an approximate threefold higher urinary MMP activity in nonprogressive DKD patients compared to those with progressive disease (p = 0.002). The urinary MMP activity:creatinine ratio was significantly higher in normoalbuminuric and microalbuminuric DKD compared to macroalbuminuric DKD. Positive correlations were observed between the rate of total MMP activity and interstitial collagenases (r = 0.75, p Conclusions: Total MMP activity can be easily measured in human urine. Surprisingly and in contrast to MMP activity in the kidney, urine MMP activity is elevated in DKD. However, there is a significantly lower MMP activity in patients with progressive DKD. ROC analysis demonstrates that single urine MMP activity estimation is superior to albuminuria in predicting DKD patients with progressive disease