Slide 1

Thank you Mr Chairman. First of all, I thank the organisers for offering me such an opportunity to talk about our recent data.

Slide 2

In recent years increasing evidence --- chronic kidney disease, CKD is a strong cardiovascular risk and therefore, the concept of cardio-renal association is well-recognised recognised. One possible factor that explains this link seems to be endothelial dysfunction. As you know, endothelial dysfunction plays an important role in both initiation and development of atherosclerosis.
In addition, accumulating evidence such as that endothelial dysfunction also could be involved in proteinuria and CKD progression. This endothelial dysfunction with reduced NO bioavailability is induced by increased oxidative stress, depletion of BH4, an essential co-factor for NO synthase, NOS and accumulated endogenous NOS inhibitor such as ADMA. Today I’ll talk about the role of ADMA in the pathogenesis of CKD.

Slide 3

Before talking about such a role of ADMA I will summarise the biology of ADMA briefly. This figure shows the structures of L-arginine, NO substrate and three kinds of dimethylarginines in vivo. L-NMMA, as well as ADMA works as a competitive inhibitor of L-arginine, thus inhibiting NO generation. Since there maybe a minor amount of L-NMMA, some ADMA and SDMA has no effect on NOS activity ADMA could be a major endogenous NOS inhibitor.

Slide 4

Plasma levels of ADMA were increased in patients with reduced NO bioavailability, such as hypertension, hypercholesterolemia, diabetes and CKD. Therefore, increased ADMA could be involved in endothelial dysfunction thus contributing to development of atherosclerosis and heart disease conditions.

Slide 5

Indeed, we previously reported that plasma levels of ADMA are significantly associated with intima-media thickness, a surrogate marker for atherosclerosis in human subjects.

Slide 6

The plasma levels of ADMA are particularly high in patients with ESRD and therefore, this maybe a reason for the high prevalence of cardiovascular disease in this population.

Slide 7

In fact, as shown in this slide Professor Zoccali clearly demonstrated that high levels of plasma levels of ADMA is related to high risks of cardiovascular events, as well as mortality in patients with ESRD.

Slide 8

This slide shows metabolism of ADMA, dimethylarginines such as ADMA and SDMA were derived from degradation product of methylated proteins which is post-transcriptionally produced by protein methyltransferase, PRMT. After proteolysis of methylated proteins 3 dimethylarginines are released from the cells. Only ADMA but not SDMA is metabolised into citrulline and dimethylarginines producing dimethylarginine dimethylaminohydrolase, DDAH. More than 80% of ADMA is degraded by the action of DDAH suggesting an important role of DDAH for regulating ADMA. Indeed, under high oxidative conditions such as diabetes, hypercholesterolemia and CKD, the enzymatic activity of DDAH is reduced thus contributing to AMDA elevation.

Slide 9

Next I’ll talk about the role of ADMA in the progression of CKD.

Slide 10

The possibility that ADMA is involved in CKD progression is suggested by two recent studies, both epidemiological studies revealed that high levels of plasma levels of ADMA consistently predicted a faster rate of renal function loss in patients with CKD.

Slide 11

However, the precise role of ADMA in the pathogenesis of renal dysfunction remains to be elucidated. There maybe two possible mechanisms by which ADMA could contribute to the progression of CKD. One is a blood pressure or a proteinuria dependent effect of ADMA and the other is a direct effect of ADMA on renal microvasculature.

Slide 12

– we previously demonstrated that plasma levels of ADMA were significantly associated with mean blood pressure levels in healthy human subjects.

Slide 13

In addition, a positive correlation between ADMA and blood pressure levels is also observed in animal models of CKD.

Slide 14

This observation suggests that ADMA could be a causal factor for hypertension in CKD. Moreover, recently a positive correlation between ADMA and proteinuria levels was also shown in patients with CKD stage I.

Slide 15

Hypertension and proteinuria are major determinants for CKD progression. So ADMA could be involved in CKD progression via a proteinuria or hypertension dependent effect.
In addition to these factors, there is evidence that ADMA is directly involved in CKD progression. This figure illustrates the microvasculature of the nephron. Peritubular capillary, PTC plexus is fed by afferent arterioles and supplies oxygen to tubular and interstitial cells.
Loss or dysfunction of peritubular capillaries may lead to chronic ischemia in tubulointerstitium and thereby, enhances tubular atrophy and renal scarring processes. This is known as chronic hypoxia hypothesis.

Slide 16

Indeed, severe PTC loss was observed in nephrectomy rats, as well as in patients with renal failure, suggesting that an essential role of PTC rose in progression of CKD.

Slide 17

Recently, Kang and his colleagues demonstrated that blockade of NO synthase by L-NAME induced more severe capillary rarefaction than that observed in nephrectomy alone and subsequently enhanced renal fibrosis in this model suggesting NO plays an important role in maintaining PTC and therefore, NO has a protective role against --- processes.

Slide 18

Therefore, we hypothesised that endogenously accumulated ADMA in CKD may cause PTC loss, thereby being involved in the progression of renal injury. To address this issue we investigated the relationship between ADMA and PTC loss in nephrectomy rats and we also investigated the effects of DDAH-elicited ADMA lowering on renal function and pathology.

Slide 19

Four weeks after nephrectomy, the rats were randomly divided into 4 groups; one group was immediately killed to evaluate the relationship between ADMA and PTC and the other rats were treated with tail vein injection of adenovirus encoding human DDAH 1 or control vector or treated with hydralazine which served as a blood pressure control model.

Slide 20

As you can see, plasma levels of ADMA are significantly related to capillary rarefaction and particularly of renal fibrosis.

Slide 21

So then we next investigated the effect of ADMA lowering by DDAH overexpression on these changes. Adenovirus infection efficiency were confirmed by X-gal staining of the liver. It revealed that almost all cells of the liver were positive for --- DDAH infection actually increased its mRNA levels, as well as the enzymatic activity in the liver and subsequently decreased plasma levels of ADMA.

Slide 22

This figure shows time course changes in blood pressure in this model. As you can see, a progressive increase in blood pressure levels was observed in control rats which was completely blocked by hydralazine treatment, as well as DDAH overexpression.

Slide 23

This figure shows the immunohistochemistry of PTC in control rats, DDAH overexpressing rats and hydralazine treatment rats. As you can see, progressive loss of PTC was observed in control or hydralazine treated rats which was significantly ameliorated by the DDAH overexpression.

Slide 24

This figure shows --- staining --- tubulointerstitial fibrosis. Progressive interstial fibrosis was also observed in control and hydralazine treated rats which was also blocked by DDAH overexpression.

Slide 25

This slide shows creatinine clearance in control and ---- progressive decrease in creatinine clearance values was observed. DDAH overexpression also brought this decrease.

Slide 26

We finally investigated the relationship between ADMA levels and proteinuria levels. As you can see, plasma levels of ADMA are significantly related to urinary protein excretion levels. In addition, DDAH overexpression significantly inhibited the increase in proteinuria in this model.

Slide 27

In this last slide I – an overall schema about the role of ADMA in cardio-renal association.
In CKD accumulated ADMA may be involved in endothelial dysfunction and hypertension thus contributing to the development of cardiovascular disease. ADMA induced endothelial dysfunction also plays an important role of CKD progression by way of PTC loss and proteinuria.
Countering ADMA by substitution of DDAH proteins or enhancement of its activity may become a novel therapeutic strategy for treatment of patients with CKD. Thank you so much for your attention.

Slide 28

Chairman: Thank you very much for this overview and this very new data regarding progression of renal disease and the role of ADMA. We have time for several questions. Yes, please can you ask your question?

Question: Savin, Milwaukee. Several of my colleagues have been looking at ADMA and nitric oxide in our isolated glomeruli and find that ADMA or other nitric oxide inhibitors actually cause the permeability barrier to break down and replacement of nitric oxide corrects this as does superoxide dismutase ---. So I’d like to add to your very nice presentation another hypothesis for a role of ADMA in proteinuria which is that by blocking nitric oxide it enhances the effects of endogenously made superoxide and other oxygen radicals. Thank you for a very nice presentation.

Dr Ueda: Thank you so much. As you mentioned, ADMA may decrease NO bioavailability, thus contributing to glomerular permeability of proteins. In this model it is not shown but glomerular capillary loss was also observed and DDAH overexpression also inhibited glomerular capillary loss. This structural change maybe induces proteinuria in this model.

Question: Thank you. In your interesting study that you’ve just published in the JASN you have compared the effect of DDAH to hydralazine right? Hydralazine is not the most nephro-protective agent. So did you have the opportunity to compare DDAH and either an ACE inhibitor or a blocker of the angiotensin II receptor?

Dr Ueda: Yes, we are trying the effect of an ACE inhibitor on ADMA DDAH axis now. I’d like to show the results in the near future.

Question: Ok thanks.

Chairman: Are there any questions? Well thank you very much if not we have to congratulate all the 3 speakers apart from me and the assistance for a lively discussion and presentation. Thank you very much.