Measurement of succinate in urine samples as a biomarker of kidney damage in diabetic subjects
The present invention relates in general to the discovery of urinary succinate as a novel biomarker of kidney disease. More specifically, the invention provides for the measurement of succinate in urine samples that has great potential for the easy and early diagnosis of kidney damage and would allow early prediction of kidney disease and therapeutic intervention.
1. A non-invasive method for early diagnosis of potential kidney damage due to renin-angiotensin system activation in a diabetic subject, comprising:
obtaining, non-invasively, a test urine sample from said diabetic subject;
pre-treating said test urine sample with perchloric acid followed by KOH to neutralize the sample;
determining a succinate level in said test urine sample by an enzymatic assay that oxidizes a stoichiometric amount of NADH corresponding to the level of succinate in the urine sample;
comparing the succinate level in said test urine sample with a succinate level in a control sample, wherein said control sample is either from healthy subjects or diabetic subjects without kidney damage; and
determining a diagnosis, wherein a diagnosis of kidney damage due to renin-angiotensin system activation is determined if:
(1) the control sample is from healthy subjects and the succinate level in said test urine sample is at least four-fold higher than the succinate level in the control sample, or
(2) the control sample is from diabetic subjects without kidney damage and the succinate level in said test urine sample is at least two-fold higher than the succinate level in the control sample.
2. The method according to claim 1 , wherein the control sample is from diabetic subjects without kidney damage, and a diagnosis of kidney damage due to renin-angiotensin system activation is determined when the succinate level in said test urine sample is at least two-fold higher than the succinate level in the control sample.
3. A method for early diagnosis of kidney damage due to renin-angiotensin system activation in a subject suffering from diabetes, comprising:
obtaining a test urine sample from said subject;
pre-treating the test urine sample with perchloric acid followed by KOH to neutralize the sample;
determining a succinate level in said test urine sample by an enzymatic assay that oxidizes a stoichiometric amount of NADH corresponding to the succinate level;
comparing the succinate level in said test urine sample with a succinate level in a corresponding healthy non-diabetic control sample; and
determining a diagnosis, wherein a diagnosis of likely kidney damage due to renin-angiotensin activation in said subject is determined if the succinate level in said test urine sample is at least 10 times higher than the succinate level in the corresponding control sample.
4. The method according to claim 3 , wherein said corresponding control sample is urine from healthy subjects.
5. A non-invasive method for early detection of kidney damage due to renin-angiotensin system activation in a diabetic subject, comprising:
obtaining a test urine sample from said diabetic subject;
pre-treating said test urine sample with perchloric acid followed by KOH to neutralize the acid treated sample;
determining a succinate/creatinine ratio in said test urine sample, wherein said determining step comprising performing an enzymatic assay that oxidizes a stoichiometric amount of NADH corresponding to the succinate level in the test urine sample;
comparing said succinate/creatinine ratio in said test urine sample with a succinate/creatinine ratio in a control sample, wherein said control sample is from a healthy subject or a diabetic subject without kidney damage; and
making a detection determination, wherein a detection of kidney damage due to renin-angiotensin system activation is determined if:
(1) the succinate/creatinine ratio in said test urine sample is at least four-fold higher than in the control sample from healthy subjects, or
(2) the succinate/creatinine ratio in said test urine sample is at least two-fold higher than in the control sample from diabetic subjects without kidney damage.
6. A method for early detection of kidney disease due to renin-angiotensin system activation in a diabetic subject comprising:
obtaining a test urine sample from said diabetic subject;
pre-treating said test urine sample with perchloric acid followed by KOH to neutralize the sample;
determining a succinate/creatinine ratio in said test urine sample, said determining step comprising performing an enzymatic assay that oxidizes NADH in a stoichiometric amount corresponding to the succinate level in the test urine sample;
comparing the succinate/creatinine ratio in the test urine sample with a succinate/creatinine ratio in a control sample, wherein said control sample is from a healthy subject; and
making a detection determination, wherein detection of kidney disease in the diabetic subject is determined if said succinate/creatinine ratio in the test urine sample is at least four-fold higher than the succinate/creatinine ratio in the control sample.
7. The method according to claim 5 , wherein said control sample is urine from diabetic subjects without kidney damage.