Urinary chemokine C-X-C motif ligand 16 and endostatin as predictors of tubulointerstitial fibrosis in patients with advanced diabetic kidney disease.

BACKGROUND: Interstitial fibrosis and tubular atrophy (IFTA) is a well-recognized risk factor for poor renal outcome in patients with diabetic kidney disease (DKD). However, a noninvasive biomarker for IFTA is currently lacking. The purpose of this study was to identify urinary markers of IFTA and to determine their clinical relevance as predictors of renal prognosis. METHODS: Seventy patients with biopsy-proven isolated DKD were enrolled in this study. We measured multiple urinary inflammatory cytokines and chemokines by multiplex enzyme-linked immunosorbent assay in these patients and evaluated their association with various pathologic features and renal outcomes. RESULTS: Patients enrolled in this study exhibited advanced DKD at the time of renal biopsy, characterized by moderate to severe renal dysfunction [mean estimated glomerular filtration rate (eGFR) 36.1 mL/min/1.73 m2] and heavy proteinuria (mean urinary protein:creatinine ratio 7.8 g/g creatinine). Clinicopathologic analysis revealed that higher IFTA scores were associated with worse baseline eGFR (P < 0.001) and poor renal outcome (P = 0.002), whereas glomerular injury scores were not. Among measured urinary inflammatory markers, C-X-C motif ligand 16 (CXCL16) and endostatin showed strong correlations with IFTA scores (P = 0.001 and P < 0.001, respectively), and patients with higher levels of urinary CXCL16 and/or endostatin experienced significantly rapid renal progression compared with other patients (P < 0.001). Finally, increased urinary CXCL16 and endostatin were independent risk factors for poor renal outcome after multivariate adjustments (95% confidence interval 1.070-3.455, P = 0.029). CONCLUSIONS: Urinary CXCL16 and endostatin could reflect the degree of IFTA and serve as biomarkers of renal outcome in patients with advanced DKD.

Matrix Metalloproteinase-7 Is a Urinary Biomarker and Pathogenic Mediator of Kidney Fibrosis.

Matrix metalloproteinase-7 (MMP-7), a secreted zinc- and calcium-dependent endopeptidase, is a transcriptional target of canonical Wnt/ß-catenin signaling. Because Wnt/ß-catenin is activated in diseased kidney, we hypothesized that urinary MMP-7 level may be used as a noninvasive surrogate biomarker for fibrotic lesions. To test this hypothesis, we conducted a cross-sectional study, measuring urinary MMP-7 levels in a cohort of 102 patients with CKD. Compared with normal subjects, patients with various kidney disorders had markedly elevated urinary levels of MMP-7. Furthermore, urinary MMP-7 levels closely correlated with renal fibrosis scores in patients. In mice, knockout of MMP-7 ameliorated the fibrotic lesions and expression of matrix genes induced by obstructive injury. Genetic ablation of MMP-7 also preserved E-cadherin protein expression and substantially reduced the expression of total and dephosphorylated ß-catenin and the de novo expression of vimentin and fibroblast-specific protein 1 in renal tubules of obstructed kidneys. In vitro, MMP-7 proteolytically degraded E-cadherin in proximal tubular cells, leading to ß-catenin liberation and nuclear translocation and induction of ß-catenin target genes by a mechanism independent of Wnt ligands. Finally, pharmacologic inhibition of MMP-7 immediately after obstructive injury reduced renal fibrosis in vivo These results suggest that MMP-7 not only can serve as a noninvasive biomarker but also is an important pathogenic mediator of kidney fibrosis.

Changes in Urinary Microbiome Populations Correlate in Kidney Transplants With Interstitial Fibrosis and Tubular Atrophy Documented in Early Surveillance Biopsies.

An unbalanced microbiome may lead to disease by creating aberrant immune responses. A recent association of cellular rejection with the development of interstitial fibrosis and tubular atrophy (IFTA) suggests the role of immune-mediated tissue injury. We hypothesized that developing IFTA correlates with altered urinary tract microbiomes (UMBs). UMBs at two serial time points, 1 and 6-8 months posttransplant, were assessed by 16S microbial ribosomal gene sequencing in 25 patients developing biopsy-proven IFTA compared to 23 transplant patients with normal biopsies and excellent function (TX) and 20 healthy nontransplant controls (HC). Streptococcus, the dominant genera in HC males, was lower in IFTA and TX males at 1 month compared to HCs. At 6-8 months, Streptococcus was further decreased in IFTA males, but normalized in TX. IFTA males and females had increases in number of genera per sample at 6-8 months. UMB composition varied substantially between individuals in all groups. Despite the wide variation in UMBs between individuals, IFTA was associated with a loss in dominant resident urinary microbes in males, and a parallel increase in nonresident, pathogenic bacteria in males and females. UMB changes may contribute to IFTA development by alteration of the host immune response.

Urine Fibrosis Markers and Risk of Allograft Failure in Kidney Transplant Recipients: A Case-Cohort Ancillary Study of the FAVORIT Trial.

BACKGROUND: Kidney tubulointerstitial fibrosis marks risk for allograft failure in kidney transplant recipients, but is poorly captured by estimated glomerular filtration rate (eGFR) or urine albumin-creatinine ratio (ACR). Whether urinary markers of tubulointerstitial fibrosis can noninvasively identify risk for allograft failure above and beyond eGFR and ACR is unknown. STUDY DESIGN: Case-cohort study. SETTING & PARTICIPANTS: The FAVORIT (Folic Acid for Vascular Outcome Reduction in Transplantation) Trial was a randomized double-blind trial testing vitamin therapy to lower homocysteine levels in stable kidney transplant recipients. We selected a subset of participants at random (n=491) and all individuals with allograft failure during follow-up (cases; n=257). PREDICTOR: Using spot urine specimens from the baseline visit, we measured 4 urinary proteins known to correlate with tubulointerstitial fibrosis on biopsy (urine α1-microglobulin [A1M], monocyte chemoattractant protein 1 [MCP-1], and procollagen type III and type I amino-terminal amino pro-peptide). OUTCOME: Death-censored allograft failure. RESULTS: In models adjusted for demographics, chronic kidney disease risk factors, eGFR, and ACR, higher concentrations of urine A1M (HR per doubling, 1.73; 95% CI, 1.43-2.08) and MCP-1 (HR per doubling, 1.60; 95% CI, 1.32-1.93) were strongly associated with allograft failure. When additionally adjusted for concentrations of other urine fibrosis and several urine injury markers, urine A1M (HR per doubling, 1.76; 95% CI, 1.27-2.44]) and MCP-1 levels (HR per doubling, 1.49; 95% CI, 1.17-1.89) remained associated with allograft failure. Urine procollagen type III and type I levels were not associated with allograft failure. LIMITATIONS: We lack kidney biopsy data, BK titers, and HLA antibody status. CONCLUSIONS: Urine measurement of tubulointerstitial fibrosis may provide a noninvasive method to identify kidney transplant recipients at higher risk for future allograft failure, above and beyond eGFR and urine ACR.

Urine Epidermal Growth Factor, Monocyte Chemoattractant Protein-1 or Their Ratio as Biomarkers for Interstitial Fibrosis and Tubular Atrophy in Primary Glomerulonephritis.

BACKGROUND/AIMS: The degree of tubular atrophy and interstitial fibrosis (IFTA) is an important prognostic factor in glomerulonephritis. Imbalance between pro-inflammatory cytokines such as monocyte chemoattractant protein- 1 (MCP-1) and protective cytokines such as epidermal growth factor (EGF) likely determine IFTA severity. In separate studies, elevated MCP-1 and decreased EGF have been shown to be associated with IFTA severity. In this study, we aim to evaluate the predictive value of urinary EGF/MCP-1 ratio compared to each biomarker individually for moderate to severe IFTA in primary glomerulonephritis (GN). METHODS: Urine samples were collected at biopsy from primary GN (IgA nephropathy, focal and segmental glomerulosclerosis, minimal change disease, membranous nephropathy). MCP-1 and EGF were analyzed by enzyme-linked immunosorbent assay. RESULTS: EGF, MCP-1 and EGF/MCP-1 ratio from primary GN, all correlated with IFTA (n=58). By univariate analysis, glomerular filtration rate, EGF, and EGF/MCP-1 ratio were associated with IFTA. By multivariate analysis, only EGF/MCP-1 ratio was independently associated with IFTA. EGF/MCP-1 ratio had a sensitivity of 88% and specificity of 74 % for IFTA. EGF/MCP-1 had good discrimination for IFTA (AUC=0.85), but the improvement over EGF alone was not significant. CONCLUSION: EGF/MCP-1 ratio is independently associated IFTA severity in primary glomerulonephritis, but the ability of EGF/MCP-1 ratio to discriminate moderate to severe IFTA may not be much better than EGF alone.

Serum and urine markers of collagen degradation reflect renal fibrosis in experimental kidney diseases.

BACKGROUND: The extent of renal fibrosis in chronic kidney disease (CKD) is the best predictor for progression of most renal diseases. To date, no established biomarkers of renal fibrosis exist. METHODS: We measured circulating and urinary-specific matrix metalloproteinase (MMP)-generated collagen type I and III degradation fragments (C1M and C3M) and an N-terminal propeptide of collagen III (Pro-C3), as markers of collagen type III production, in three rat models of CKD and fibrosis: renal mass reduction (5/6 nephrectomy), progressive glomerulonephritis (chronic anti-Thy1.1 nephritis) and adenine crystal-induced nephropathy. Healthy rats served as controls. RESULTS: In all three models, the animals developed significant CKD and renal fibrosis. Compared with healthy rats, serum C1M and C3M significantly increased in rats with 5/6 nephrectomy and adenine nephropathy (2- to 3-fold), but not with chronic anti-Thy1.1 nephritis. Urinary C1M and C3M levels increased 9- to 100-fold in all three models compared with controls. Urinary degradation markers correlated closely with renal deposition of collagen type I and type III. Pro-C3 was significantly increased only in the urine of 5/6 nephrectomy rats. CONCLUSIONS: In particular, urinary markers of MMP-driven collagen degradation, rather than collagen production markers, may represent a novel, specific and non-invasive diagnostic approach to assess kidney fibrosis.

miR-29c in urinary exosomes as predictor of early renal fibrosis in lupus nephritis.

BACKGROUND: Despite overall improvement in prognosis, 10-30% of patients with lupus nephritis (LN) will develop end-stage renal disease. To date, renal biopsy is still the 'gold standard' test used to predict renal outcome. However, due to its invasive nature, new non-invasive biomarkers are required. Urinary exosomes, microvesicles released by every epithelial cell facing the urinary space, represent an ideal source of markers for renal dysfunction and injury. Here, we sought to evaluate miR-29c expression levels in urinary exosomes as a novel biomarker of renal fibrosis in LN. METHODS: Urinary exosomes were isolated from 32 samples of patients with biopsy-proven LN, 15 non-lupus chronic kidney diseases and 20 healthy controls. Electronic microscopy and western blot were used to characterize the exosomes. Expression levels of miR-29c were detected by RT-PCR quantitative and correlated with clinical and histological parameters along with the expression levels of Smad2/3, TGF-ß and MMP2/9. For comparison, miRNA expression was also evaluated in the urinary pellet. RESULTS: MiR-29c levels in urinary exosomes showed a negatively strong correlation with the histological chronicity index (r = -0.898, P = 0.001) and glomerular sclerosis (r = -0.555, P = 0.007). No correlation with eGFR and creatinine levels was found. MiR-29c expression levels could predict the degree of chronicity in patients with LN with an area under the curve (AUC) of 0.946 (P < 0.001) and with high sensitivity and specificity (94% and 82%). Smad3 and MMP2 expression in urinary exosomes correlated negatively with miR-29c expression (r = -0.737 and -0.856, respectively). In the urinary pellet, no miR-29c expression was detected; however, upregulation of Smad3 and MMP2 was observed (3.54- and 5.85-fold increase). CONCLUSIONS: Overall, miR-29c correlated with the degree of renal chronicity but not with renal function, suggesting it could be used as a novel non-invasive marker of early progression to fibrosis in patients with LN.

Total plasma clearance versus urinary plasma clearance of (51)Cr-EDTA in patients with cirrhosis with and without fluid retention.

BACKGROUND AND AIM: In patients with fluid retention, the total plasma clearance of (51)Cr-EDTA (ClP) may overestimate the glomerular filtration rate (GFR). The present study was therefore undertaken in order to compare ClP with the urinary plasma clearance of (51)Cr-EDTA (ClU) in patients with cirrhosis with and without fluid retention. MATERIAL AND METHODS: A total of 136 patients with cirrhosis (24 without fluid retention, 112 with ascites) received a quantitative intravenous injection of (51)Cr-EDTA followed by plasma and quantitative urinary samples for 5 hours. ClP was determined from the injected dose relative to the plasma concentration-time area, extrapolated to infinity. ClU was determined as urinary excretion relative to the plasma concentration-time area up to voiding. RESULTS: In patients without fluid retention, the difference between ClP and ClU (ClP - ClU = ClΔ) was mean 4.5 mL/min/1.73 m(2). In patients with ascites, ClΔ was significantly higher (17.6 mL/min/1.73 m(2), p < 0.0001). ClΔ increased with lower values of GFR (r = - 0.458, p < 0.001). Repeated measurements of ClU in a subgroup of patients with fluid retention (n = 25) gave almost identical values. Different types of corrections of one-pool clearance were almost identical with ClP, except for higher clearance values, which were somewhat underestimated by the former. CONCLUSION: In patients with fluid retention and ascites ClP and corrected one-pool clearance overestimates GFR substantially. Although ClU may underestimate GFR slightly, patients with ascites should collect urine quantitatively in order to obtain a reliable measurement of GFR.

MicroRNA-29c in urinary exosome/microvesicle as a biomarker of renal fibrosis.

Micro (mi)RNAs are frequently dysregulated in the development of renal fibrosis. Exosomes are small membrane vesicles that could be isolated from urine secreted from all nephron segments. Here we sought to observe for the first time whether miRNA in urine exosome could serve as a potential biomarker of renal fibrosis. Urine samples were collected from 32 chronic kidney disease (CKD) patients who underwent kidney biopsy and 7 controls. Exosome was isolated and confirmed by immunogold staining of exosome marker. Members of miR-29, miR-200, and RNU6B as endogenous control were detected by RT quantitative PCR. Electronic microscopy verified a typical shape of exosome with average size of 65.1 nm and labeled it with anti-CD9 and anti-aquaporin 2 antibody. Members of miR-29 and miR-200 are readily measured with reduced levels compared with controls (P < 0.05) and can robustly distinguish CKD from controls [area under the curve (AUC) varied from 0.902 to 1 by receiver operating characteristics analysis]. miR-29c correlated with both estimated glomerular filtration rate (r = 0.362; P < 0.05) and degree of tubulointerstitial fibrosis (r = -0.359; P < 0.05) for CKD patients. Moreover, miRNA in exosome was decreased in mild fibrosis group compared with moderated to severe group. miR-29a and miR-29c could predict degree of tubulointerstitial fibrosis with AUC of 0.883 and 0.738 (P < 0.05). The sensitivity and specificity for distinguishing mild from moderate to severe fibrosis were 93.8 and 81.3% with the use of miR-29a and 68.8 and 81.3% for miR-29c. Overall, miR-29c in urinary exosome correlates with both renal function and degree of histological fibrosis, suggesting it as a novel, noninvasive marker for renal fibrosis.

Urinary miR-21, miR-29, and miR-93: novel biomarkers of fibrosis.

BACKGROUND: MicroRNAs (miRNAs) play important roles in the progression of renal fibrosis. We studied the urinary levels of miR-21, miR-29 family and miR-93, which are downstream mediators of the transforming growth factor-ß(1) (TGF-ß(1)), in patients with immunoglobulin A (IgA) nephropathy. METHODS: We studied the urinary miRNA levels of 43 IgA nephropathy patients and 13 healthy controls. RESULTS: The IgA nephropathy group had significantly lower urinary miR-29b and miR-29c, but higher miR-93 levels than controls. Proteinuria significantly correlated with urinary levels of miR-29b (r = -0.388, p = 0.003) and miR-29c (r = -0.409, p = 0.002). Glomerular filtration rate significantly correlated with urinary levels of miR-21 (r = 0.338, p = 0.028), miR-29b (r = 0.333, p = 0.031) and miR-29c (r = 0.304, p = 0.050). Urinary miR-93 level significantly correlated with glomerular scarring (r = -0.392, p = 0.010). Urinary miRNA level of SMAD3, but not TGF-ß(1), correlated with urinary miR-21 (r = 0.624, p < 0.001), miR-29b (r = 0.566, p < 0.001), miR-29c (r = 0.619, p < 0.001) and miR-93 (r = 0.332, p = 0.032). CONCLUSIONS: Urinary miR-29b and miR-29c levels correlated with proteinuria and renal function, while urinary miR-93 level correlated with glomerular scarring. More importantly, urinary levels of these miRNA targets significantly correlated with urinary SMAD3 level. Our results suggest that these miRNA targets are regulated by the TGF-ß(1)/SMAD3 pathway and they may play important roles in the development of progressive renal fibrosis in IgA nephropathy.

Accelerated renal fibrosis in cardiorenal syndrome is associated with long-term increase in urine neutrophil gelatinase-associated lipocalin levels.

BACKGROUND: Cardiac events are the main cause of death among patients with end-stage renal failure. Even a mild renal disease is currently considered a major risk factor for cardiovascular complications following myocardial infarction (MI). The aim of the present study was to detect histological, sera and urine characteristics of kidney injury in cardiorenal syndrome (CRS) compared to chronic kidney disease (CKD) with an intact cardiac function. METHODS: We employed a rat model for CRS, in which an acute MI (AMI) was induced 4 weeks after establishment of subtotal nephrectomy. Four weeks later, left ventricular function was assessed by echocardiography and changes in renal performance were examined using histological and biochemical parameters. RESULTS: Increased interstitial fibrosis as well as renal inflammation were observed in renal sections derived from CRS rats, compared to subtotal nephrectomy (CKD)-only animals. Moreover, we found that even though AMI on the background of CKD was not associated with a further decrease in creatinine clearance or a further increase in sera BUN levels compared to CKD only, a significant long-term elevation in urine neutrophil gelatinase-associated lipocalin (Ngal) levels was detectable post-MI induction. CONCLUSIONS: AMI in the CKD setting is associated with accelerated renal fibrosis and long-term elevated urine Ngal values, suggesting that cardiac dysfunction contributes to accelerated intrinsic kidney injury in CKD. The data indicate that elevated urine Ngal may potentially serve as an early non-invasive laboratory parameter for a left ventricular dysfunction-related renal injury.

Renal accumulation of prooxidant mineral elements and CKD in domestic cats.

Felids have a high incidence of chronic kidney disease (CKD), for which the most common renal lesion is chronic interstitial nephritis (CIN). CIN can be induced by tissue oxidative stress, which is determined by the cellular balance of pro- and anti-oxidant metabolites. Fish-flavoured foods are more often fed to cats than dogs, and such foods tend to have higher arsenic content. Arsenic is a pro-oxidant metallic element. We propose that renal accumulation of pro-oxidant elements such as arsenic and depletion of anti-oxidant elements such as zinc, underpin the high incidence of CIN in domestic cats. Total arsenic and other redox-reactive metal elements were measured in kidneys (after acid-digestion) and urine (both by inductively-coupled plasma-mass spectrometry) of domestic cats (kidneys, n = 56; urine, n = 21), domestic dogs (kidneys, n = 54; urine, n = 28) and non-domesticated Scottish Wildcats (kidneys, n = 17). Renal lesions were graded by severity of CIN. In our randomly sampled population, CIN was more prevalent in domestic cat versus domestic dog (51%, n = 32 of 62 cats; 15%, 11 of 70 dogs were positive for CIN, respectively). CIN was absent from all Scottish wildcats. Tissue and urinary (corrected for creatinine) arsenic content was higher in domestic cats, relative to domestic dogs and wildcats. Urine arsenic was higher in domestic cats and dogs with CIN. Arsenobetaine, an organic and relatively harmless species of arsenic, was the primary form of arsenic found in pet foods. In summary, the kidneys of domestic cats appear to have greater levels of pro-oxidant trace elements, as compared to dogs and wildcats. Since there was no difference in renal arsenic levels in cats with or without CIN, renal arsenic accumulation does not appear a primary driver of excess CIN in cats. Given clear differences in renal handling of pro vs. anti-oxidant minerals between cats and dogs, further in vivo balance studies are warranted. These may then inform species-specific guidelines for trace element incorporation into commercial diets.

Effect of prednisolone on glyoxalase 1 in an inbred mouse model of aristolochic acid nephropathy using a proteomics method with fluorogenic derivatization-liquid chromatography-tandem mass spectrometry.

Prednisolone is involved in glucose homeostasis and has been used for treatment for aristolochic acid (AA) nephropathy (AAN), but its effect on glycolysis in kidney has not yet been clarified. This study aims to investigate the effect in terms of altered proteins after prednisolone treatment in a mice model of AAN using a proteomics technique. The six-week C3H/He female mice were administrated AA (0.5 mg/kg/day) for 56 days. AA+P group mice were then given prednisolone (2 mg/kg/day) via oral gavage for the next 14 days, and AA group mice were fed water instead. The tubulointerstitial damage was improved after prednisolone treatment comparing to that of AA group. Kidney homogenates were harvested to perform the proteomics analysis with fluorogenic derivatization-liquid chromatography-tandem mass spectrometry method (FD-LC-MS/MS). On the other hand, urinary methylglyoxal and D-lactate levels were determined by high performance liquid chromatography with fluorescence detection. There were 47 altered peaks and 39 corresponding proteins on day 14 among the groups, and the glycolysis-related proteins, especially glyoxalase 1 (GLO1), fructose-bisphosphate aldolase B (aldolase B), and triosephosphate isomerase (TPI), decreased in the AA+P group. Meanwhile, prednisolone decreased the urinary amount of methylglyoxal (AA+P: 2.004 ± 0.301 µg vs. AA: 2.741 ± 0.630 µg, p < 0.05), which was accompanied with decrease in urinary amount of D-lactate (AA+P: 54.07 ± 5.45 µmol vs. AA: 86.09 ± 8.44 µmol, p < 0.05). Prednisolone thus alleviated inflammation and interstitial renal fibrosis. The renal protective mechanism might be associated with down-regulation of GLO1 via reducing the contents of methylglyoxal derived from glycolysis. With the aid of proteomics analysis and the determination of methylglyoxal and its metabolite-D-lactate, we have demonstrated for the first time the biochemical efficacy of prednisolone, and urinary methylglyoxal and its metabolite-D-lactate might be potential biomarkers for AAN.

Urinary miRNA-27b-3p and miRNA-1228-3p correlate with the progression of Kidney Fibrosis in Diabetic Nephropathy.

Diabetic Nephropathy (DN) is a chronic complication of diabetes and the primary cause of end stage renal disease. Differential diagnosis for DN requires invasive histological investigation, thus there is need for non-invasive biomarkers to discriminate among different histological lesions in diabetic patients. With the aim to identify a pattern of differentially expressed miRNAs in kidney biopsies of DN patients, we assayed miRNA expression in kidney biopsies from DN patients, diabetic patients with membranous nephropathy and patients with normal histology. Nine miRNAs were differentially expressed among the three groups, and 2 miRNAs (miR-27b-3p and miR-1228-3p) showed interaction with an ubiquitin-conjugating E2 enzyme variant (UBE2v1). UBE2v1 mediates the formation of lysine 63-linked ubiquitin chains, a mechanism we previously showed as involved in DN kidney fibrosis. Both miRNAs were validated as down-regulated in biopsies and urines of DN patients, possibly affected by DNA methylation. Interestingly, the urinary levels of both miRNAs could also discriminate among different degrees of renal fibrosis. Finally, we showed that the combined urinary expression of both miRNAs was also able to discriminate DN patients from other glomerulonephritides in diabetic patients. In conclusion we identified two miRNAs potentially useful as candidate biomarkers of tubular-interstitial fibrosis in diabetic patients with DN.

Soluble klotho as a marker of renal fibrosis and podocyte injuries in human kidneys.

Klotho deficiency is relevant to renal fibrosis and podocyte injury in vivo and in vitro. We examined whether histological findings of renal biopsy specimens were associated with the levels of soluble klotho in humans. We investigated renal biopsy specimens of 67 patients and detailed microscopic findings were reviewed. Soluble serum/urinary klotho and urinary angiotensinogen were assessed by enzyme-linked immunosorbent assays, and tissue klotho expression was assessed by immunohistochemical staining. The median age of the study participants was 35.6 years. High serum klotho levels (≥14 pg/mL) were associated with decreased odds ratios (ORs) of interstitial fibrosis (OR = 0.019, P = 0.003) and segmental sclerosis (OR = 0.190, P = 0.022) in multivariable logistic regression analysis. Patients with a lower urinary klotho-to-creatinine ratio (UKCR) were significantly more likely to have diffuse foot process effacement (OR = 0.450, P = 0.010). The area under the receiver-operating characteristic curve (AUC) of serum klotho for predicting interstitial fibrosis was 0.920 (95% CI, 0.844-0.996), and the best cut-off value of serum klotho was 138.1 pg/mL. The AUC of UKCR for predicting diffuse foot process effacement was 0.754 (95% CI, 0.636-0.872), and the best cut-off value of UKCR was 96.7 pg/mgCr. Urinary angiotensinogen-to-creatinine ratio was not associated with serum klotho, UKCR, or any pathological finding. Our data suggested that soluble serum and urinary klotho levels represent a potential biomarker to predict renal fibrosis and podocyte injury in humans.

A PEG-based method for the isolation of urinary exosomes and its application in renal fibrosis diagnostics using cargo miR-29c and miR-21 analysis.

PURPOSE: To assess a new and highly specific, but low-cost, easily performed and suitable for large-scale applications method for renal fibrosis (RF) diagnostics. METHODS: Thirty-five RF and twenty non-RF patients were enrolled in the study. An appropriate polyethylene glycol (PEG) was used to isolate urinary exosomes. The efficiency of isolation process was evaluated by the morphology and size observation, as well as the detection of specific markers (CD63, CD9). The expression level of exosomal miR-29c, miR-21 and the endogenous control snRNA-U6 were detected by qRT-PCR. The diagnostic potency of urinary exosomal miR-29c and miR-21 was estimated by the ROC method. Spearman's rank-order correlations analysis was used to assess the correlation between the miRNAs and clinical parameters, including pathological index. RESULTS: PEG-based method for isolation urinary exosome was effective and could be completed with a relatively low-speed centrifugal machine. Exosomal miR-29c and miR-21 were detected in all samples. The analysis of miRNAs in urinary exosomes revealed significant dys-regulation of miR-29c and miR-21 associated with RF. Exosomal miR-29c and miR-21 could predict degree of RF with AUC of 0.8333 and 0.7639 (P < 0.05). Correlation analysis showed that the level of miR-29c had a significant negative relationship with eGFR and the interstitial relative area. CONCLUSIONS: The PEG-based method for isolation urinary exosome is an inexpensive and easily performed approach. The application for cargo miRNA analysis is feasible. Urinary exosomal miR-29c may present a promising diagnostic approach.

Short-term oral gavage administration of adenine induces a model of fibrotic kidney disease in rats.

INTRODUCTION: The adenine model of kidney disease typically involves dietary delivery of adenine over several weeks. This model can be variable in its disease progression and can result in significant mortality. In the current study, the amount of adenine delivered to rats was controlled by utilizing oral gavage administration over a short period in an attempt to induce robust renal pathology while addressing variability and viability of the animals. METHODS: Adenine (150 or 200 mg/kg) was administered via oral gavage for 10 consecutive days, and assessed over a total of 20 days. RESULTS: Both adenine dose groups manifested pathophysiological features of kidney disease such as proteinuria, elevated serum creatinine and BUN, and tubulointerstitial fibrosis. The animals also displayed a decline in glomerular filtration rate. Renal mRNA expression of genes associated with injury, inflammation, and fibrosis (i.e., Col1a1, Acta2, Serpine1, Timp1, Fn-Eda, Tgfb1, Ccl2, Nlrp3, Aqp1 and Ccnd1) were elevated as were urinary biomarkers that have translational utility (i.e., clusterin, KIM-1, MCP-1, OPN, NGAL, B2M, calbindin, and cystatin C). All disease endpoints were more pronounced in the 200 mg/kg group, however, while measures of tissue fibrosis were sustained, there was partial recovery by day 20 in functional readouts. No mortality was observed in either dose group. DISCUSSION: Short-term delivery of adenine via precise gavage delivery induced a robust model with hallmarks of fibrotic kidney disease, had limited variance between animals, and no animal morbidity within the 20 days studied. This model represents a methodical alternative to long-term dietary dosing of adenine.

The effect of sildenafil on rats with adenine-Induced chronic kidney disease.

The erectile dysfunction drug sildenafil has cardiopulmonary protective actions, and a nephroprotective action in cisplatin and ischemia-reperfusion-induced acute kidney injury. Here, we assessed its possible ameliorative action in a model of chronic kidney disease (CKD) using adenine feeding. Eight groups of rats were treated with saline (controls), adenine (0.25% w/w in feed daily for 5 weeks), and oral sildenafil (0.1, 0.5 or 2.5 mg/kg), either alone, or concomitantly with adenine. Urine was collected 24 h after the end of the treatments from all rats and blood pressure measured, followed by collection of blood and kidneys for the measurement of several functional, biochemical and histopathological parameters. Adenine treatment reduced body weight, creatinine renal clearance, and increased water intake and urine output, as well as the plasma concentrations of urea and creatinine, neutrophil gelatinase-associated lipocalin, and N-acetyl-ß-D-glucosaminidase activity, and albumin in urine. Adenine also increased the concentrations of the uremic toxins indoxyl sulfate, uric acid and phosphate, and a number of proteins and inflammatory cytokines, and decreased that of several anti - oxidant indices. Renal histopathological markers of damage (inflammation and fibrosis) were significantly increased by adenine. Sildenafil, given simultaneously with adenine, induced a dose - dependent improvements in most of the above parameters, suggesting its possible use as adjunct treatment for CKD in humans.

[Clinical implication of urine test for markers of endothelial dysfunction and angiogenesis factors in assessment of tubulointerstitial fibrosis in chronic glomerulonephritis].

AIM: To evaluate contribution of endothelial dysfunction and impairment of endothelial proliferation/ regeneration to mechanisms of development of tubulointerstitial fibrosis (TIF) in chronic glomerulonephritis (CGN) basing on urinary levels of markers of endothelial activation/impairment and angiogenesis factors. MATERIAL AND METHODS: A total of 67 CGN patients entered the study: 19 patients with moderate urinary syndrome (group 1), 37 patients with nephrotic syndrome (group 2), 11 patients with nephrotic syndrome and persistent renal failure (RF). A control group consisted of 12 healthy subjects. The examination covered excretion with urine of Willebrand factor (WF), plasminogen activator inhibitor I (PAL-I), fibrin degradation products (FDP), vascular endothelial growth factor (VEGF). These values were compared with severity of fibrous changes in renal interstitium estimated by biopsy morphometry. RESULTS: CGN patients had signs of affection of parietal effects of vascular endothelium. In particular, increased excretion of functionally active WF, PAI-I and FDP correlating with activity/severity of CGN. The changes were especially noticeable in patients with progressive forms of CGN (with NS and RF). Patients with morphologically verified TIF (interstitial area more than 20%) excretion of endothelial dysfunction markers was higher than in CGN patients free of TIF In a progressive course of nephritis endothelial dysfunction deteriorates by endothelial proliferation/regeneration impairment as shown by reduced urinary excretion of angiogenic factor VEGF and parallel elevation of functionally active WF in urine of patients with severe forms of CGN. Combined contribution of endothelial dysfunction and angiogenesis impairment to mechanisms of TIF development is seen from these values relations with severity of creatinemia and fibrous alterations in tubulointerstitial tissues of the kidney. CONCLUSION: The results point to participation of endothelium in mechanisms promoting development of TIF and RF in CGN both in terms of endothelial dysfunction and impairment of endothelial repair capacity. Clinicomorphological comparisons confirm the significance of WF, PAI-I and VEGF in assessment of local-renal endothelial changes and severity of fibrosis in renal tissue in CGN. Due to availability of the study material, perspectives of fibrogenesis monitoring in the kidneys with the tests appear which is essential for making prognosis and treatment policy in CGN patients.

Dysregulation of urinary miR-21 and miR-200b associated with interstitial fibrosis and tubular atrophy (IFTA) in renal transplant recipients.

OBJECTIVE: microRNAs (miRNAs) are endogenous gene expression regulators that are involved in renal function and physiopathology, making them potential biomarkers in the realm of transplantation. Here, we aimed to evaluate urinary miRNA expression levels and assess their possible values in the prospective evaluation of renal allograft function. DESIGN AND METHODS: The expression levels of four miRNAs involved in renal fibrosis were quantified in urine cell pellets of 47 kidney recipients (with stable allograft function (SGF) and biopsy proven interstitial fibrosis and tubular atrophy; IFTA) and 15 healthy controls by real-time quantitative-PCR (RT-qPCR). The hierarchical clustering and receiver operating characteristic (ROC) analysis of miRNAs were performed in the study groups. RESULTS: Significant differences were observed in the urinary expression of miR-21 (P<0.001), miR-142-3p (P<0.001), and miR-200b (P=0.021) between IFTA and controls. The hierarchical clustering of miRNA expression clustered most of the SGF and IFTA patients in separated groups. Estimated GFR was significantly correlated with urinary levels of miR-21 (r=0.585, P=0.003) and miR-200 (r=-0.447, P=0.033). ROC analysis confirmed the ability of urinary miR-21 and miR-200b to discriminate the IFTA recipients with an area under the ROC curve of ≥0.81 (P<0.001) and high sensitivity and specificity. CONCLUSION: Taken together, our findings indicated that the aberrant urinary miR-21 and miR-200b expression levels were accompanied with renal allograft dysfunction and IFTA. Therefore, they can be considered as potential diagnostic biomarkers for renal allograft monitoring.