Preanalytical considerations in quantifying circulating miRNAs that predict end-stage kidney disease in diabetes.

Our previous study identified 8 risk and 9 protective plasma miRNAs associated with progression to end-stage kidney disease (ESKD) in diabetes. This study aimed to elucidate preanalytical factors that influence the quantification of circulating miRNAs. Using the EdgeSeq platform, which quantifies 2,002 miRNAs in plasma, including ESKD-associated miRNAs, we compared miRNA profiles in whole plasma versus miRNA profiles in RNA extracted from the same plasma specimens. Less than half of the miRNAs were detected in standard RNA extraction from plasma. Detection of individual and concentrations of miRNAs were much lower when RNA extracted from plasma was quantified by RNA sequencing (RNA-Seq) or quantitative reverse transcription PCR (qRT-PCR) platforms compared with EdgeSeq. Plasma profiles of miRNAs determined by the EdgeSeq platform had excellent reproducibility in assessment and had no variation with age, sex, hemoglobin A1c, BMI, and cryostorage time. The risk ESKD-associated miRNAs were detected and measured accurately only in whole plasma and using the EdgeSeq platform. Protective ESKD-associated miRNAs were detected by all platforms except qRT-PCR; however, correlations among concentrations obtained with different platforms were weak or nonexistent. In conclusion, preanalytical factors have a profound effect on detection and quantification of circulating miRNAs in ESKD in diabetes. Quantification of miRNAs in whole plasma and using the EdgeSeq platform may be the preferable method to study profiles of circulating cell-free miRNAs associated with ESKD and possibly other diseases.

Integrated analysis of blood DNA methylation, genetic variants, circulating proteins, microRNAs, and kidney failure in type 1 diabetes.

Variation in DNA methylation (DNAmet) in white blood cells and other cells/tissues has been implicated in the etiology of progressive diabetic kidney disease (DKD). However, the specific mechanisms linking DNAmet variation in blood cells with risk of kidney failure (KF) and utility of measuring blood cell DNAmet in personalized medicine are not clear. We measured blood cell DNAmet in 277 individuals with type 1 diabetes and DKD using Illumina EPIC arrays; 51% of the cohort developed KF during 7 to 20 years of follow-up. Our epigenome-wide analysis identified DNAmet at 17 CpGs (5'-cytosine-phosphate-guanine-3' loci) associated with risk of KF independent of major clinical risk factors. DNAmet at these KF-associated CpGs remained stable over a median period of 4.7 years. Furthermore, DNAmet variations at seven KF-associated CpGs were strongly associated with multiple genetic variants at seven genomic regions, suggesting a strong genetic influence on DNAmet. The effects of DNAmet variations at the KF-associated CpGs on risk of KF were partially mediated by multiple KF-associated circulating proteins and KF-associated circulating miRNAs. A prediction model for risk of KF was developed by adding blood cell DNAmet at eight selected KF-associated CpGs to the clinical model. This updated model significantly improved prediction performance (c-statistic = 0.93) versus the clinical model (c-statistic = 0.85) at P = 6.62 × 10-14. In conclusion, our multiomics study provides insights into mechanisms through which variation of DNAmet may affect KF development and shows that blood cell DNAmet at certain CpGs can improve risk prediction for KF in T1D.

Neuroblastoma suppressor of tumorigenicity 1 is a circulating protein associated with progression to end-stage kidney disease in diabetes.

Circulating proteins associated with transforming growth factor-ß (TGF-ß) signaling are implicated in the development of diabetic kidney disease (DKD). It remains to be comprehensively examined which of these proteins are involved in the pathogenesis of DKD and its progression to end-stage kidney disease (ESKD) in humans. Using the SOMAscan proteomic platform, we measured concentrations of 25 TGF-ß signaling family proteins in four different cohorts composed in total of 754 Caucasian or Pima Indian individuals with type 1 or type 2 diabetes. Of these 25 circulating proteins, we identified neuroblastoma suppressor of tumorigenicity 1 (NBL1, aliases DAN and DAND1), a small secreted protein known to inhibit members of the bone morphogenic protein family, to be most strongly and independently associated with progression to ESKD during 10-year follow-up in all cohorts. The extent of damage to podocytes and other glomerular structures measured morphometrically in 105 research kidney biopsies correlated strongly with circulating NBL1 concentrations. Also, in vitro exposure to NBL1 induced apoptosis in podocytes. In conclusion, circulating NBL1 may be involved in the disease process underlying progression to ESKD, and its concentration in circulation may identify subjects with diabetes at increased risk of progression to ESKD.

A dominant negative ADIPOQ mutation in a diabetic family with renal disease, hypoadiponectinemia, and hyperceramidemia.

Adiponectin, encoded by ADIPOQ, is an insulin-sensitizing, anti-inflammatory, and renoprotective adipokine that activates receptors with intrinsic ceramidase activity. We identified a family harboring a 10-nucleotide deletion mutation in ADIPOQ that cosegregates with diabetes and end-stage renal disease. This mutation introduces a frameshift in exon 3, resulting in a premature termination codon that disrupts translation of adiponectin's globular domain. Subjects with the mutation had dramatically reduced circulating adiponectin and increased long-chain ceramides levels. Functional studies suggest that the mutated protein acts as a dominant negative through its interaction with non-mutated adiponectin, decreasing circulating adiponectin levels, and correlating with metabolic disease.

Results of untargeted analysis using the SOMAscan proteomics platform indicates novel associations of circulating proteins with risk of progression to kidney failure in diabetes.

This study applies a large proteomics panel to search for new circulating biomarkers associated with progression to kidney failure in individuals with diabetic kidney disease. Four independent cohorts encompassing 754 individuals with type 1 and type 2 diabetes and early and late diabetic kidney disease were followed to ascertain progression to kidney failure. During ten years of follow-up, 227 of 754 individuals progressed to kidney failure. Using the SOMAscan proteomics platform, we measured baseline concentration of 1129 circulating proteins. In our previous publications, we analyzed 334 of these proteins that were members of specific candidate pathways involved in diabetic kidney disease and found 35 proteins strongly associated with risk of progression to kidney failure. Here, we examined the remaining 795 proteins using an untargeted approach. Of these remaining proteins, 11 were significantly associated with progression to kidney failure. Biological processes previously reported for these proteins were related to neuron development (DLL1, MATN2, NRX1B, KLK8, RTN4R and ROR1) and were implicated in the development of kidney fibrosis (LAYN, DLL1, MAPK11, MATN2, endostatin, and ROR1) in cellular and animal studies. Specific mechanisms that underlie involvement of these proteins in progression of diabetic kidney disease must be further investigated to assess their value as targets for kidney-protective therapies. Using multivariable LASSO regression analysis, five proteins (LAYN, ESAM, DLL1, MAPK11 and endostatin) were found independently associated with risk of progression to kidney failure. Thus, our study identified proteins that may be considered as new candidate prognostic biomarkers to predict risk of progression to kidney failure in diabetic kidney disease. Furthermore, three of these proteins (DLL1, ESAM, and MAPK11) were selected as candidate biomarkers when all SOMAscan results were evaluated.

Association of Coding Variants in Hydroxysteroid 17-beta Dehydrogenase 14 (HSD17B14) with Reduced Progression to End Stage Kidney Disease in Type 1 Diabetes.

BACKGROUND: Rare variants in gene coding regions likely have a greater impact on disease-related phenotypes than common variants through disruption of their encoded protein. We searched for rare variants associated with onset of ESKD in individuals with type 1 diabetes at advanced kidney disease stage. METHODS: Gene-based exome array analyses of 15,449 genes in five large incidence cohorts of individuals with type 1 diabetes and proteinuria were analyzed for survival time to ESKD, testing the top gene in a sixth cohort (n=2372/1115 events all cohorts) and replicating in two retrospective case-control studies (n=1072 cases, 752 controls). Deep resequencing of the top associated gene in five cohorts confirmed the findings. We performed immunohistochemistry and gene expression experiments in human control and diseased cells, and in mouse ischemia reperfusion and aristolochic acid nephropathy models. RESULTS: Protein coding variants in the hydroxysteroid 17-ß dehydrogenase 14 gene (HSD17B14), predicted to affect protein structure, had a net protective effect against development of ESKD at exome-wide significance (n=4196; P value=3.3 × 10-7). The HSD17B14 gene and encoded enzyme were robustly expressed in healthy human kidney, maximally in proximal tubular cells. Paradoxically, gene and protein expression were attenuated in human diabetic proximal tubules and in mouse kidney injury models. Expressed HSD17B14 gene and protein levels remained low without recovery after 21 days in a murine ischemic reperfusion injury model. Decreased gene expression was found in other CKD-associated renal pathologies. CONCLUSIONS: HSD17B14 gene is mechanistically involved in diabetic kidney disease. The encoded sex steroid enzyme is a druggable target, potentially opening a new avenue for therapeutic development.

Circulating proteins protect against renal decline and progression to end-stage renal disease in patients with diabetes.

Diabetic kidney disease (DKD) and its major clinical manifestation, progressive renal decline that leads to end-stage renal disease (ESRD), are a major health burden for individuals with diabetes. The disease process that underlies progressive renal decline comprises factors that increase risk as well as factors that protect against this outcome. Using untargeted proteomic profiling of circulating proteins from individuals in two independent cohorts with type 1 and type 2 diabetes and varying stages of DKD followed for 7 to 15 years, we identified three elevated plasma proteins-fibroblast growth factor 20 (OR, 0.69; 95% CI, 0.54 to 0.88), angiopoietin-1 (OR, 0.72; 95% CI, 0.57 to 0.91), and tumor necrosis factor ligand superfamily member 12 (OR, 0.75; 95% CI, 0.59 to 0.95)-that were associated with protection against progressive renal decline and progression to ESRD. The combined effect of these three protective proteins was demonstrated by very low cumulative risk of ESRD in those who had baseline concentrations above median for all three proteins, whereas the cumulative risk of ESRD was high in those with concentrations below median for these proteins at the beginning of follow-up. This protective effect was shown to be independent from circulating inflammatory proteins and clinical covariates and was confirmed in a third cohort of diabetic individuals with normal renal function. These three protective proteins may serve as biomarkers to stratify diabetic individuals according to risk of progression to ESRD and might also be investigated as potential therapeutics to delay or prevent the onset of ESRD.

Comprehensive Search for Novel Circulating miRNAs and Axon Guidance Pathway Proteins Associated with Risk of ESKD in Diabetes.

BACKGROUND: Mechanisms underlying the pro gression of diabetic kidney disease to ESKD are not fully understood. METHODS: We performed global microRNA (miRNA) analysis on plasma from two cohorts consisting of 375 individuals with type 1 and type 2 diabetes with late diabetic kidney disease, and targeted proteomics analysis on plasma from four cohorts consisting of 746 individuals with late and early diabetic kidney disease. We examined structural lesions in kidney biopsy specimens from the 105 individuals with early diabetic kidney disease. Human umbilical vein endothelial cells were used to assess the effects of miRNA mimics or inhibitors on regulation of candidate proteins. RESULTS: In the late diabetic kidney disease cohorts, we identified 17 circulating miRNAs, represented by four exemplars (miR-1287-5p, miR-197-5p, miR-339-5p, and miR-328-3p), that were strongly associated with 10-year risk of ESKD. These miRNAs targeted proteins in the axon guidance pathway. Circulating levels of six of these proteins-most notably, EFNA4 and EPHA2-were strongly associated with 10-year risk of ESKD in all cohorts. Furthermore, circulating levels of these proteins correlated with severity of structural lesions in kidney biopsy specimens. In contrast, expression levels of genes encoding these proteins had no apparent effects on the lesions. In in vitro experiments, mimics of miR-1287-5p and miR-197-5p and inhibitors of miR-339-5p and miR-328-3p upregulated concentrations of EPHA2 in either cell lysate, supernatant, or both. CONCLUSIONS: This study reveals novel mechanisms involved in progression to ESKD and points to the importance of systemic factors in the development of diabetic kidney disease. Some circulating miRNAs and axon guidance pathway proteins represent potential targets for new therapies to prevent and treat this condition.

Effect of TNFα stimulation on expression of kidney risk inflammatory proteins in human umbilical vein endothelial cells cultured in hyperglycemia.

We recently identified a kidney risk inflammatory signature (KRIS), comprising 6 TNF receptors (including TNFR1 and TNFR2) and 11 inflammatory proteins. Elevated levels of these proteins in circulation were strongly associated with risk of the development of end-stage kidney disease (ESKD) during 10-year follow-up. It has been hypothesized that elevated levels of these proteins in circulation might reflect (be markers of) systemic exposure to TNFα. In this in vitro study, we examined intracellular and extracellular levels of these proteins in human umbilical vein endothelial cells (HUVECs) exposed to TNFα in the presence of hyperglycemia. KRIS proteins as well as 1300 other proteins were measured using the SOMAscan proteomics platform. Four KRIS proteins (including TNFR1) were down-regulated and only 1 protein (IL18R1) was up-regulated in the extracellular fraction of TNFα-stimulated HUVECs. In the intracellular fraction, one KRIS protein was down-regulated (CCL14) and 1 protein was up-regulated (IL18R1). The levels of other KRIS proteins were not affected by exposure to TNFα. HUVECs exposed to a hyperglycemic and inflammatory environment also showed significant up-regulation of a distinct set of 53 proteins (mainly in extracellular fraction). In our previous study, circulating levels of these proteins were not associated with progression to ESKD in diabetes.

A profile of multiple circulating tumor necrosis factor receptors associated with early progressive kidney decline in Type 1 Diabetes is similar to profiles in autoimmune disorders.

This study comprehensively evaluated the association between known circulating tumor necrosis factor (TNF) superfamily ligands and receptors and the development of early progressive kidney decline (PKD) leading to end-stage kidney disease (ESKD) in Type 1 diabetes. Participants for the study were from the Macro-Albuminuria Study (198 individuals), and the Micro-Albuminuria Study (148 individuals) of the Joslin Kidney Study. All individuals initially had normal kidney function and were followed for seven-fifteen years to determine the slope of the estimate glomerular filtration rate and to ascertain onset of ESKD. Plasma concentrations of 25 TNF superfamily proteins were measured using proximity extension assay applied in the OLINK proteomics platform. In the both studies risk of early PKD, determined as estimated glomerular filtration rate loss greater than or equal to three ml/min/1.73m2/year, was associated with elevated circulating levels of 13 of 19 TNF receptors examined. In the Macro-Albuminuria Study, we obtained similar findings for risk of progression to ESKD. These receptors comprised: TNF-R1A, -R1B, -R3, -R4, -R6, -R6B, -R7, -R10A, -R10B, -R11A, -R14, -R21, and -R27. Serial measurements showed that circulating levels of these TNF receptors had increased before the onset of PKD. In contrast, none of the six measured TNF ligands showed association with risk of early PKD. Of significance, the disease process that underlies PKD leading to ESKD in Type 1 diabetes has a profile also seen in autoimmune disorders. The mechanisms of this enrichment may be causally related to the development of PKD in Type 1 diabetes and must be investigated further. Thus, some of these receptors may be used as new risk predictors of ESKD.

Profibrotic Circulating Proteins and Risk of Early Progressive Renal Decline in Patients With Type 2 Diabetes With and Without Albuminuria.

OBJECTIVE: The role of fibrosis in early progressive renal decline in type 2 diabetes is unknown. Circulating WFDC2 (WAP four-disulfide core domain protein 2) and matrix metalloproteinase 7 (MMP-7; Matrilysin) are postulated to be biomarkers of renal fibrosis. This study examined an association of circulating levels of these proteins with early progressive renal decline. RESEARCH DESIGN AND METHODS: Individuals with type 2 diabetes enrolled in the Joslin Kidney Study with an estimated glomerular filtration rate (eGFR) ≥60 mL/min/1.73 m2 were monitored for 6-12 years to ascertain fast early progressive renal decline, defined as eGFR loss ≥5 mL/min/1.73 m2/year. RESULTS: A total of 1,181 individuals were studied: 681 without and 500 with albuminuria. Median eGFR and albumin-to-creatinine ratio (ACR) at baseline were 97 mL/min/1.73 m2 and 24 mg/g, respectively. During follow-up, 152 individuals experienced fast early progressive renal decline: 6.9% in those with normoalbuminuria and 21% with albuminuria. In both subgroups, the risk of renal decline increased with increasing baseline levels of WFDC2 (P < 0.0001) and MMP-7 (P < 0.0001). After adjustment for relevant clinical characteristics and known biomarkers, an increase by one quartile in the fibrosis index (combination of levels of WFDC2 and MMP-7) was associated with higher risk of renal decline (odds ratio 1.63; 95% CI 1.30-2.04). The association was similar and statistically significant among patients with and without albuminuria. CONCLUSIONS: Elevation of circulating profibrotic proteins is associated with the development of early progressive renal decline in type 2 diabetes. This association is independent from albuminuria status and points to the importance of the fibrotic process in the development of early renal decline.

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.

New Frontiers: Approaches to Understand the Mechanistic Basis of Renal Toxicity.

A scientific session entitled "New Frontiers: Approaches to Understand the Mechanistic Basis of Renal Toxicity" focused on novel biomarkers to monitor kidney injury both preclinically and clinically, as well as providing mechanistic insight of the induced injury. Further, the role and impact of kidney membrane transporters in drug-induced kidney toxicity provided additional considerations when understanding kidney injury and the complex role of drug transporters in either sensitivity or resistance to drug-induced injury. The onset of nephropathy in diabetic patients was also presented, focusing on the quest to discover novel biomarkers that would differentiate diabetic populations more susceptible to nephropathy and renal failure. The session highlighted exciting new research areas and novel biomarkers that will enhance our understanding of kidney injury and provide tools for ensuring patient safety clinically.

Circulating miRNA Profiles Associated With Hyperglycemia in Patients With Type 1 Diabetes.

We investigated plasma microRNA (miRNA) profiles associated with variation of hyperglycemia, measured as hemoglobin A1c (HbA1c), in two panels of patients with type 1 diabetes (T1D). Using the HTG Molecular Diagnostics EdgeSeq platform, 2,083 miRNAs were measured in plasma from 71 patients included in a screening panel. Quantitative real-time PCR was used to measure the candidate miRNAs in plasma from 95 patients included in an independent replication panel. We found 10 miRNAs replicated in both panels and 4 with high statistical significance. The strongest positive correlations with HbA1c were found with miR-125b-5p (rs = 0.40, P = 6.0 × 10-5) and miR-365a-3p (rs = 0.35, P = 5.9 × 10-4). The strongest negative correlations were found with miR-5190 (rs = -0.30, P = 0.003) and miR-770-5p (rs = -0.27, P = 0.008). Pathway analysis revealed that 50 Kyoto Encyclopedia of Genes and Genomes pathways were significantly enriched by genes targeted by these four miRNAs. The axon guidance signaling pathway was enriched (P < 1 × 10-7) by genes targeted by all four miRNAs. In addition, three other pathways (Rap1 signaling, focal adhesion, and neurotrophin signaling) were also significantly enriched but with genes targeted by only by three of the identified miRNAs. In conclusion, our study identified four circulating miRNAs that were influenced by variation in hyperglycemia. Dysregulation of these miRNAs, which are associated with hyperglycemia in patients with T1D, may contribute to the development of diabetes complications. However, there are multitudes of possible mechanisms/pathways through which dysregulation of these miRNAs may impact risk of diabetes complications.

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.

Fast renal decline to end-stage renal disease: an unrecognized feature of nephropathy in diabetes.

A new model of diabetic nephropathy in type 1 diabetes emerged from our studies of Joslin Clinic patients. The dominant feature is progressive renal decline, not albuminuria. This decline is a unidirectional process commencing while patients have normal renal function and, in the majority, progressing steadily (linearly) to end-stage renal disease (ESRD). While an individual's rate of renal decline is constant, the estimated glomerular filtration rate (eGFR) slope varies widely among individuals from -72 to -3.0 ml/min/year. Kidney Disease: Improving Global Outcomes guidelines define rapid progression as rate of eGFR declines > 5 ml/min/year, a value exceeded by 80% of patients in Joslin's type 1 diabetes ESRD cohort. The extraordinary range of slopes within the rapid progression category prompted us to partition it into "very fast," "fast" and "moderate" decline. We showed, for the first time, that very fast and fast decline from normal eGFR to ESRD within 2 to 10 years constitutes 50% of the Joslin cohort. In this review we present data about frequency of fast decliners in both diabetes types, survey some mechanisms underlying fast renal decline, discuss methods of identifying patients at risk and comment on the need for effective therapeutic interventions. Whether the initiating mechanism of fast renal decline affects glomerulus, tubule, interstitium or vasculature is unknown. Since no animal model mimics progressive renal decline, studies in humans are needed. Prospective studies searching for markers predictive of the rate of renal decline yield findings that may make detection of fast decliners feasible. Identifying such patients will be the foundation for developing effective individualized methods to prevent or delay onset of ESRD in diabetes.

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.