Key points
Increased activation of the renin‐angiotensin‐aldosterone system (RAAS) and elevated growth factor production are of crucial importance in the development of renal fibrosis leading to ...diabetic kidney disease.
The aim of this study was to provide evidence for the antifibrotic potential of RAAS inhibitor (RAASi) treatment and to explore the exact mechanism of this protective effect.
We found that RAASi ameliorate diabetes‐induced renal interstitial fibrosis and decrease profibrotic growth factor production.
RAASi prevents fibrosis by acting directly on proximal tubular cells, and inhibits hyperglycaemia‐induced growth factor production and thereby fibroblast activation.
These results suggest a novel therapeutic indication and potential of RAASi in the treatment of renal fibrosis.
In diabetic kidney disease (DKD) increased activation of renin‐angiotensin‐aldosterone system (RAAS) contributes to renal fibrosis. Although RAAS inhibitors (RAASi) are the gold standard therapy in DKD, the mechanism of their antifibrotic effect is not yet clarified. Here we tested the antifibrotic and renoprotective action of RAASi in a rat model of streptozotocin‐induced DKD. In vitro studies on proximal tubular cells and renal fibroblasts were also performed to further clarify the signal transduction pathways that are directly altered by hyperglycaemia. After 5 weeks of diabetes, male Wistar rats were treated for two more weeks per os with the RAASi ramipril, losartan, spironolactone or eplerenone. Proximal tubular cells were cultured in normal or high glucose (HG) medium and treated with RAASi. Platelet‐derived growth factor (PDGF) or connective tissue growth factor (CTGF/CCN2)‐induced renal fibroblasts were also treated with various RAASi. In diabetic rats, reduced renal function and interstitial fibrosis were ameliorated and elevated renal profibrotic factors (TGFβ1, PDGF, CTGF/CCN2, MMP2, TIMP1) and alpha‐smooth muscle actin (αSMA) levels were decreased by RAASi. HG increased growth factor production of HK‐2 cells, which in turn induced activation and αSMA production of fibroblasts. RAASi decreased tubular PDGF and CTGF expression and reduced production of extracellular matrix (ECM) components in fibroblasts. In proximal tubular cells, hyperglycaemia‐induced growth factor production increased renal fibroblast transformation, contributing to the development of fibrosis. RAASi, even in non‐antihypertensive doses, decreased the production of profibrotic factors and directly prevented fibroblast activation. All these findings suggest a novel therapeutic role for RAASi in the treatment of renal fibrosis.
Key points
Increased activation of the renin‐angiotensin‐aldosterone system (RAAS) and elevated growth factor production are of crucial importance in the development of renal fibrosis leading to diabetic kidney disease.
The aim of this study was to provide evidence for the antifibrotic potential of RAAS inhibitor (RAASi) treatment and to explore the exact mechanism of this protective effect.
We found that RAASi ameliorate diabetes‐induced renal interstitial fibrosis and decrease profibrotic growth factor production.
RAASi prevents fibrosis by acting directly on proximal tubular cells, and inhibits hyperglycaemia‐induced growth factor production and thereby fibroblast activation.
These results suggest a novel therapeutic indication and potential of RAASi in the treatment of renal fibrosis.
Delayed graft function after kidney transplantation is common and increases morbidity and health care costs. There is evidence that endotrophin, a specific fragment of pro-collagen type VI, promotes ...the inflammatory response in kidney diseases. We tested the hypothesis that pretransplant endotrophin in kidney transplant recipients may be associated with the risk of delayed graft function. Pretransplant plasma endotrophin was assessed using an enzyme-linked immunosorbent assay in three independent cohorts with 806 kidney transplant recipients. The primary outcome was delayed graft function, i.e., the necessity of at least one dialysis session within one-week posttransplant. In the discovery cohort median pretransplant plasma endotrophin was higher in 32 recipients (12%) who showed delayed graft function when compared to 225 recipients without delayed graft function (58.4 ng/mL IQR 33.4-69.0; N = 32; vs. 39.5 ng/mL IQR 30.6-54.5; N = 225; P = 0.009). Multivariable logistic regression, fully adjusted for confounders showed, that pretransplant plasma endotrophin as a continuous variable was independently associated with delayed graft function in both validation cohorts, odds ratio 2.09 95% CI 1.30-3.36 and 2.06 95% CI 1.43-2.97. Pretransplant plasma endotrophin, a potentially modifiable factor, was independently associated with increased risk of delayed graft function and may be a new avenue for therapeutic interventions.
We evaluated biomarkers related to kidney fibrosis for the outcome of patients with IgA nephropathy (IgAN). Clinical parameters (estimated glomerular filtration rate, hypertension, proteinuria) and ...histological findings were assessed in 134 patients with IgAN at the time of diagnosis and followed up prospectively (mean follow-up time, 56.5 months). We measured biomarkers of collagen and laminin turnover in serum and urine collected at the time of kidney biopsy using a novel enzyme-linked immunosorbent assay. Linear discriminant analysis and logistic regression models were used to predict the patient's kidney outcome. Five serum and urine biomarkers of laminin and collagen turnover (sLG1M, sPRO-C3, sPRO-C6, uPRO-C6/Cr, uC3M/Cr) could significantly differentiae IgAN patients with a worse prognosis. Clinical parameters (glomerular filtration rate (GFR), proteinuria) distinguished patients at risk of IgAN progression with a specificity of 87.3% and a sensitivity of 45.2% (area under the curve-AUC 0.751). The addition of the biomarkers significantly increased the prognostic ability with a specificity of 85.1% and a sensitivity of 73.3% (AUC 0.905). We have identified three serum (sLG1M, sPRO-C3, sPRO-C6) and two urinary markers (uPRO-C6/Cr, u-C3M /Cr) that significantly improve the prognostic ability of markers of kidney function to identify an IgAN patient's risk of progressing to ESKD.
Activity and chronicity of kidney involvement in ANCA-associated vasculitis (AAV) can be currently reliably evaluated only by kidney biopsy. In this study, we measured a panel of serum and urinary ...biomarkers collected at the time of kidney biopsy and hypothesized that they could reflect specific histopathological parameters in the biopsy and help to predict prognosis.
We examined a cohort of 45 patients with AAV and 10 healthy controls. Biomarker levels (DKK-3, CD163, EGF, PRO-C6 and C3M) were measured in this study by ELISA. Biopsies were scored with a scoring system for AAV (focal x crescentic x sclerotic x mixed class) and interstitial fibrosis was quantified.
Levels of urinary DKK-3, CD163, EGF, PRO-C6 and C3M significantly differed among biopsy classes in AAV, with urinary DKK-3 and PRO-C6 levels being highest in the sclerotic class and lowest in the focal class, urinary CD163 levels highest in the crescentic class and urinary C3M levels highest in the focal class. Moreover, the urinary biomarkers were able to discriminate focal biopsy class from the other classes. Urinary DKK-3, EGF, PRO-C6 and C3M levels measured at the time of biopsy were also significantly related to the extent of fibrosis and to the final kidney function at the end of follow-up.
This small pilot study suggests that selected urinary biomarkers of fibrosis and inflammation may reflect changes in the kidney biopsy and be prognostic of kidney outcome in patients with AAV.
Kidney fibrosis is the hallmark of chronic kidney disease (CKD) and is characterized by an imbalanced extracellular matrix (ECM) remodeling. Collagen type III is one of the main ECM components of the ...interstitial matrix of the kidney. We hypothesized that measuring three biomarkers of collagen type III reflecting different aspects of this protein turnover (C3M, C3C, and PRO-C3) may provide different information about the fibrotic burden in patients with IgA nephropathy (IgAN). We examined a cohort of 134 patients with IgAN. The three collagen type III biomarkers were measured in serum (S) and in urine (U) samples taken on the same day before kidney biopsy was performed. Biopsies were evaluated for interstitial fibrosis and tubular atrophy, according to the Banff and MEST-C scores. S-PRO-C3 and S-C3C correlated with the degree of fibrosis in the biopsy, whereas U-C3M/Cr had an inverse correlation with fibrosis. U-C3M/Cr had the highest discrimination ability for advanced fibrosis, which was maintained after adjustment for the other collagen type III biomarkers, proteinuria, and serum creatinine. The data presented in this study indicate that measuring the different fragments of the same ECM protein and in different matrices provides a variety of information regarding pathological kidney tissue alterations in patients with IgAN.
Early prediction of kidney graft function may assist clinical management, and for this, reliable non-invasive biomarkers are needed. We evaluated endotrophin (ETP), a novel non-invasive biomarker of ...collagen type VI formation, as a prognostic marker in kidney transplant recipients. ETP levels were measured with the PRO-C6 ELISA in the plasma (P-ETP) of 218 and urine (U-ETP/Cr) of 172 kidney transplant recipients, one (D1) and five (D5) days, as well as three (M3) and twelve (M12) months, after transplantation. P-ETP and U-ETP/Cr at D1 (P-ETP AUC = 0.86,
< 0.0001; U-ETP/Cr AUC = 0.70,
= 0.0002) were independent markers of delayed graft function (DGF) and P-ETP at D1 had an odds ratio of 6.3 (
< 0.0001) for DGF when adjusted for plasma creatinine. The results for P-ETP at D1 were confirmed in a validation cohort of 146 transplant recipients (AUC = 0.92,
< 0.0001). U-ETP/Cr at M3 was negatively associated with kidney graft function at M12 (
= 0.007). This study suggests that ETP at D1 can identify patients at risk of delayed graft function and that U-ETP/Cr at M3 can predict the future status of the allograft. Thus, measuring collagen type VI formation could aid in predicting graft function in kidney transplant recipients.
After a potential biological incident the sampling strategy and sample analysis are crucial for the outcome of the investigation and identification. In this study, we have developed a simple sandwich ...ELISA based on commercial components to quantify BSA (used as a surrogate for ricin) with a detection range of 1.32-80 ng/mL. We used the ELISA to evaluate different protein swabbing procedures (swabbing techniques and after-swabbing treatments) for two swab types: a cotton gauze swab and a flocked nylon swab. The optimal swabbing procedure for each swab type was used to obtain recovery efficiencies from different surface materials. The surface recoveries using the optimal swabbing procedure ranged from 0-60% and were significantly higher from nonporous surfaces compared to porous surfaces. In conclusion, this study presents a swabbing procedure evaluation and a simple BSA ELISA based on commercial components, which are easy to perform in a laboratory with basic facilities. The data indicate that different swabbing procedures were optimal for each of the tested swab types, and the particular swab preference depends on the surface material to be swabbed.
Abstract Background and Aims People who survive Acute Kidney Injury (AKI) experience increased risk of long-term adverse outcomes, including a significantly elevated rate of readmission to hospital. ...This may be related to the pathophysiological consequences of AKI, including extracellular matrix (ECM) remodeling in the kidneys and other organs. Tools to monitor the long-term health risk of patients after AKI are needed to improve patient outcome. Here, we investigated the potential of the novel ECM remodeling biomarker LG1M, reflecting laminin degradation mediated by MMP-9, as a prognostic marker for readmission after AKI. Method We measured LG1M in plasma samples collected 1 year after the episode of AKI using the nordicLG1MTM ELISA assay in plasma samples from 791 patients from the AKI Risk in Derby (ARID) study, who were then followed prospectively until year 3. 390 of the patients had been hospitalized for an episode of AKI and 401 patients who did not sustain AKI were included as controls (non-AKI). Correlation of LG1M levels with age, serum creatinine, eGFR, PCR, ACR, and CRP was tested with Spearman rank correlation (all variables measured at year 1). Uni- and multivariate Cox regression analysis was used to analyze the association of LG1M with the risk of readmission. Results LG1M levels were significantly higher in the AKI group (median IQR = 31.9 25.7-42.8 ng/mL) compared to the control group (median IQR = 30.3 24.9-37.2 ng/mL, P < 0.01). In the AKI group, LG1M correlated with age (r = 0.16, P < 0.01), serum creatinine (r = 0.33, P < 0.0001), eGFR (r = −0.35, P < 0.0001), and ACR (r = 0.23, P < 0.0001). In the control group, LG1M only correlated with serum creatinine (r = 0.22, P < 0.0001) and eGFR (r = −0.22, P < 0.0001). By year 3, 265 out of 390 patients in the AKI group and 258 out of 401 patients in the control group were readmitted (Chi-squared test, P = 0.28). In a univariate Cox model with readmission as outcome, LG1M was significant associated with readmission in the AKI group (HR 95% CI = 1.007 1.001-1.013, P < 0.05) but not in the control group (HR 95% CI = 1.005 0.997-1.013, P = 0.23). When adjusting for age, sex, ACR, and eGFR measured at year 1 as well as baseline CKD and diabetes status, LG1M lost it significance in the AKI group (HR 95% CI = 1.004 0.998-1.011, P = 0.22) and was still non-significant in the control group (HR 95% CI = 1.002 0.994-1.011, P = 0.57). Conclusion In this study, we showed that the novel biomarker LG1M is prognostic for readmission in patients after AKI. Our findings may indicate that this biomarker, reflecting laminin degradation, can be used as a novel post-AKI biomarker that can help assess patients’ long-term health risks.
Abstract Background and Aims Endotrophin is a pro-fibrotic and pro-inflammatory molecule derived from the post-translational processing of type VI collagen. Evidence for an association of increased ...circulating levels of endotrophin with increased risk of kidney and cardiovascular-related outcome and all-cause mortality in persons with type 2 diabetes have recently emerged. Here we want to investigate endotrophin as an early marker of kidney-related outcome risk in persons with type 2 diabetes with normal levels of albuminuria. Method Endotrophin was measured using the nordicPRO-C6TM assay (Nordic Bioscience, Herlev, Denmark) in serum of 1088 individuals with type 2 diabetes from the Joslin Diabetes Study. 635 individuals had normal to mildly increased albuminuria (<30 mg/g), 379 had moderately increased albuminuria (30-300 mg/g) and 74 had severely increased albuminuria (>300 mg/g). All patients had eGFR >60 and where hence classified as CKD stage 1 and 2. Risk of kidney disease progression was defined as a 40% decline in eGFR within 10 years. Results In the cohort (n = 1088), a total of 128 patients progressed to 40% eGFR decline. High levels of circulating endotrophin were associated with an increased risk of kidney disease progression in unadjusted logistic regression analysis (OR for T3 vs T1+T2 (PRO-C6): 2.1 95% CI: 1.4-3.0, p = 0.0001) and after adjustment for eGFR, HbA1c, age and sex (OR for T3 vs T1+T2 (PRO-C6): 1.8 95% CI: 1.1-2.6, p = 0.005). When looking at patients classified in albuminuria stages, endotrophin was significantly associated with kidney disease progression in the individuals with normal to mildly increased albuminuria (39 events) (OR for T3 vs T1+T2 (PRO-C6): 2.6 95% CI: 1.3-4.9, p = 0.005 in unadjusted analysis and 2.3 95% CI: 1.1-4.5, p = 0.02 after adjustment for eGFR, HbA1c, age and sex, but it was not significantly associated to kidney progression in the groups with moderately (49 events) and severely (40 events) increased albuminuria. Conclusion This study sheds further light on the potential of endotrophin as an early marker of long-term adverse kidney outcome in persons with type 2 diabetes. The association of circulating endotrophin with increased risk of kidney disease progression had already been reported both in CKD and DKD patients (for shorter-term outcome), and this study further expands on the applicability of endotrophin as a biomarker in persons with type 2 diabetes without an overt kidney disease, who will nevertheless progress to develop CKD in a 10-year time horizon.
Abstract
Background and Aims
Glomerulonephritis is one of the most common causes of chronic kidney disease (CKD). Causes of glomerular diseases include lupus nephritis (LN), focal segmental ...glomerulonephritis (FSGS) and minimal change disease (MCD). The hallmark of CKD is renal fibrosis characterized by an imbalanced turnover of extracellular matrix (ECM) components and CKD is associated with increased inflammation. The aim for this study was to screen a biomarker panel based on extracellular matrix remodeling biomarkers as well as fibrotic activity and inflammation biomarkers. Additionally, to identify biomarkers that are differentially expressed in glomerular diseases compared to healthy and that are differently expressed between the different etiologies.
Method
The study included 79 healthy controls (age (median years): 35, sex (%women): 51), 48 patients with LN (age: 39, sex: 63), 13 patients with FSGS (age: 44, sex: 23), and 14 patients with MCD (age: 51, sex: 47). The following biomarkers were measured in serum and/or urine samples from all 154 subjects:
• Interstitial matrix remodeling biomarkers; formation of collagen type III (PRO-C3) and VI (PRO-C6), and degradation of collagen type III (C3M).
• Basement membrane remodeling biomarkers; MMP-mediated degradation of collagen type IV (C4M, C4G, TUM), laminin (LG1M) and perlecan (LG3).
• Two biomarkers of fibrotic activity; acetylated N-terminal of alpha-smooth muscle actin (αSMA) and transforming growth factor β (TGF-β).
• An inflammation biomarker of calprotectin (CPa9-HNE) reflecting neutrophil activity.
Results
Overall, all three diseases showed the same trend of change (either no change, increase or decrease compared to healthy controls) in biomarker levels. There was a significant increase of PRO-C3, PRO-C6, TUM, LG1M, αSMA, and CPa9-HNE in serum, and a significant decrease of C3M and C4M serum in patients compared to heathy controls. Four of the biomarkers were differently expressed between the different etiologies. In serum, C4G levels were significantly higher LN and MCD compared to FSGS, LG1M levels were significantly higher in LN compared to MCD, and CPa9-HNE levels were significantly higher in MCD compared to LN (all, P<0.05). In urine, TUM levels were significantly higher in LN compared to FSGS and MCD (P<0.05).
Conclusion
The data presented in this study indicate that both biomarkers of interstitial matrix and basement membrane remodeling as well as biomarkers of fibrotic activity and inflammation reflect the changes that take place during development of glomerular diseases. Some of these markers may be able to distinguish etiologies including the hardly differentiated diseases FSGS and MCD. This needs to be tested in larger studies.
PDF
