The complexity of kidney disease and diagnosing it - cystatin C, selective glomerular hypofiltration syndromes and proteome regulation.

Estimation of kidney function is often part of daily clinical practice, mostly done by using the endogenous glomerular filtration rate (GFR)-markers creatinine or cystatin C. A recommendation to use both markers in parallel in 2010 has resulted in new knowledge concerning the pathophysiology of kidney disorders by the identification of a new set of kidney disorders, selective glomerular hypofiltration syndromes. These syndromes, connected to strong increases in mortality and morbidity, are characterized by a selective reduction in the glomerular filtration of 5-30 kDa molecules, such as cystatin C, compared to the filtration of small molecules <1 kDa dominating the glomerular filtrate, for example water, urea and creatinine. At least two types of such disorders, shrunken or elongated pore syndrome, are possible according to the pore model for glomerular filtration. Selective glomerular hypofiltration syndromes are prevalent in investigated populations, and patients with these syndromes often display normal measured GFR or creatinine-based GFR-estimates. The syndromes are characterized by proteomic changes promoting the development of atherosclerosis, indicating antibodies and specific receptor-blocking substances as possible new treatment modalities. Presently, the KDIGO guidelines for diagnosing kidney disorders do not recommend cystatin C as a general marker of kidney function and will therefore not allow the identification of a considerable number of patients with selective glomerular hypofiltration syndromes. Furthermore, as cystatin C is uninfluenced by muscle mass, diet or variations in tubular secretion and cystatin C-based GFR-estimation equations do not require controversial race or sex terms, it is obvious that cystatin C should be a part of future KDIGO guidelines.

Addition of cystatin C predicts cardiovascular death better than creatinine in intensive care.

OBJECTIVE: Decreased kidney function increases cardiovascular risk and predicts poor survival. Estimated glomerular filtration rate (eGFR) by creatinine may theoretically be less accurate in the critically ill. This observational study compares long-term cardiovascular mortality risk by the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) creatinine equation; Caucasian, Asian, paediatric and adult cohort (CAPA) cystatin C equation and the CKD-EPI combined creatinine/cystatin C equation. METHODS: The nationwide study includes 22 488 intensive care patients in Uppsala, Karolinska and Lund University Hospitals, Sweden, between 2004 and 2015. Creatinine and cystatin C were analysed with accredited methods at admission. Reclassification and model discrimination with C-statistics was used to compare creatinine and cystatin C for cardiovascular mortality prediction. RESULTS: During 5 years of follow-up, 2960 (13 %) of the patients died of cardiovascular causes. Reduced eGFR was significantly associated with cardiovascular death by all eGFR equations in Cox regression models. In each creatinine-based GFR category, 17%, 19% and 31% reclassified to a lower GFR category by cystatin C. These patients had significantly higher cardiovascular mortality risk, adjusted HR (95% CI), 1.55 (1.38 to 1.74), 1.76 (1.53 to 2.03) and 1.44 (1.11 to 1.86), respectively, compared with patients not reclassified. Harrell's C-statistic for cardiovascular death for cystatin C, alone or combined with creatinine, was 0.73, significantly higher than for creatinine (0.71), p<0.001. CONCLUSIONS: A single cystatin C at admission to the intensive care unit added significant predictive value to creatinine for long-term cardiovascular death risk assessment. Cystatin C, alone or in combination with creatinine, should be used for estimating GFR for long-term risk prediction in critically ill.

Strong Associations Between Early Tubular Damage and Urinary Cytokine, Chemokine, and Growth Factor Levels in Elderly Males and Females.

Acute tubular necrosis is associated with high mortality rates and it is important to develop new biomarkers for tubular damage. The aim of this study was to investigate the effect of early tubular damage on a large number of urinary cytokines, chemokines, and growth factors. We selected 90 urine samples from the Prospective Investigation of the Vasculature in Uppsala Seniors Study (41 males and 49 females). The tubular damage markers cystatin C, neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule-1 (KIM-1) were analyzed in the urine samples and urinary cytokine levels were analyzed with 2 multiplex assays (proximity extension assay). After adjustment for sex, body mass index, estimated glomerular filtration rate, smoking, and multiplicity testing using the false discovery rate approach, there remained 26 cytokines that correlated significantly with urine cystatin C, 27 cytokines that correlated with NGAL, and 66 cytokines that correlated with KIM-1. Tubular damage shows a strong association with urinary cytokines, chemokines, and growth factors. Our findings indicate that multiplex proteomics could be a promising new approach to explore the complex effects of tubular damage.

Strong Associations between Plasma Osteopontin and Several Inflammatory Chemokines, Cytokines, and Growth Factors.

Osteopontin is a member of the proinflammatory cytokine network, a complex system that involves many chemokines, cytokines, and growth factors. The aim of the present study was to study the associations between osteopontin and a large number of chemokines, cytokines, and growth factors. We analyzed plasma and urine osteopontin in 652 men from the Uppsala Longitudinal Study of Adult Men (ULSAM) study cohort and compared the levels with the levels of eighty-five chemokines, cytokines, and growth factors. We found significant associations between plasma osteopontin and 37 plasma biomarkers in a model adjusted for age, and 28 of those plasma biomarkers were significant in a model also adjusting for cardiovascular risk factors. There were no significant associations after Bonferroni adjustment between urine osteopontin and any of the studied plasma cytokine biomarkers. This study shows that circulating osteopontin participates in a protein-protein interaction network of chemokines, cytokines, and growth factors. The network contains responses, pathways, and receptor binding interactions relating to cytokines, regulation of the immune system, and also regulation of apoptosis and intracellular signal transduction.

Albumin Urinary Excretion Is Associated with Increased Levels of Urinary Chemokines, Cytokines, and Growth Factors Levels in Humans.

The aim of the present study was to study the associations between urine albumin excretion, and a large number of urinary chemokines, cytokines, and growth factors in a normal population. We selected 90 urine samples from individuals without CVD, diabetes, stroke or kidney disease belonging to the Prospective Investigation of the Vasculature in Uppsala Seniors Study (41 males and 49 females, all aged 75 years). Urinary cytokine levels were analyzed with two multiplex assays (proximity extension assays) and the cytokine levels were correlated with urine albumin. After adjustment for sex, body mass index (BMI), estimated glomerular filtration rate (eGFR), smoking and multiplicity testing, 11 biomarkers remained significantly associated with urine albumin: thrombospondin 2, interleukin 6, interleukin 8, hepatocyte growth factor, matrix metalloproteinase-12 (MMP-12), C-X-C motif chemokine 9, tumor necrosis factor receptor superfamily member 11B, osteoprotegerin, growth-regulated alpha protein, C-X-C motif chemokine 6, oncostatin-M (OSM) and fatty acid-binding protein, intestinal, despite large differences in molecular weights. In this study, we found associations between urinary albumin and both small and large urine proteins. Additional studies are warranted to identify cytokine patterns and potential progression markers in various renal diseases.

Cystatin C predicts long term mortality better than creatinine in a nationwide study of intensive care patients.

Decreased glomerular filtration rate (GFR) is linked to poor survival. The predictive value of creatinine estimated GFR (eGFR) and cystatin C eGFR in critically ill patients may differ substantially, but has been less studied. This study compares long-term mortality risk prediction by eGFR using a creatinine equation (CKD-EPI), a cystatin C equation (CAPA) and a combined creatinine/cystatin C equation (CKD-EPI), in 22,488 patients treated in intensive care at three University Hospitals in Sweden, between 2004 and 2015. Patients were analysed for both creatinine and cystatin C on the same blood sample tube at admission, using accredited laboratory methods. During follow-up (median 5.1 years) 8401 (37%) patients died. Reduced eGFR was significantly associated with death by all eGFR-equations in Cox regression models. However, patients reclassified to a lower GFR-category by using the cystatin C-based equation, as compared to the creatinine-based equation, had significantly higher mortality risk compared to the referent patients not reclassified. The cystatin C equation increased C-statistics for death prediction (p < 0.001 vs. creatinine, p = 0.013 vs. combined equation). In conclusion, this data favours the sole cystatin C equation rather than the creatinine or combined equations when estimating GFR for risk prediction purposes in critically ill patients.

Day-to-day variation of the kidney proximal tubular injury markers urinary cystatin C, KIM1, and NGAL in patients with chronic kidney disease.

Background: It is important to know the intraindividual variation of biomarkers to be able to distinguish a change of a biomarker due to the course of the disease from the normal biological variation of the marker. The purpose of this study was to investigate the day-to-day variability of urine markers in nephrology patients.Materials: 23 nephrology patients were included in the study. First morning urine samples were collected daily for ten consecutive days and analyzed for U-cystatin C, U-KIM1, U-NGAL and U-creatinine. The day-to-day variation was calculated as concentrations of the markers and as creatinine ratios. Values deviating more than the 90th percentile of the normal intraindividual variation was used to define a disease/treatment specific change.Results: The day-to-day coefficient of variation (CV) for individual patients varied between 9.6 and 100.3% for NGAL (mean 45.6%) and between 8.8 and 107.3% for the NGAL/creatinine ratio (mean 43.8%). The corresponding values for KIM1 were between 10.9 and 60.2% (mean 30.1%) and for the ratio between 8.7 and 59.8% (mean 23.4%) and for cystatin C 3.8-67.4% (mean 25.0%) and for the cystatin C/creatinine ratio 5.9-78.4% (mean 24.8%).Conclusions: The similar intraindividual CV values between the renal tubules damage markers and their corresponding creatinine ratios speaks against using creatinine ratio. Using the 90th percentiles of the CV values as a limit for clinical change means that NGAL has to change by 83.3%, KIM1 by 45.5% and Cystatin C by 46.3% before the change can be considered clinically significant in patients with chronic kidney disease.

Associations Between Apolipoprotein A1, High-Density Lipoprotein Cholesterol, and Urinary Cytokine Levels in Elderly Males and Females.

There exists a close relationship between cardiovascular diseases and chronic kidney disease. Apolipoprotein A1 and high-density lipoprotein (HDL) cholesterol are widely used as cardiovascular risk markers but they also have anti-inflammatory properties. The aim of this study was to investigate any associations between HDL levels and cytokine levels in urine. We randomly selected 90 urine samples from the Prospective Investigation of the Vasculature in Uppsala Seniors Study (41 males and 49 females). The samples were analyzed with 2 multiplex assays, Multiplex Inflammation I and Cardiovascular II kits (Olink Bioscience, Uppsala, Sweden). We analyzed the correlations between 158 cytokines in urine with apolipoprotein A1, HDL cholesterol, apolipoprotein B, and low-density lipoprotein cholesterol. There were strong correlations for apolipoprotein A1 and HDL cholesterol with individual cytokines. After adjustment for multiplicity testing, there were 33 significant correlations between apolipoprotein A1 and cytokine levels and 14 of these were also significantly correlated with HDL cholesterol. The strongest associations were observed for IL-1α, SPON2, RAGE, PAR-1, TRAIL-R2, IL-4RA, TNFRSF11A, and SCF. A total of 28 out of 33 correlations were negative, indicating a negative relationship between apolipoprotein A1 and urinary cytokines. The study shows a negative correlation between apolipoprotein A1 and HDL cholesterol and urinary cytokine levels. The finding is in agreement with the anti-inflammatory properties of HDL.

Reference Intervals for Fecal Calprotectin in Pregnant Women Using a Particle Enhanced Turbidimetric Assay.

BACKGROUND: Fecal calprotectin is widely used as a marker for inflammatory bowel diseases (IBD). IBD often affects women during their reproductive years, but there are no established reference intervals during pregnancy. The aim of the present study was to define reference values during pregnancy and in the postpartum period to allow comparisons between patient results and reference values. METHODS: Fecal samples were collected from 84 healthy females during pregnancy week 26 to 28 and a second sample was collected six months after delivery. The samples were weighed, extracted, and centrifugated to remove debris. The extracted samples were then analyzed on a chemistry analyzer using a particle enhanced turbidimetric immunoassay reagent. RESULTS: The calculated reference interval during pregnancy was < 127 µg/g (90% confidence interval, 90 - 164 µg/g) and the corresponding reference interval during the postpartum period was < 143 µg/g (60 - 226 µg/g). There were no significant statistical differences between F-calprotectin values analyzed at the two sampling times. CONCLUSIONS: The reference values are slightly higher than the cutoff values of 50 - 100 µg/g often used as General cutoff for fecal calprotectin.

Lower creatinine concentration values and lower inter-laboratory variation among Swedish hospital laboratories in 2014 compared to 1996: results from the Equalis external quality assessment program.

Background Creatinine measurement for estimation of glomerular filtration rate (GFR) is a frequently used laboratory test. Differences in analytic creatinine methods have caused large inter-laboratory variation. International and national standardization efforts have been made in the last decade. Methods This study describes the results of the standardization efforts in Sweden by summarizing data for creatinine concentration in blood plasma in the Equalis quality assessment program during 1996-2014. Results Non-compensated Jaffe methods dominated in 1996-2001 (91 of 103 laboratories; 90%) and were then gradually replaced by either compensated Jaffe methods or enzymatic creatinine methods. In 2014 a majority of Swedish hospital laboratories (139 of 159; 87%) used enzymatic methods. The reported mean creatinine value by the Swedish laboratories was about 10 µmol/L higher than the isotope dilution mass spectrometry (IDMS) assured reference value in 2003, but consistent with the reference value from 2009 to 2014. The inter-laboratory CV was 7%-9% for creatinine values until 2007, and thereafter gradually decreased to about 4%-5% in 2014. Conclusions The introduction of enzymatic methods in Swedish laboratories has contributed to achieving a low inter-laboratory variation. Also, the reported values are lower for enzymatic methods compared to Jaffe methods, and the values obtained with enzymatic methods were consistent with IDMS certified values established at reference laboratories. Thus, many Swedish hospital laboratories reported 10 µmol/L lower, and more true, creatinine concentrations in 2012 than in 2003, which may cause bias in longitudinal studies.

Evaluation of Nova StatStrip and FreeStyle Precision Pro blood ketone tests using 3-hydroxybutyrate doped samples.

BACKGROUND: The most clinically useful blood ketone in the diagnosis, management, and recovery of diabetes ketoacidosis in both adults and children is 3-hydroxybutyrate. In the absence of laboratory routine methods, several point-of-care methods are in use, but very few clinical evaluations are published. METHODS: This study evaluates linearity and reproducibility of two handheld point-of-care meters for blood 3-hydroxybutyrate measurement for hospital use, Nova StatStrip, and FreeStyle Precision Pro. Whole blood from healthy volunteers was spiked with different concentrations of a 3-hydroxybutyrate solution and tested on the point-of-care instruments. The results were compared with plasma 3-hydroxybutyrate that was analyzed with a laboratory enzymatic end point spectrophotometric reference method. RESULTS: Blood 3-hydroxybutyrate on StatStrip was linear with the reference method up to approximately 4 mmol/L, and FreeStyle was linear up to 6 mmol/L. At higher concentrations, the point-of-care instruments gave falsely too low results, especially the StatStrip meter. The FreeStyle meter had better precision and less bias than StatStrip. CONCLUSION: In the acute setting of diabetes ketoacidosis, blood 3-hydroxybutyrate in the higher ranges should be interpreted with caution as the point-of-care meters are less accurate there.

Normal values for calprotectin in stool samples of infants from the population-based longitudinal born into life study.

Faecal calprotectin is a protein used as a diagnostic marker for inflammatory bowel diseases. We determined upper limits for normal calprotectin values for neonatal, 6, 12 and 24 months old children using a turbidimetric immunoassay in a cohort of Swedish children. The advantage of the method is that opposite to previously used enzyme-linked immunosorbent assay (ELISA) method, it enables measuring single samples, and thus, shortens the analysis time significantly. There were 72 samples (41.7% female) collected neonatally, 63 samples (34.9% female) at 6 months, 60 samples (40.0% female) at 12 months and 51 samples (43.1% female) at 24 months. The upper limits for normal values were 233, 615, 136 and 57 µg mg-1 for infants aged 0, 6, 12 and 24 months, respectively.

Seasonal variability of NT-proBNP in Swedish primary care patients.

The aim of this study was to determine if there is a seasonal variation in the widely used heart failure marker NT-proBNP. The study included all primary care requests for NT-proBNP in the county of Uppsala, Sweden, between January 2007 and December 2015. For seasonal variation, the NT-proBNP results for individual months were compared. The NT-proBNP values were highest in July to September, but there was also a minor peak in December-January. In conclusion, a seasonal periodicity for NT-proBNP was demonstrated in primary care patients. The data could be useful for practitioners for evaluation of NT-proBNP results and monitoring of patients with heart failure.

Reference Intervals for Fecal Calprotectin in Adults Using Two Different Extraction Methods in the Uppsala-SCAPIS Cohort.

BACKGROUND: Fecal calprotectin measurement is generally recommended to exclude inflammatory bowel disease (IBD) in patients with suspected IBD. A problem with the fecal calprotectin assays so far has been the rather long test-turnaround times. Recently a particle enhanced turbidimetric immunoassay (PETIA) for fecal calprotectin with assay times of approximately 10 minutes has been introduced on the European market. The aim of this study was to define reference intervals for adults with this new fecal calprotectin PETIA using two different extraction methods. METHODS: Samples were collected from 382 healthy individuals from the Swedish CArdioPulmonary bioImage Study (SCAPIS) Uppsala cohort in the age range 50 - 65 years. 202 samples were processed with CALEX® Cap extraction device (BÜHLMANN, Schönenbuch, Switzerland) and 180 samples were extracted using weighed samples. The extracted samples were analyzed on a Mindray BS-380 using the fCal Turbo PETIA reagent (BÜHLMANN). RESULTS: The calculated reference values for the Calex device were < 199 µg/g for the whole cohort, < 184 µg/g for females, and < 215 µg/g for males, while the corresponding values for weighed samples were < 153 µg/g for the whole cohort, < 141 µg/g for females, and < 215 µg/g for males. There were no significant statistical differences for calprotectin levels in males and females. CONCLUSIONS: The CALEX device yielded slightly higher calprotectin values. As there were no significant gender differences, the study indicates gender independent reference intervals of < 199 µg/g feces for the CALEX device and < 153 µg/g feces for weighed samples in patients in the 50 - 65 year age range.

Urinary Osteopontin Predicts Incident Chronic Kidney Disease, while Plasma Osteopontin Predicts Cardiovascular Death in Elderly Men.

BACKGROUND AND OBJECTIVES: The matricellular protein osteopontin is involved in the pathogenesis of both kidney and cardiovascular disease. However, whether circulating and urinary osteopontin levels are associated with the risk of these diseases is less studied. DESIGN SETTING PARTICIPANTS AND MEASUREMENTS: A community-based cohort of elderly men (Uppsala Longitudinal Study of Adult Men [ULSAM]; n = 741; mean age: 77 years) was used to study the associations between plasma and urinary osteopontin, incident chronic kidney disease, and the risk of cardiovascular death during a median of 8 years of follow-up. RESULTS: There was no significant cross-sectional correlation between plasma and urinary osteopontin (Spearman ρ = 0.07, p = 0.13). Higher urinary osteopontin, but not plasma osteopontin, was associated with incident chronic kidney disease in multivariable models adjusted for age, cardiovascular risk factors, baseline glomerular filtration rate, urinary albumin/creatinine ratio, and the inflammatory markers interleukin 6 and high-sensitivity C-reactive protein (odds ratio for 1 standard deviation [SD] of urinary osteopontin, 1.42, 95% CI 1.00-2.02, p = 0.048). Conversely, plasma osteopontin, but not urinary osteopontin, was independently associated with cardiovascular death (multivariable hazard ratio per SD increase, 1.35, 95% CI 1.14-1.58, p < 0.001, and 1.00, 95% CI 0.79-1.26, p = 0.99, respectively). The addition of plasma osteopontin to a model with established cardiovascular risk factors significantly increased the C-statistics for the prediction of cardiovascular death (p < 0.002). CONCLUSIONS: Higher urinary osteopontin specifically predicts incident chronic kidney disease, while plasma osteopontin specifically predicts cardiovascular death. Our data put forward osteopontin as an important factor in the detrimental interplay between the kidney and the cardiovascular system. The clinical implications, and why plasma and urinary osteopontin mirror different pathologies, remain to be established.

Albumin adjustment of total calcium does not improve the estimation of calcium status.

BACKGROUND: There is a longstanding controversy as to whether plasma measurements of total calcium should be adjusted for albumin concentration, and if so which formulas are the most appropriate. METHODS: Ionised calcium, total calcium and albumin results, analysed at the same time at Uppsala University Hospital Laboratory between February 2005 and June 2013, were retrieved from a laboratory information system. The dataset included results from 20,003 patients. Total calcium was albumin-modified by a locally derived formula, based on 3106 patients from the dataset, and formulas from the literature. The agreement between the reference method ionised calcium and unadjusted total calcium and the seven different albumin-modifying calcium formulas, respectively, were compared with intra-class correlation coefficients (ICC). RESULTS: Total calcium showed substantial agreement to ionised calcium, ICC 0.85 (95% CI 0.84-0.86) for the whole validation cohort. Albumin-modified calcium by different formulas showed significantly less or equal agreement, however the locally determined formula performed better than formulas taken from the literature. Also, total calcium classified the patient as hypo-normo- or hypercalcemic right in 82% of the patients. The albumin-modified calcium did not classify patients significantly better except in the subgroup hypoalbuminemia (<30 g/L) where the local formula classified the patients slightly better than total calcium. CONCLUSIONS: Albumin modification of total calcium determinations is unlikely to add valuable information, and this practice should be abandoned. Ionised calcium should be used more frequently when aberrant results for total calcium are followed up, or in patients with known hypoalbuminemia.

Seasonal variations of urate in a Swedish adult population.

Seasonality in the incidence and prevalence of gout has previously been reported but the cause of this seasonality in gout is not explained. The aim of this study was to evaluate possible seasonal variations of urate in a large unselected Swedish adult population. We analyzed 170,915 urate test results from patients at a tertiary care hospital between 2000 and 2016. The results were divided according to sex and sampling month of the year. The median urate values were overall higher in males compared to females and both males and females had peak urate concentrations in the summer months (June-August). There is a seasonal pattern for urate concentrations in a large Swedish population similar to the previously reported seasonality for gout. This may be clinically important and could contribute to the circannual variation of gout. The seasonal pattern should be recognized when evaluating patient results both in clinical practice and in research studies.

Reference values for 34 frequently used laboratory tests in 80-year-old men and women.

OBJECTIVES: Reference values are usually based on blood samples from healthy individuals in the age range 20-50 years. Most patients seeking health care are older than this reference population. Many reference intervals are age dependent and there is thus a need to have appropriate reference intervals also for elderly individuals. METHODS: We analyzed a group of frequently used laboratory tests in an 80-year-old population (n=531, 266 females and 265 males). The 2.5th and 97.5th percentiles for these markers were calculated according to the International Federation of Clinical Chemistry guidelines on the statistical treatment of reference values. RESULTS: Reference values are reported for serum alanine transaminase (ALT), albumin, alkaline phosphatase, pancreatic amylase, apolipoprotein A1, apolipoprotein B, apolipoprotein B/apolipoprotein A1 ratio, aspartate aminotransferase (AST), AST/ALT ratio, bilirubin, calcium, calprotectin, cholesterol, HDL-cholesterol, creatinine kinase (CK), creatinine, creatinine estimated GFR, C-reactive protein, cystatin C, cystatin C estimated GFR, gamma-glutamyltransferase (GGT), iron, iron saturation, lactate dehydrogenase (LDH), magnesium, phosphate, transferrin, triglycerides, urate, urea, zinc, hemoglobin, platelet count and white blood cell count. The upper reference limit for creatinine and urea was significantly increased while the lower limit for iron and albumin was decreased in this elderly population in comparison with the population in the Nordic Reference Interval Project (NORIP). CONCLUSIONS: Reference values calculated from the whole population and a subpopulation without cardiovascular disease showed strong concordance. Several of the reference interval limits were outside the 90% confidence interval of NORIP.

Urinary KIM-1, but not urinary cystatin C, should be corrected for urinary creatinine.

OBJECTIVES: The interest for tubular damage markers such as urinary cystatin C (U-CystC) and kidney injury molecule-1 (U-KIM-1) grows, especially for the diagnosis of acute kidney injury. The trend to measure proteins in spot urine samples instead of 24-h urine collections calls for adjustment of urine dilution with urinary creatinine (UCr). However, it is not known whether UCr adjustment provides a more true value of basal U-CystC and U-KIM-1 levels than absolute values. DESIGN & METHODS: This study examines the rationale for UCr correction for U-CystC and U-KIM-1 by exploring the linear relations between U-CystC and U-KIM-1 and UCr, respectively, and the biological day to day variation of absolute concentrations and UCr adjusted values of the two biomarkers. RESULTS: Both U-CystC and U-KIM-1 concentrations correlated positively with UCr (R=0.37, P<0.001 and R=0.62, P<0.001, respectively) in 378 participants in a community cohort, which indicated a rationale for adjustment with UCr. However, U-CystC/Cr ratio associated negatively with UCr (R=- 0.31, P<0.001), which could indicate a certain amount of 'over-adjustment'. Morning urine collected for 10 consecutive days from 13 healthy volunteers showed a biological day to day variation of 82% for U-CystC, 75% for U-cystC/Cr ratio, 70% for U-KIM-1 and 46% for U-KIM-1/Cr ratio. CONCLUSIONS: This study supports the use of U-KIM-1/Cr ratio in clinical population studies. Data supporting the use of U-CysC/U-Cr ratio were less convincing and the possible confounding of UCr has to be acknowledged in clinical settings.

Associations between urinary kidney injury biomarkers and cardiovascular mortality risk in elderly men with diabetes.

AIM: Three urinary biomarkers, kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and cystatin C, have been suggested as clinically relevant highly specific biomarkers of acute kidney tubular damage. Yet, the utility of these biomarkers in the prognostication of diabetic nephropathy has been less studied. Therefore, we aimed to investigate the longitudinal association between these urinary biomarkers and cardiovascular mortality in patients with diabetes. METHODS: The study sample consisted of participants with diabetes in the community-based Uppsala Longitudinal Study of Adult Men (n = 91; mean age 77.8 years). During follow-up (median 8.3 years, interval 0.7-13.4 years), 33 participants died of cardiovascular causes. RESULTS: In a multivariable Cox regression model adjusting for age, glomerular filtration rate, and urinary albumin/creatinine ratio, higher urinary KIM-1/creatinine was associated with an increased risk for cardiovascular mortality (HR per SD increase 1.51, 95% confidence intervals 1.03-2.24, P = 0.03). Neither urinary NGAL/creatinine nor urinary cystatin C/creatinine were independently associated with an increased cardiovascular mortality risk. CONCLUSION: In elderly men with diabetes, higher urinary KIM-1/creatinine was associated with an increased long-term risk of cardiovascular mortality independently of established markers of diabetic nephropathy. Our data provide support for kidney tubular damage as an important aspect of diabetic nephropathy that merits further investigation.