METHODS

1. URINARY BIOMARKERS IN EXPERIMENTAL DIABETES FRANKLIN FUENMAYOR, MD1; GANESAN RAMESH, PhD1;DAVID M POLLOCK, PhD2; JENNIFER S POLLOCK, PhD2;JOHN J WHITE, MD1,2 1MEDICINE, SECTION OF NEPHROLOGY & 2MEDICINE, SECTION EXPERIMENTAL MEDICINE GEORGIA HEALTH SCIENCES UNIVERSITY INTRODUCTION GENERAL STUDY DESIGN SUMMARY AND CONCLUSIONS URINARY BIOMARKERS AT 4 AND 10 WEEKS Diabetes is a global epidemic that is associated with increased risk of cardiovascular disease, kidney disease, and premature death (1). Diabetic kidney disease (DKD) is the leading cause of end‐stage renal disease in the United States and its incidence is increasing (2). Currently available therapies for DKD are limited. Early detection of DKD and treatment with agents blocking the renin‐angiotensin system is associated with slower progression of disease (3-5). Once established, DKD leads to progressive renal failure and the need for renal replacement therapy (6). Currently, our only established marker for DKD is an increase in urinary albumin excretion, or microalbumin, which is thought to represent early glomerular damage. Although the focus has largely been on the glomerulus, DKD is also associated with tubulointerstitial injury which may precede apparent glomerulopathy (7). Recently, urinary tubular biomarkers useful in detecting acute kidney injury (AKI) have garnered interest in chronic renal diseases (8,9). Higher levels of NGAL and KIM-1 were associated with a significant and greater decline in kidney function, but not after adjustment for other known progression factors. KIM-1, NAG, N-gal and netrin-1 are accepted biomarkers representing acute renal tubular injury in human and animal models (11-16), but these biomarkers have not been validated in human or in animal models of chronic kidney disease. Likewise, the role of tubulointerstitial injury in DKD has not been adequately addressed. In order to develop therapies aimed at the tubulointerstitial damage in DKD, validated animal models are urgently needed. Therefore, the purpose of this study is to evaluate the role of kidney tubular injury biomarkers in animal models of chronic disease. Specifically, we measured urinary levels of NAG, KIM-1 and N-gal in a rat model of type 1 diabetes (Streptozotocin) and compared with a model of hypertension known to develop significant tubulointerstitial injury, the DOCA salt model. 4 weeks 10 weeks 24 hour urinary excretion of NAG, KIM-1, N-gal, and netrin-1 were measured at 4 weeks in all groups and at 10 weeks in CTL and STZ rats. At 4 weeks, levels of NAG, N-gal, and netrin-1 are significantly elevated in STZ rats compared to CTL. After 10 weeks, all measured biomarkers are elevated in STZ rats compared to CTL. NAG and Netrin-1 appeared to be the most sensitive early markers based on their fold-increase compared to baseline values (Figure 2). NAG and KIM-1 exhibited a progressive increase over time whereas N-gal and KIM-1 levels were similar at 4 and 10 weeks. Likewise, NAG, KIM-1, N-gal, and netrin-1 are all increased in DOCA rats to a similar degree compared to baseline at 4 weeks . There was strong correlation between urinary N-gal and protein excretion (r2 = 0.663 p < 0.0001). This is the first study to evaluate the role of multiple urinary tubular biomarkers in an experimental model of type 1 diabetes. In our study, levels of all biomarkers measured were significantly elevated at 10 weeks duration. More importantly, NAG, N-gal, and netrin-1 were elevated early prior to the development of significant proteinuria. These results likely represent early tubulointerstitial injury not yet seen histopathologically. Our findings support the hypothesis that tubulointerstitial injury occurs early in the course of diabetic nephropathy and may occur before the onset of glomerular injury. These findings highlight the fact that our only established marker of diabetic nephropathy, albuminuria, may represent tubular rather than glomerular damage as the majority of filtered albumin is reabsorbed by healthy proximal tubules. Future studies should extend to other models of diabetic nephropathy better suited to establishing mechanism of renal tubular injury. Understanding the mechanisms and establishing the role of tubular injury biomarkers in diabetic nephropathy will be key in the development of therapies targeting tubulointerstial injury and hopefully slowing the progression of the most common cause of end-stage kidney disease in the US. STZ Biomarkers Biomarkers 4 weeks Total (24 hour) excretion and spot values corrected per mg of urinary creatinine. *P < 0.05 compared to 4 week CTL; **P < 0.05 compared to 10 week CTL DOCA Biomarkers CHANGE INBIOMARKER EXCRETION DIABETIC vs CONTROL Characteristics of Sham (CTL), Diabetic (STZ), and Hypertensive Rats (DOCA) • REFERENCES Excretory data were derived from 24-h urine collections in metabolic cages within 24h of sacrifice. Plasma was obtained under anesthesia immediately prior to sacrifice. Values are means ± SEM; *P < 0.05 compared to 4 weeks CTL; **P < 0.05 compared to 10 weeks CTL • 1. Bruno G, Landi A. Epidemiology and costs of diabetes. Transplant Proc. 2011;43:327‐9. • 2. U S Renal Data System, USRDS 2010 Annual Data Report: Atlas of Chronic Kidney Disease and End‐Stage Renal Disease in the United States, National Institutes of Health, National Institute of Diabetes and • Digestive and Kidney Diseases, Bethesda, MD, 2010. • 3. 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Regression of microalbuminuria in type 1 diabetes is associated with lower levels of urinary tubular injury biomarkers, kidney injury molecule-1, and N-acetyl-beta-D-glucosaminidase. Kidney Int 2011; 79: 464–470. • Nielsen SE, Andersen S, Zdunek D et al. Tubular markers do not predict the decline in glomerular filtration rate in type 1 diabetic patients with overt nephropathy. Kidney Int. 2011;79:1113-8. • 10. Fassett RG, Venuthurupalli SK, Gobe GC et al. Biomarkers in chronic kidney disease: a review. Kidney Int. 2011 doi:10.1038/ki.2011.198 [Epub ahead of print]. • 11. Wang W, Reeves WB, Ramesh G. Netrin-1 an kidney injury. Netrin-1 protects against ischemia-reperfusion injury of the kidney. Am J Physiol Renal Physiol. 2008;294:F739-47. • 14. Reeves WB, Kwon O, Ramesh G. Netrin-1 and kidney injury. II. Netrin-1 is an early biomarker of acute kidney injury. Am J Physiol Renal Physiol. 2008;294:F731-8. • 15. Basnakian AG. Netrin-1: a potential universal biomarker for acute kidney injury. Am J Physiol Renal Physiol. 2008;294:F729-30. • 16. Ramesh G, Krawczeski CD, Woo JG et al. Urinary netrin-1 is an early predictive biomarker of acute kidney injury after cardiac surgery. Clin J Am Soc Nephrol. 2010;5:395-401. • 15. Vaidya VS, Ozer JS, Dieterle F et al. Kidney injury molecule-1 outperforms traditional biomarkers of kidney injury in preclinical biomarker qualification studies. Nat Biotechnol. 2010;28:478-85. Values are normalized to baseline values. Data expressed as fold increase over baseline ± SEM Change in Biomarker Excretion Hypertensive Rats vs Control METHODS SD rats were made diabetic by i.v. administration of streptozotocin (STZ) (65 mg/kg) (n = 7). Controls (CTL) received normal saline (n = 9). Rats were studied for 10 weeks. Rats were placed in metabolic cages at baseline, 4 weeks, and 10 weeks. Urinary kidney injury molecule-1 (KIM-1) and N-acetyl--D-glucosaminidase (NAG) were measured by ELISA. For a positive control, we used the DOCA salt model, which is known to develop significant tubulointerstitial injury. These rats underwent uninephrectomy and were implanted with a 200 mg time-released DOCA pellet and given normal saline as drinking water (n = 7) x 4 weeks. Systolic blood pressure in conscious rats during the 10 week course of study. Values are means ± SEM at 2 week time intervals. *P < 0.05 compared to CTL at the same time period Values are normalized to baseline values. Data expressed as fold increase over baseline ± SEM