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
URINARY BIOMARKERS AT 4 AND 10 WEEKS
GENERAL STUDY DESIGN
INTRODUCTION
4 weeks
Diabetes is a global epidemic that is associated with increased risk of
10 weeks
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
ST
Z
Biomarkers
Biomarkers
4 weeks
and treatment with agents blocking the renin‐angiotensin system is
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
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
associated with slower progression of disease (3-5). Once established,
DKD leads to progressive renal failure and the need for renal
DOCA
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
Biomarkers
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
Characteristics of Sham (CTL), Diabetic (STZ), and Hypertensive Rats (DOCA)
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
CTL 4 wks
STZ 4 wks
DOCA 4 wks
STZ 10 wks
Weight (g)
337.3 ± 5.8
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**
KIM-1, NAG, N-gal and netrin-1 are accepted biomarkers representing
Urine Flow (mL)
15.5 ± 0.7
153.6 ± 20.9* 114.3 ± 13.0*
17.7 ± 1.1
200.8 ± 15.1**
acute renal tubular injury in human and animal models (11-16), but these
CrCl (ml/min)
X
X
0.62 ± 0.1**
1.39 ± 0.2
1.82 ± 0.3
biomarkers have not been validated in human or in animal models of
U protein (mg)
25.1 ± 7.7
29.0 ± 7.6
315.3 ± 51.8*
32.3 ± 6.4
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.
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
308.5 ± 13.1* 271.0 ± 8.0*
CTL 10 wks
CHANGE INBIOMARKER EXCRETION DIABETIC vs CONTROL
399.9 ± 9.4 319.6 ± 12.8**
Values are normalized to baseline values. Data expressed as fold increase over
baseline ± SEM
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
CHANGE IN BIOMARKER EXCRETION HYPERTENSIVE RATS VS CONTROL
injury, the DOCA salt model.
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.
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.
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
the role of kidney tubular injury biomarkers in animal models of chronic
SUMMARY AND CONCLUSIONS
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
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