Discovery of new biomarker for diabetic nephropathy

Research shows that analysis of the levels of the renal protein angiotensinogen in urine can provide an earlier indication of the appearance of kidney damage in diabetics.

By Karina Toledo

Agência FAPESP – A new study has revealed that analysis of the levels of a protein known as angiotensinogen, produced in the kidneys and detected in the urine, may enable the earlier detection of diabetic nephropathy, one of the most serious complications of diabetes.

Due to changes in the renal blood vessels, the disease causes the kidneys to lose their ability to adequately filter the blood and thus allows important urinary proteins to escape into the body. If untreated, this condition can lead to chronic kidney insufficiency.

Currently, a diagnosis is made by analyzing albumin levels in the urine. However, when this protein is detected in tests, it means that the kidney tissue has already been damaged.

“We believe that analysis of renal angiotensinogen in the urine can help to identify the problem at an earlier stage, while there is still time to reverse the damage,” said Ovidiu Constantin Baltatu, a professor at Camilo Castelo Branco University (Unicastelo) and coordinator of the research funded by FAPESP.

Preclinical tests conducted on rats benefited from a partnership between researchers from the Max Delbrück Center for Molecular Medicine in Germany and the Institute of Biomedical Sciences (ICB) at the University of São Paulo (USP). The group is currently looking for new partners to conduct the clinical testing required to characterize and validate the new biomarker.

According to Baltatu, the project’s initial objective was to investigate whether diabetes affects men and women differently. “Gender studies are fairly recent, appearing over the last 10 or 15 years, and their focus is on coming up with personalized treatments,” said the Romanian-born researcher.

The line of research began when Baltatu was living in Berlin, Germany, and studying gender differences related to hypertensive cardiomyopathy and nephropathy at the Max Delbrück Center.

“We were able to demonstrate that the male hormones, or androgens, stimulate activity in the renin-angiotensin system (the set of peptides, enzymes and receptors involved in controlling blood pressure) and contribute to the development of hypertension and, consequently, hypertensive cardiomyopathy and nephropathy,” said Baltatu.

The findings – reported in articles published in the journals Hypertension and Journal of The American Society of Nephrology – raised the question of whether the same would occur in the case of nephropathy caused by diabetes.

To confirm this suspicion, in experiments conducted in Brazil, the scientists induced a condition similar to type 1 (insulin-dependent) diabetes in rats by injecting them with the antibiotic streptozotocin.

Streptozotocin causes the destruction of cells that are responsible for producing insulin in the pancreas, and in only a few days, the animals present a sustained increase in the levels of glucose in their blood. Twelve weeks after injection, the researchers were already able to detect the presence of albumin in the rodents’ urine.

The animals were separated into six groups: control males (who received no injection to induce diabetes), diabetic males, diabetic males treated with flutamide (an antiandrogen drug), control females, diabetic females and diabetic females treated with flutamide.

“One of the first differences that we noted was that the albuminuria levels were much higher in the males than in the females, a sign that the disease was progressing more quickly in the males,” Baltatu said.

However, in contrast to what had been observed in the study on hypertensive nephropathy, flutamide was able to protect only the males from disease progression, and not the females. “This shows that there are different mechanisms behind the development of hypertensive nephropathy and diabetic nephropathy,” he said.

Gene expression

The next step was to analyze blood pressure and the circulating levels of the renin-angiotensin system enzymes and their precursor protein: angiotensinogen.

“Angiotensinogen is converted to angiotensin-I through the action of the enzyme renin. Angiotensin-I is then converted into angiotensin-II – one of the strongest vasoconstrictor substances ever described – by the action of angiotensin-converting enzyme,” explained Baltatu.

In the hypertension study, the group had observed that androgens had elevated the levels of circulating renin. In the case of the diabetes, however, there are often low levels of plasma renin, which this was confirmed in the diabetic rat groups.

“However, beyond this circulating renin-angiotensin or endocrine system, there are also local systems in each organ. We extracted the renal tissue of the rats to analyze gene expression to see how local enzymes were being produced. We noted that in the males, renal angiotensinogen synthesis was significantly increased,” explained Baltatu.

In comparing the renal angiotensinogen levels with the albuminuria levels of the rats, the researchers established that there was a strong correlation.

“Our hypothesis is that the increased production of angiotensinogen in the kidneys leads to a higher local level of angiotensin-II, and this leads to nephropathy and explains the increase in albuminuria. We think that renal angiotensinogen, however, can reveal diabetic nephropathy before high levels of albumin appear in tests,” said the Unicastelo researcher.

Although the correlation between angiotensinogen and albumin levels has only been established in male rats, Baltatu speculates that the biomarker may be effective in diagnosing men as well as women.

“It is possible that the correlation was not noted in females because they already had low levels of albuminuria. We need to perform another study – which could already be a clinical test – to study women who have a higher level of albuminuria (a more advanced stage of the disease). The objective will be to clarify whether the angiotensinogen cut-off amounts for diagnosing nephropathy would be the same for men and women,” said Baltatu.