METHODS

URINARY BIOMARKERS IN EXPERIMENTAL DIABETESFRANKLIN 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 SUMMARY AND CONCLUSIONS

REFERENCES

URINARY BIOMARKERS AT 4 AND 10 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

GENERAL STUDY DESIGN

STZ

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

CHANGE INBIOMARKER EXCRETION DIABETIC vs CONTROL

Values are normalized to baseline values. Data expressed as fold increase over baseline ± SEM

CHANGE IN BIOMARKER EXCRETION HYPERTENSIVE RATS VS CONTROL

Values are normalized to baseline values. Data expressed as fold increase over baseline ± SEM

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

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

CTL 4 wks STZ 4 wks DOCA 4 wks CTL 10 wks STZ 10 wks

Weight (g) 337.3 ± 5.8 308.5 ± 13.1* 271.0 ± 8.0* 399.9 ± 9.4 319.6 ± 12.8**

Glucose 99 ± 4.0 456 ± 29.5* X 96 ± 5.1 485 ± 22.7**

Food intake (g) 25.6 ± 0.8 39.1 ± 2.3* 16.0 ± 0.6* 22.3 ± 0.6 45.5 ± 1.9**

H2O in (mL) 40.1 ± 1.7 165.5 ± 19.3* 126.2 ± 14.6* 35.1 ± 0.8 221.1 ± 13.7**

Urine Flow (mL) 15.5 ± 0.7 153.6 ± 20.9* 114.3 ± 13.0* 17.7 ± 1.1 200.8 ± 15.1**

CrCl (ml/min) X X 0.62 ± 0.1** 1.39 ± 0.2 1.82 ± 0.3

U protein (mg) 25.1 ± 7.7 29.0 ± 7.6 315.3 ± 51.8* 32.3 ± 6.4 77.5 ± 13.4**

4 weeks 10 weeks   CTL STZ DOCA CTL STZ

KIM-1 pg/24h 102 141 ± 8 158 ± 22 906 ± 88 129 ± 3 179 ± 16KIM-1 pg/mg Cr 102 8.7 ± 0.49 11 ± 1.8 116 ± 21 6.6 ± 0.26 8.2 ± 0.21

NAG IU/24h 337 ± 25 3167 ± 473 2298 ± 210 366 ± 28 4042 ± 353NAG IU/mg Cr 21.0 ± 2.1 212 ± 45 306 ± 73 18.5 ± 1.2 176 ± 38

N-gal pg/24h 104 142 ± 18 409 ± 72 830 ± 146 164 ± 16 443 ± 60N-gal pg/mg Cr 103 44 ± 6 141 ± 35 559 ± 156 41 ± 3 90 ± 16

10 weeks

DOCA

4 weeks

Biomarkers Biomarkers

Biomarkers

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.

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.

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.

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