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
GENERAL STUDY DESIGN
4 weeks 10 weeks
KIM-1 pg/24h 102 KIM-1 pg/mg Cr 102 NAG IU/24h NAG IU/mg Cr N-gal pg/24h 104 N-gal pg/mg Cr 103

INTRODUCTION
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 endstage 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 reninangiotensin 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
Weight (g) Glucose Food intake (g) H2O in (mL) Urine Flow (mL) CrCl (ml/min) U protein (mg)

URINARY BIOMARKERS AT 4 AND 10 WEEKS

4 weeks CTL 141 8 8.7 0.49 337 25 21.0 2.1 142 18 44 6 STZ 158 22 11 1.8 3167 473 212 45 409 72 141 35 DOCA 906 88 116 21 2298 210 306 73 830 146 559 156 10 weeks CTL 129 3 6.6 0.26 366 28 18.5 1.2 164 16 41 3 STZ 179 16 8.2 0.21 4042 353 176 38 443 60 90 16

SUMMARY AND CONCLUSIONS

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.

ST Z

Biomarkers 4 weeks

Biomarkers

DOCA

Biomarkers

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

Characteristics of Sham (CTL), Diabetic (STZ), and Hypertensive Rats (DOCA)

CTL 4 wks 337.3 5.8 99 4.0 25.6 0.8 40.1 1.7 15.5 0.7 X 25.1 7.7 STZ 4 wks DOCA 4 wks CTL 10 wks STZ 10 wks

CHANGE INBIOMARKER EXCRETION DIABETIC vs CONTROL

308.5 13.1* 271.0 8.0* 456 29.5* 39.1 2.3* X 16.0 0.6*

399.9 9.4 319.6 12.8** 96 5.1 22.3 0.6 35.1 0.8 17.7 1.1 1.39 0.2 32.3 6.4 485 22.7** 45.5 1.9** 221.1 13.7** 200.8 15.1** 1.82 0.3 77.5 13.4**

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 Ngal 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.

165.5 19.3* 126.2 14.6* 153.6 20.9* 114.3 13.0* X 29.0 7.6 0.62 0.1** 315.3 51.8*

REFERENCES
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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 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