A signature of circulating inflammatory proteins and development of end-stage renal disease in diabetes.

Chronic inflammation is postulated to be involved in the development of end-stage renal disease in diabetes, but which specific circulating inflammatory proteins contribute to this risk remain unknown. To study this, we examined 194 circulating inflammatory proteins in subjects from three independent cohorts with type 1 and type 2 diabetes. In each cohort, we identified an extremely robust kidney risk inflammatory signature (KRIS), consisting of 17 proteins enriched in tumor necrosis factor-receptor superfamily members, that was associated with a 10-year risk of end-stage renal disease. All these proteins had a systemic, non-kidney source. Our prospective study findings provide strong evidence that KRIS proteins contribute to the inflammatory process underlying end-stage renal disease development in both types of diabetes. These proteins point to new therapeutic targets and new prognostic tests to identify subjects at risk of end-stage renal disease, as well as biomarkers to measure responses to treatment of diabetic kidney disease.

Variations in Risk of End-Stage Renal Disease and Risk of Mortality in an International Study of Patients With Type 1 Diabetes and Advanced Nephropathy.

OBJECTIVE: Patients with type 1 diabetes and diabetic nephropathy are targets for intervention to reduce high risk of end-stage renal disease (ESRD) and deaths. This study compares risks of these outcomes in four international cohorts. RESEARCH DESIGN AND METHODS: In the 1990s and early 2000s, Caucasian patients with type 1 diabetes with persistent macroalbuminuria in chronic kidney disease stages 1-3 were identified in the Joslin Clinic (U.S., 432), Finnish Diabetic Nephropathy Study (FinnDiane) (Finland, 486), Steno Diabetes Center Copenhagen (Denmark, 368), and INSERM (France, 232) and were followed for 3-18 years with annual creatinine measurements to ascertain ESRD and deaths unrelated to ESRD. RESULTS: During 15,685 patient-years, 505 ESRD cases (rate 32/1,000 patient-years) and 228 deaths unrelated to ESRD (rate 14/1,000 patient-years) occurred. Risk of ESRD was associated with male sex; younger age; lower estimated glomerular filtration rate (eGFR); higher albumin/creatinine ratio, HbA1c, and systolic blood pressure; and smoking. Risk of death unrelated to ESRD was associated with older age, smoking, and higher baseline eGFR. In adjusted analysis, ESRD risk was highest in Joslin versus reference FinnDiane (hazard ratio [HR] 1.44, P = 0.003) and lowest in Steno (HR 0.54, P < 0.001). Differences in eGFR slopes paralleled risk of ESRD. Mortality unrelated to ESRD was lowest in Joslin (HR 0.68, P = 0.003 vs. the other cohorts). Competing risk did not explain international differences in the outcomes. CONCLUSIONS: Despite almost universal renoprotective treatment, progression to ESRD and mortality in patients with type 1 diabetes with advanced nephropathy are still very high and differ among countries. Finding causes of these differences may help reduce risk of these outcomes.

Markers of early progressive renal decline in type 2 diabetes suggest different implications for etiological studies and prognostic tests development.

To identify determinants of early progressive renal decline in type 2 diabetes a range of markers was studied in 1032 patients enrolled into the 2nd Joslin Kidney Study. eGFR slopes estimated from serial measurements of serum creatinine during 5-12 years of follow-up were used to define early renal decline. At enrollment, all patients had normal eGFR, 58% had normoalbuminuria and 42% had albuminuria. Early renal decline developed in 6% and in 18% patients, respectively. As determinants, we examined baseline values of clinical characteristics, circulating markers: TNFR1, KIM-1, and FGF23, and urinary markers: albumin, KIM-1, NGAL, MCP-1, EGF (all normalized to urinary creatinine) and the ratio of EGF to MCP-1. In univariate analysis, all plasma and urinary markers were significantly associated with risk of early renal decline. When analyzed together, systolic blood pressure, TNFR1, KIM-1, the albumin to creatinine ratio, and the EGF/MCP-1 ratio remained significant with the latter having the strongest effect. Integration of these markers into a multi-marker prognostic test resulted in a significant improvement of discriminatory performance of risk prediction of early renal decline, compared with the albumin to creatinine ratio and systolic blood pressure alone. However, the positive predictive value was only 50% in albuminuric patients. Thus, markers in plasma and urine indicate that the early progressive renal decline in Type 2 diabetes has multiple determinants with strong evidence for involvement of tubular damage. However, new, more informative markers are needed to develop a better prognostic test for such decline that can be used in a clinical setting.

Improved clinical trial enrollment criterion to identify patients with diabetes at risk of end-stage renal disease.

Design of Phase III trials for diabetic nephropathy currently requires patients at a high risk of progression defined as within three years of a hard end point (end-stage renal disease, 40% loss of estimated glomerular filtration rate, or death). To improve the design of these trials, we used natural history data from the Joslin Kidney Studies of chronic kidney disease in patients with diabetes to develop an improved criterion to identify such patients. This included a training cohort of 279 patients with type 1 diabetes and 134 end points within three years, and a validation cohort of 221 patients with type 2 diabetes and 88 end points. Previous trials selected patients using clinical criteria for baseline urinary albumin-to-creatinine ratio and estimated glomerular filtration rate. Application of these criteria to our cohort data yielded sensitivities (detection of patients at risk) of 70-80% and prognostic values of only 52-63%. We applied classification and regression trees analysis to select from among all clinical characteristics and markers the optimal prognostic criterion that divided patients with type 1 diabetes according to risk. The optimal criterion was a serum tumor necrosis factor receptor 1 level over 4.3 ng/ml alone or 2.9-4.3 ng/ml with an albumin-to-creatinine ratio over 1900 mg/g. Remarkably, this criterion produced similar results in both type 1 and type 2 diabetic patients. Overall, sensitivity and prognostic value were high (72% and 81%, respectively). Thus, application of this criterion to enrollment in future clinical trials could reduce the sample size required to achieve adequate statistical power for detection of treatment benefits.

Increased plasma kidney injury molecule-1 suggests early progressive renal decline in non-proteinuric patients with type 1 diabetes.

Progressively decreasing glomerular filtration rate (GFR), or renal decline, is seen in patients with type 1 diabetes (T1D) and normoalbuminuria or microalbuminuria. Here we examined the associations of kidney injury molecule-1 (KIM-1) in plasma and urine with the risk of renal decline and determine whether those associations are independent of markers of glomerular damage. The study group comprised patients with T1D from the 2nd Joslin Kidney Study of which 259 had normoalbuminuria and 203 had microalbuminuria. Serial measurements over 4 to 10 years of follow-up (median 8 years) of serum creatinine and cystatin C were used jointly to estimate eGFRcr-cys slopes and time of onset of CKD stage 3 or higher. Baseline urinary excretion of IgG2 and albumin were used as markers of glomerular damage, and urinary excretion of KIM-1 and its plasma concentration were used as markers of proximal tubular damage. All patients had normal renal function at baseline. During follow-up, renal decline (eGFRcr-cys loss 3.3% or more per year) developed in 96 patients and 62 progressed to CKD stage 3. For both outcomes, the risk rose with increasing baseline levels of plasma KIM-1. In multivariable models, elevated baseline plasma KIM-1 was strongly associated with risk of early progressive renal decline, regardless of baseline clinical characteristics, serum TNFR1 or markers of glomerular damage. Thus, damage to proximal tubules may play an independent role in the development of early progressive renal decline in non-proteinuric patients with T1D.

Cardiac autonomic neuropathy and early progressive renal decline in patients with nonmacroalbuminuric type 1 diabetes.

BACKGROUND AND OBJECTIVES: Cardiac autonomic neuropathy predicts future adverse renal outcomes in the general population. This study sought to determine its relationship with early progressive renal decline in type 1 diabetes. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: A subset of participants with normoalbuminuria (n=204) or microalbuminuria (n=166) from the First Joslin Kidney Study underwent assessment for cardiac autonomic neuropathy using heart rate variability during baseline visits performed from January 1991 to April 1992. Cardiac autonomic neuropathy was defined as an R-R variation (mean circular resultant) <20. Participants also had baseline and follow-up measurement of eGFR. Early progressive renal decline was evaluated according to two definitions: early GFR loss (slope of eGFR estimated by cystatin C <-3.3%/year) and incident advanced CKD (stage ≥3, defined by eGFR [calculated by Modification of Diet in Renal Disease method] <60 ml/min per 1.73 m(2)). Association with baseline cardiac autonomic neuropathy was assessed by adjusted logistic regression and Cox proportional hazards. RESULTS: Among the 370 participants, 47 (13%) had baseline cardiac autonomic neuropathy, 51 (14%) had early GFR loss, and 68 (18%) had incident advanced CKD over a median 14-year follow-up. Early GFR loss occurred in 15 (32%) of the 47 patients with baseline autonomic neuropathy and in 32 (10%) of the 323 without baseline autonomic neuropathy (P<0.001). Baseline autonomic neuropathy was strongly associated with odds of early GFR loss (adjusted odds ratio, 4.09; 95% confidence interval, 1.65 to 10.12; P=0.002). Incident advanced CKD was observed in 22 (47%) of those with baseline autonomic neuropathy and 46 (14%) of those without baseline autonomic neuropathy (P<0.001). Autonomic neuropathy was independently associated with incident advanced CKD (adjusted hazard ratio, 2.76; 95% confidence interval, 1.44 to 5.30; P=0.002). CONCLUSIONS: Cardiac autonomic neuropathy was a strong independent predictor of the long-term risk of early progressive renal decline in type 1 diabetes. Future research should explore the mechanisms by which autonomic neuropathy may be associated with renal function loss.

Blood kidney injury molecule-1 is a biomarker of acute and chronic kidney injury and predicts progression to ESRD in type I diabetes.

Currently, no blood biomarker that specifically indicates injury to the proximal tubule of the kidney has been identified. Kidney injury molecule-1 (KIM-1) is highly upregulated in proximal tubular cells following kidney injury. The ectodomain of KIM-1 is shed into the lumen, and serves as a urinary biomarker of kidney injury. We report that shed KIM-1 also serves as a blood biomarker of kidney injury. Sensitive assays to measure plasma and serum KIM-1 in mice, rats, and humans were developed and validated in the current study. Plasma KIM-1 levels increased with increasing periods of ischemia (10, 20, or 30 minutes) in mice, as early as 3 hours after reperfusion; after unilateral ureteral obstruction (day 7) in mice; and after gentamicin treatment (50 or 200 mg/kg for 10 days) in rats. In humans, plasma KIM-1 levels were higher in patients with AKI than in healthy controls or post-cardiac surgery patients without AKI (area under the curve, 0.96). In patients undergoing cardiopulmonary bypass, plasma KIM-1 levels increased within 2 days after surgery only in patients who developed AKI (P<0.01). Blood KIM-1 levels were also elevated in patients with CKD of varous etiologies. In a cohort of patients with type 1 diabetes and proteinuria, serum KIM-1 level at baseline strongly predicted rate of eGFR loss and risk of ESRD during 5-15 years of follow-up, after adjustment for baseline urinary albumin-to-creatinine ratio, eGFR, and Hb1Ac. These results identify KIM-1 as a blood biomarker that specifically reflects acute and chronic kidney injury.

Early progressive renal decline precedes the onset of microalbuminuria and its progression to macroalbuminuria.

OBJECTIVE Progressive decrease in the glomerular filtration rate (GFR), or renal decline, in type 1 diabetes (T1D) is observed in patients with macroalbuminuria. However, it is unknown whether this decline begins during microalbuminuria (MA) or normoalbuminuria (NA). RESEARCH DESIGN AND METHODS The study group (second Joslin Kidney Study) comprises patients with T1D and NA (n = 286) or MA (n = 248) who were followed for 4-10 years (median 8 years). Serial measurements (median 6, range 3-16) of serum creatinine and cystatin C were used jointly to estimate GFR (eGFRcr-cys) and assess its trajectories during follow-up. RESULTS Renal decline (progressive eGFRcr-cys loss of at least 3.3% per year) occurred in 10% of the NA and 35% of the MA (P < 0.001). In both groups, the strongest determinants of renal decline were baseline serum concentrations of uric acid (P < 0.001) and tumor necrosis factor receptor 1 or 2 (TNFR-1 or -2, P < 0.001). Other significant risk factors included baseline HbA1c, age/diabetes duration, and systolic blood pressure. Relative impacts of these determinants were similar in NA and MA. Renal decline was not associated with sex or baseline serum concentration of TNF-α, IL-6, IL-8, IP-10, MCP-1, VCAM, ICAM, Fas, or FasL. CONCLUSIONS Renal decline in T1D begins during NA and it is determined by multiple factors, similar to MA. Thus, this early decline is the primary disease process leading to impaired renal function in T1D. Changes in albumin excretion rate, such as the onset of MA or its progression to macroalbuminuria, are either caused by or develop in parallel to the early renal decline.

Risk of ESRD and all cause mortality in type 2 diabetes according to circulating levels of FGF-23 and TNFR1.

INTRODUCTION: Recent studies demonstrated that circulating fibroblast growth factor (FGF)-23 was associated with risk of end stage renal disease (ESRD) and mortality. This study aims to examine whether the predictive effect of FGF-23 is independent from circulating levels of tumor necrosis factor receptor 1 (TNFR1), a strong predictor of ESRD in Type 2 diabetes (T2D). METHODS: We studied 380 patients with T2D who were followed for 8-12 years and were used previously to examine the effect of TNFR1. Baseline plasma FGF-23 was measured by immunoassay. RESULTS: During follow-up, 48 patients (13%) developed ESRD and 83 patients (22%) died without ESRD. In a univariate analysis, baseline circulating levels of FGF-23 and TNFR1 were significantly higher in subjects who subsequently developed ESRD or died without ESRD than in those who remained alive. In a Cox proportional hazard model, baseline concentration of FGF-23 was associated with increased risk of ESRD, however its effect was no longer significant after controlling for TNFR1 and other clinical characteristics (HR 1.3, p = 0.15). The strong effect of circulating level of TNFR1 on risk of ESRD was not changed by including circulating levels of FGF-23 (HR 8.7, p<0.001). In the Cox multivariate model, circulating levels of FGF-23 remained a significant independent predictor of all-cause mortality unrelated to ESRD (HR 1.5, p<0.001). CONCLUSIONS: We demonstrated that the effect of circulating levels of FGF-23 on the risk of ESRD is accounted for by circulating levels of TNFR1. We confirmed that circulating levels of FGF-23 have an independent effect on all-cause mortality in T2D.

Serum concentration of cystatin C and risk of end-stage renal disease in diabetes.

OBJECTIVE: Patients with diabetes have a high risk of end-stage renal disease (ESRD). We examined whether prediction of this outcome, according to chronic kidney disease (CKD) staging by creatinine-based estimates of the glomerular filtration rate (eGFRcreat), is improved by further staging with serum cystatin C-based estimates (eGFRcyst). RESEARCH DESIGN AND METHODS: Patients with diabetes in CKD stages 1-3 were selected from three cohorts: two from Joslin Diabetes Center, one with type 1 diabetes (N = 364) and one with type 2 diabetes (N = 402), and the third from the Finnish Diabetic Nephropathy (FinnDiane) Study of type 1 (N = 399). Baseline serum concentrations of creatinine and cystatin C were measured in all patients. Follow-up averaged 8-10 years and onsets of ESRD (n = 246) and death unrelated to ESRD (n = 159) were ascertained. RESULTS: Although CKD staging by eGFRcyst was concordant with that by eGFRcreat for 62% of Joslin patients and 73% of FinnDiane patients, those given a higher stage by eGFRcyst than eGFRcreat had a significantly higher risk of ESRD than those with concordant staging in all three cohorts (hazard ratio 2.3 [95% CI 1.8-3.1]). Similarly, patients at a lower stage by eGFRcyst than by eGFRcreat had a lower risk than those with concordant staging (0.30 [0.13-0.68]). Deaths unrelated to ESRD followed the same pattern, but differences were not as large. CONCLUSIONS: In patients with diabetes, CKD staging based on eGFRcyst significantly improves ESRD risk stratification based on eGFRcreat. This conclusion can be generalized to patients with type 1 and type 2 diabetes and to diabetic patients in the U.S. and Finland.

Circulating TNF receptors 1 and 2 predict ESRD in type 2 diabetes.

Levels of proinflammatory cytokines associate with risk for developing type 2 diabetes but whether chronic inflammation contributes to the development of diabetic complications, such as ESRD, is unknown. In the 1990s, we recruited 410 patients with type 2 diabetes for studies of diabetic nephropathy and recorded their characteristics at enrollment. During 12 years of follow-up, 59 patients developed ESRD (17 per 1000 patient-years) and 84 patients died without ESRD (24 per 1000 patient-years). Plasma markers of systemic inflammation, endothelial dysfunction, and the TNF pathway were measured in the study entry samples. Of the examined markers, only TNF receptors 1 and 2 (TNFR1 and TNFR2) associated with risk for ESRD. These two markers were highly correlated, but ESRD associated more strongly with TNFR1. The cumulative incidence of ESRD for patients in the highest TNFR1 quartile was 54% after 12 years but only 3% for the other quartiles (P<0.001). In Cox proportional hazard analyses, TNFR1 predicted risk for ESRD even after adjustment for clinical covariates such as urinary albumin excretion. Plasma concentration of TNFR1 outperformed all tested clinical variables with regard to predicting ESRD. Concentrations of TNFRs moderately associated with death unrelated to ESRD. In conclusion, elevated concentrations of circulating TNFRs in patients with type 2 diabetes at baseline are very strong predictors of the subsequent progression to ESRD in subjects with and without proteinuria.

Circulating TNF receptors 1 and 2 predict stage 3 CKD in type 1 diabetes.

Elevated plasma concentrations of TNF receptors 1 and 2 (TNFR1 and TNFR2) predict development of ESRD in patients with type 2 diabetes without proteinuria, suggesting these markers may contribute to the pathogenesis of renal decline. We investigated whether circulating markers of the TNF pathway determine GFR loss among patients with type 1 diabetes. We followed two cohorts comprising 628 patients with type 1 diabetes, normal renal function, and no proteinuria. Over 12 years, 69 patients developed estimated GFR less than 60 mL/min per 1.73 m(2) (16 per 1000 person-years). Concentrations of TNFR1 and TNFR2 were strongly associated with risk for early renal decline. Renal decline was associated only modestly with total TNFα concentration and appeared unrelated to free TNFα. The cumulative incidence of estimated GFR less than 60 mL/min per 1.73 m(2) for patients in the highest TNFR2 quartile was 60% after 12 years compared with 5%-19% in the remaining quartiles. In Cox proportional hazards analysis, patients with TNFR2 values in the highest quartile were threefold more likely to experience renal decline than patients in the other quartiles (hazard ratio, 3.0; 95% confidence interval, 1.7-5.5). The risk associated with high TNFR1 values was slightly less than that associated with high TNFR2 values. TNFR levels were unrelated to baseline free TNFα level and remained stable over long periods within an individual. In conclusion, early GFR loss in patients with type 1 diabetes without proteinuria is strongly associated with circulating TNF receptor levels but not TNFα levels (free or total).

Genome-wide association scan for diabetic nephropathy susceptibility genes in type 1 diabetes.

OBJECTIVE: Despite extensive evidence for genetic susceptibility to diabetic nephropathy, the identification of susceptibility genes and their variants has had limited success. To search for genes that contribute to diabetic nephropathy, a genome-wide association scan was implemented on the Genetics of Kidneys in Diabetes collection. RESEARCH DESIGN AND METHODS: We genotyped approximately 360,000 single nucleotide polymorphisms (SNPs) in 820 case subjects (284 with proteinuria and 536 with end-stage renal disease) and 885 control subjects with type 1 diabetes. Confirmation of implicated SNPs was sought in 1,304 participants of the Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Interventions and Complications (EDIC) study, a long-term, prospective investigation of the development of diabetes-associated complications. RESULTS: A total of 13 SNPs located in four genomic loci were associated with diabetic nephropathy with P < 1 x 10(-5). The strongest association was at the FRMD3 (4.1 protein ezrin, radixin, moesin [FERM] domain containing 3) locus (odds ratio [OR] = 1.45, P = 5.0 x 10(-7)). A strong association was also identified at the CARS (cysteinyl-tRNA synthetase) locus (OR = 1.36, P = 3.1 x 10(-6)). Associations between both loci and time to onset of diabetic nephropathy were supported in the DCCT/EDIC study (hazard ratio [HR] = 1.33, P = 0.02, and HR = 1.32, P = 0.01, respectively). We demonstratedexpression of both FRMD3 and CARS in human kidney. CONCLUSIONS: We identified genetic associations for susceptibility to diabetic nephropathy at two novel candidate loci near the FRMD3 and CARS genes. Their identification implicates previously unsuspected pathways in the pathogenesis of this important late complication of type 1 diabetes.

A genome-wide linkage scan for genes controlling variation in renal function estimated by serum cystatin C levels in extended families with type 2 diabetes.

We performed a variance components linkage analysis of renal function, measured as glomerular filtration rate (GFR), in 63 extended families with multiple members with type 2 diabetes. GFR was estimated from serum concentrations of cystatin C and creatinine in 406 diabetic and 428 nondiabetic relatives. Results for cystatin C were summarized because they are superior to creatinine results. GFR aggregates in families with significant heritability (h(2)) in diabetic (h(2) = 0.45, P < 1 x 10(-5)) and nondiabetic (h(2) = 0.36, P < 1 x 10(-3)) relatives. Genetic correlation (r(G) = 0.35) between the GFR of diabetic and nondiabetic relatives was less than one (P = 0.01), suggesting that genes controlling GFR variation in these groups are different. Linkage results supported this interpretation. In diabetic relatives, linkage was strong on chromosome 2q (logarithm of odds [LOD] = 4.1) and suggestive on 10q (LOD = 3.1) and 18p (LOD = 2.2). In nondiabetic relatives, linkage was suggestive on 3q (LOD = 2.2) and 11p (LOD = 2.1). When diabetic and nondiabetic relatives were combined, strong evidence for linkage was found only on 7p (LOD = 4.0). In conclusion, partially distinct sets of genes control GFR variation in relatives with and without diabetes on chromosome 2q, possibly on 10q and 18p in the former, and on 7p in both. None of these genes overlaps with genes controlling variation in urinary albumin excretion.

No association of plasma prothrombin concentration or the G20210A mutation with incident cardiovascular disease: results from the Cardiovascular Health Study.

Prothrombin is a key factor in blood clotting, a process intimately involved in thrombotic disease. We assessed prothrombin levels and G20210A genotype in a case-control study within the Cardiovascular Health Study. Cases included angina, myocardial infarction, stroke, and the presence of MRI-detectable infarcts (n approximately 250 each). Population-based controls free of clinical cardiovascular disease (CVD) (n approximately 500) and a subset free of clinical and subclinical CVD (n approximately 250) were used for comparison. The 20210 A allele, frequency 2.9%, was associated with higher mean prothrombin levels: 166.3 vs. 139.5 microg/ml (P <0.001). Significant correlates of prothrombin included gender, plasma lipids, other vitamin K-dependent proteins, and inflammatory markers, but not race, smoking, hypertension, diabetes, measures of subclinical CVD, or markers of procoagulant activity. Compared to controls, neither genotype nor prothrombin level was associated with any CVD case group. We conclude that, in the elderly, neither prothrombin level nor 20210 genotype were associated with either CVD risk factors or events. This is consistent with the lack of association of prothrombin levels with measures of underlying CVD or procoagulant markers.