Yusuke Sakamaki

Renal tubular Sirt1 attenuates diabetic albuminuria by epigenetically suppressing Claudin-1 overexpression in podocytes

Sirtuin 1 (Sirt1), a NAD+-regulated deacetylase with numerous known positive effects on cellular and whole-body metabolism, is expressed in the renal cortex and medulla. It is known to have protective effects against age-related disease, including diabetes. Here we investigated the protective role of Sirt1 in diabetic renal damage. We found that Sirt1 in proximal tubules (PTs) was downregulated before albuminuria occurred in streptozotocin-induced or obese (db/db) diabetic mice. PT-specific SIRT1 transgenic and Sirt1 knockout mice showed prevention and aggravation of the glomerular changes that occur in diabetes, respectively, and nondiabetic knockout mice exhibited albuminuria, suggesting that Sirt1 in PTs affects glomerular function. Downregulation of Sirt1 and upregulation of the tight junction protein Claudin-1 by SIRT1-mediated epigenetic regulation in podocytes contributed to albuminuria. We did not observe these phenomena in 5/6 nephrectomized mice. We also demonstrated retrograde interplay from PTs to glomeruli using nicotinamide mononucleotide (NMN) from conditioned medium, measurement of the autofluorescence of photoactivatable NMN and injection of fluorescence-labeled NMN. In human subjects with diabetes, the levels of SIRT1 and Claudin-1 were correlated with proteinuria levels. These results suggest that Sirt1 in PTs protects against albuminuria in diabetes by maintaining NMN concentrations around glomeruli, thus influencing podocyte function.

KLF4-dependent epigenetic remodeling modulates podocyte phenotypes and attenuates proteinuria

The transcription factor Kruppel-like factor 4 (KLF4) has the ability, along with other factors, to reprogram somatic cells into induced pluripotent stem (iPS) cells. Here, we determined that KLF4 is expressed in kidney glomerular podocytes and is decreased in both animal models and humans exhibiting a proteinuric. Transient restoration of KLF4 expression in podocytes of diseased glomeruli in vivo, either by gene transfer or transgenic expression, resulted in a sustained increase in nephrin expression and a decrease in albuminuria. In mice harboring podocyte-specific deletion of Klf4, adriamycin-induced proteinuria was substantially exacerbated, although these animals displayed minimal phenotypical changes prior to adriamycin administration. KLF4 overexpression in cultured human podocytes increased expression of nephrin and other epithelial markers and reduced mesenchymal gene expression. DNA methylation profiling and bisulfite genomic sequencing revealed that KLF4 expression reduced methylation at the nephrin promoter and the promoters of other epithelial markers; however, methylation was increased at the promoters of genes encoding mesenchymal markers, suggesting selective epigenetic regulation of podocyte gene expression. Together, these results suggest that KLF4 epigenetically modulates podocyte phenotype and function and that the podocyte epigenome can be targeted for direct intervention and reduction of proteinuria.

Developmental activity of the renin-angiotensin system during the "critical period" modulates later L-NAME-induced hypertension and renal injury.

The incidence of hypertension and hypertensive renal disease is increasing worldwide, and new strategies to prevent these diseases need to be investigated. The aims of this study were 1) to examine if transient exposure to an angiotensin receptor blocker (ARB) during an early period in hypertension development confers protection against subsequent worsening of hypertension and renal injury induced by the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), and 2) conversely, to examine the effects of transient exposure to angiotensin II (Ang II) during the same period. First, spontaneously hypertensive rats (SHR) were treated transiently from age 3 to 10 weeks with an ARB (candesartan cilexetil), a calcium channel antagonist or a vasodilator, then taken off treatment for 2 months. Administration of L-NAME at age 18 weeks caused severe hypertension and renal injury. However, the rats that had been exposed to the ARB not only had a lower blood pressure, but also failed to show signs of renal injury or increase of oxidative stress. Furthermore, the elevation of components of the renin-angiotensin-aldosterone system was also suppressed in these rats. In the second study, Wistar-Kyoto rats (WKY) and SHR were exposed to Ang II from age 4 to 8 weeks. The follow-up showed that the blood pressures in the WKY remained elevated compared to controls, while the SHR had heightened increases in blood pressure, renal renin mRNA, and urinary 8-hyroxydeoxyguanosine after L-NAME administration. Together, these experiments demonstrate that transient treatment of rats during an early phase in the development of hypertension with an ARB suppresses the renin-angiotensin-aldosterone system and confers long-term protection against subsequent L-NAME–induced renal injury and increases in renal oxidative stress. Conversely, developmental exposure to Ang II during this “critical” period had the opposite effect, predisposing rats to higher blood pressure, renal injury, and oxidative stress after L-NAME administration.

Regression of glomerulosclerosis in response to transient treatment with angiotensin II blockers is attenuated by blockade of matrix metalloproteinase-2

Understanding mechanisms that contribute to the regression of glomerulosclerosis is important for developing new strategies to treat chronic kidney disease. We reported that transient high-dose treatment with an angiotensin receptor blocker causes regression of renal arteriolar hypertrophy and hypertension in spontaneously hypertensive rats. To extend those findings to another form of kidney disease, we examined the short- and long-term effects of transient high-dose angiotensin receptor blocker treatment in a mouse model of adriamycin-induced glomerulosclerosis. A 2-week course of candesartan caused a dose-dependent regression of established glomerulosclerotic lesions sustained for over 6 months following cessation of treatment. Highly sensitive in situ zymography and activity assays showed that glomerular matrix metalloproteinase (MMP)-2 activity was increased after high-dose angiotensin blocker therapy. Treatment of cultured podocytes with candesartan resulted in an increase in MMP-2 activity. The regression of glomerulosclerosis was partially attenuated in mice pretreated with the MMP inhibitor doxycycline, as well as in MMP-2 knockout mice. Our results suggest that transient high-dose angiotensin receptor blocker treatment effectively induced sustained regression of glomerulosclerosis by a mechanism mediated, in part, by changes in MMP-2 activity.

“Pulse” Treatment With High-Dose Angiotensin Blocker Reverses Renal Arteriolar Hypertrophy and Regresses Hypertension

One ultimate goal of hypertension therapy is to cause permanent reversal (“regression”) of already established hypertension. Our aim was to examine whether high-dose “pulse” treatment with a renin-angiotensin system inhibitor could cause regression of established hypertension and to link this action to reversal of arteriolar hypertrophy and changes in vascular matrix metalloproteinase activities. First, 16-week-old male spontaneously hypertensive rats (n=60) were pulse treated for 2 weeks with high-dose angiotensin-converting enzyme inhibitor (enalapril), angiotensin receptor blocker (candesartan), calcium channel blocker (nifedipine), or vasodilator (hydralazine) with or without salt restriction, and the long-term effects on blood pressure were examined. Second, spontaneously hypertensive rats were treated with angiotensin receptor blocker or calcium channel blocker, and the effects on renal gene expressions, arteriolar structure, and vascular matrix metalloproteinase were compared. Treatment of spontaneously hypertensive rats with different antihypertensive agents caused apparently similar reductions in blood pressure during the course of the pulse treatment, within the limitations of the tail-cuff method. After cessation of medications, blood pressure in the rats treated with renin-angiotensin system inhibitor remained reduced by >30 to 40 mm Hg for 4 months. No such effect was seen with calcium channel blocker or vasodilator. The 2-week angiotensin receptor blocker treatment induced a marked reversal of the arteriolar hypertrophy specifically in the small (30 to 100 &mgr;m) renal arterioles, together with increased expression and activity of matrix metalloproteinase-13. In conclusion, transient high-dose pulse treatment with angiotensin receptor blocker caused changes in vascular matrix metalloproteinase activity, specific reversal of renal arteriolar hypertrophy, and regression of hypertension in spontaneously hypertensive rats.

Renin-angiotensin blockade resets podocyte epigenome through Kruppel-like Factor 4 and attenuates proteinuria

Proteinuria is a central component of chronic kidney disease and an independent risk factor for cardiovascular disease. Kidney podocytes have an essential role as a filtration barrier against proteinuria. Kruppel-like Factor 4 (KLF4) is expressed in podocytes and decreased in glomerular diseases leading to methylation of the nephrin promoter, decreased nephrin expression and proteinuria. Treatment with an angiotensin receptor blocker (ARB) reduced methylation of the nephrin promoter in murine glomeruli of an adriamycin nephropathy model with recovery of KLF4 expression and a decrease in albuminuria. In podocyte-specific KLF4 knockout mice, the effect of ARB on albuminuria and the nephrin promoter methylation was attenuated. In cultured human podocytes, angiotensin II reduced KLF4 expression and caused methylation of the nephrin promoter with decreased nephrin expression. In patients, nephrin promoter methylation was increased in proteinuric kidney diseases with decreased KLF4 and nephrin expression. KLF4 expression in ARB-treated patients was higher in patients with than without ARB treatment. Thus, angiotensin II can modulate epigenetic regulation in podocytes and ARB inhibits these actions in part via KLF4 in proteinuric kidney diseases. This study provides a new concept that renin-angiotensin system blockade can exert therapeutic effects through epigenetic modulation of the kidney gene expression.

Absence of Gelatinase (MMP-9) or Collagenase (MMP-13) Attenuates Adriamycin-Induced Albuminuria and Glomerulosclerosis

Background/Aims: The role of matrix metalloproteinases (MMPs) in the pathogenesis of glomerular injury appears to be complex. To investigate the role of individual MMPs, we examined the course of Adriamycin-induced albuminuria and glomerulosclerosis in mice lacking either a gelatinase (MMP-9) or a collagenase (MMP-13). Methods: Adriamycin was administered to MMP-9 or MMP-13 knockout (KO) mice. Glomerular injury was assessed by the quantification of albuminuria, the glomerular injury score and type IV collagen immunostaining. Results: Treatment of mice with Adriamycin (18 mg/kg i.v.) resulted in marked albuminuria and glomerulosclerosis reaching a peak at 4–8 weeks. The albuminuria and glomerulosclerosis were significantly (p < 0.05) attenuated in both the MMP-9 KO and MMP-13 KO mice compared to controls. In contrast, treatment of wild-type mice with the broad-spectrum MMP inhibitor doxycycline did not have a beneficial effect on the albuminuria and glomerulosclerosis. Conclusion: These results support a role for both gelatinase (MMP-9) and collagenase (MMP-13) in the pathogenesis of glomerular injury in the Adriamycin-induced glomerulosclerosis model. MMP inhibitors with high specificity towards MMP-9 and/or MMP-13 may be potential future candidates to provide more effective therapies to inhibit the development of glomerulosclerosis.

Comparison of health costs associated with treatment of hypertension with a calcium channel blocker and angiotensin-converting enzyme inhibitor in the United States and Japan

Hypertension is prevalent in over 25% of populations in developed countries, and poses an increasing economic burden on health resources. Therefore it is predicted that future medical treatment of hypertension will be increasingly affected by cost considerations. Several classes of antihypertensive drugs are used as first-line agents for the treatment of hypertension, but the economic impact of using these agents in different countries remains to be addressed. In this study, we compared health costs associated with treatment of hypertension using the calcium channel blocker amlodipine and the angiotensin-converting enzyme inhibitor enalapril in the US and Japan. Pharmaceutical costs and hospitalization costs were analyzed from established databases. The data for the prevalence of myocardial infarction and stroke were derived from the Framingham study and the Hisayama study. Analysis of the economic impacts using relative risk differences between the calcium channel blocker and angiotensin-converting enzyme inhibitor together with regional hospitalization costs resulted in an apparent 11.2 billion yen health cost reduction in favor of the calcium channel blocker in the case of Japan, in contrast to an apparent 5.7 billion yen reduction in favor of the angiotensin-converting enzyme inhibitor in the case of the US. The trends in Japan for 2000 and 2004 were similar. These results suggest that there are regional differences in the health costs associated with different classes of hypertensive agents, which could affect national policies on the choice of antihypertensive drugs. It is predicted that future treatment of hypertension will be increasingly tailored to the epidemiologic profiles and medical costs of individual communities.

Differential Effects of Transient Treatment of Spontaneously Hypertensive Rats with Various Antihypertensive Agents on the Subsequent Development of Diabetic Nephropathy

Background/Aims: We have previously shown that treatment of spontaneously hypertensive rats (SHR) with an angiotensin receptor blocker (ARB) during the ‘critical period’ from age 3 to 10 weeks confers protection against L-NAME-induced renal injury later in life. The aim of this study was to examine the effects of transient prepubertal exposure to ARB on the development of nephropathy in streptozotocin-induced diabetic SHR and to compare the results with other antihypertensive agents including a mineralocorticoid receptor antagonist (MR-ant). Methods: Male SHR (n = 43) were transiently treated with candesartan (ARB), potassium canreonate (the active metabolite of the MR-ant spironolactone) or hydralazine (vasodilator) between 3 and 10 weeks of age with untreated rats serving as controls. An additional group was treated continuously with candesartan throughout the study. Rats were injected with streptozotocin to induce diabetes at age 16 weeks and followed until age 8 months. Results: Diabetic control rats showed signs of diabetic nephropathy including albuminuria and mesangial expansion. These changes were significantly suppressed in rats exposed to ARB or MR-ant. Systolic blood pressure was significantly reduced compared to controls in the ARB (transient) and ARB (sustained) groups, but not in the MR-ant or vasodilator groups. Conclusion: Transient prepubertal exposure to ARB or MR-ant, but not vasodilator, confers protection against the later development of diabetic nephropathy and involves blood pressure-independent protective mechanisms.

Renal thrombotic microangiopathy in a patient with septic disseminated intravascular coagulation

BackgroundThe mechanism for the development of thrombotic microangiopathy (TMA) during sepsis has only been partially elucidated. TMA is recognized as a disease caused by various factors, and may be involved in the emergence of organ damage in severe sepsis. Here we report a case of TMA that followed disseminated intravascular coagulation (DIC) due to severe infection in a patient with a reduced ADAMTS-13 activity level.Case presentationAn 86-year-old Japanese woman was admitted to our hospital because of low back pain and fever. A careful evaluation led to a diagnosis of acute obstructive pyelonephritis due to a ureteral stone. Proteus mirabilis was isolated from both blood and urine cultures. The patient developed systemic inflammatory response syndrome and DIC, and was treated with antibiotics and daily continuous hemodiafiltration. Although infection and the coagulation abnormalities due to DIC were successfully controlled, renal failure persisted and her consciousness level deteriorated progressively in association with severe thrombocytopenia and microangiopathic hemolytic anemia. We therefore suspected the presence of TMA and started plasma exchange, which resulted in an impressive improvement in consciousness as well as the laboratory abnormalities. The ADAMTS-13 activity was 44% and the patient tested negative for the ADAMTS-13 inhibitor prior to the initiation of plasma exchange. A renal biopsy was performed to determine the etiology of acute renal injury, which revealed findings that were interpreted to be compatible with the sequelae of TMA. The follow-up studies performed after the successful treatment of TMA showed that her plasma ADAMTS-13 activity level remained persistently low. It is surmised that septic DIC occurring in the presence of preexisting reduced ADAMTS-13 activity have led to the development of secondary TMA in the present case.ConclusionThe present case suggests that TMA can be superimposed on sepsis-induced DIC, and plasma exchange is expected to be beneficial in such situations. Clinicians should consider the possibility of secondary TMA that follows sepsis-induced DIC in certain indicative clinical settings.