Hideki Inoue

Different Mechanisms for the Progression of CKD with ACE Gene Polymorphisms.

Background/Aims: The blockade of the renin-angiotensin-aldosterone system is the major target of efforts to prevent the progression of chronic kidney disease (CKD). Dual blockade with angiotensin-converting enzyme (ACE) inhibitor and angiotensin II receptor blocker has been reported to show additive renoprotection. However, three types of insertion/deletion (I/D) polymorphism have been reported, and it is unclear whether the dual blockade is effective for all the ACE genotypes. Methods: We treated 93 CKD patients with or without dual blockade and analyzed the effects on blood pressure (BP), proteinuria, progression of CKD and the relationship to I/D ACE polymorphisms. Results: After long-term medication (average 33 ± 2 months), BP decreased in all the genotype groups. However, urinary protein excretion decreased only in the II and DI groups (II: –27.1%, DI: –20.5%, DD: +0.8%). In the II and DI groups, amelioration of the progression of renal failure was correlated with reductions in BP and urinary protein excretion. However, the progression rate of renal failure was not correlated with proteinuria in the DD group. Conclusion: Proteinuria and BP are key factors for the progression of CKD in II/DI patients, while controlling the BP rather than reducing the proteinuria appears to be crucial in DD patients.

Angiotensin-converting enzyme inhibitor withdrawal and ACE gene polymorphism

AIMS Withdrawal of angiotensin-converting enzyme (ACE) inhibitors may affect the progression of chronic renal failure and an insertion/deletion (I/D) polymorphism of the ACE gene may influence it. METHODS We retrospectively collected patients with chronic glomerulonephritis and benign nephrosclerosis who discontinued ACE inhibitor use. The relationship between the decline of renal function after the withdrawal and the influencing factors such as ACE gene polymorphism, blood pressure and proteinuria were evaluated using multiple regression analysis. RESULTS Forty-two patients (initial serum creatinine 0.5 - 6.5 mg/dl) had been treated and discontinued ACE inhibitor use. Only patients with the II or DI genotypes of the ACE gene developed the deterioration of renal function, starting at 2 months after the withdrawal. Stepwise regression analysis revealed that the level of proteinuria after the withdrawal, presence of the insertion of ACE gene and serum creatinine level at the time of withdrawal mainly influenced the decline of renal function after the withdrawal (adjusted R2 = 0.48). CONCLUSION Withdrawal of ACE inhibitor causes the deterioration of renal function in patients with the II or DI genotypes, high proteinuria after the withdrawal, and high serum creatinine level at the withdrawal, which probably causes the rebound increase in serum ACE activity.

Low pH stimulates vasopressin V2 receptor promoter activity and enhances downregulation induced by V1a receptor stimulation

Arginine vasopressin (AVP) plays a key role in the urine concentration mechanism via the vasopressin V2 receptor (V2R) and aquaporin 2 (AQP2) in the kidney. It is well known that V2R is localized on the basolateral side and the V1a receptor (V1aR) is distributed on the luminal side of the collecting ducts. Previously, we reported an increase of V1aR mRNA and a decrease of V2R mRNA in the collecting ducts under chronic metabolic acidosis. However, the regulatory mechanism of V2R in acidic conditions has not yet been determined. In the present study, we investigated the effect of changes in pH on V2R promoter activity, using the LLC-PK(1) cell line stably expressing rat V1aR (LLC-PK(1)/rV1aR). The rV2R promoter activity was significantly increased at 12 h after the incubation in low-pH conditions, which was sustained for 24 h. mRNA and protein expressions of V2R were also increased in low-pH conditions. V1aR stimulation suppressed rV2R promoter activity in a pH-dependent manner. PKA and JNK inhibitors suppressed rV2R promoter activity in both neutral and low-pH conditions without FBS. However, a JNK inhibitor prevented the increase of V2R promoter activity only in low-pH conditions in the presence of FBS. In summary, V2R expression is increased at transcriptional, mRNA, and protein levels in LLC-PK(1)/rV1aR cells under low-pH conditions. Acidic condition-induced V2R enhancement was suppressed by V1aR stimulation, suggesting the crucial role of V1aR in water and electrolyte homeostasis in acidosis.

Long-term observation of renal function on combination therapy with prostaglandin and angiotensin-converting enzyme inhibitor for chronic kidney disease

AIMS Recently, we reported the effectiveness of PAC therapy, a combination therapy with prostaglandin (PG) and angiotensin-converting enzyme inhibitor (ACE-I), as a new tool for the prevention of chronic kidney disease. In the current study, we continually treated these patients with or without PG and analyzed the survival rate of renal function by Kaplan-Meier method and Cox regression analysis. MATERIAL AND METHODS 52 patients (serum creatinine 2.9 A+/- 1.9 mg/dl) were followed-up for 48 months. 26 patients continued to receive ACE-I monotherapy and the remaining 26 patients were treated by PAC therapy. Primary end-point was defined as a decrease in 1/Cr by 0.2 (dl/mg), initiation of renal replacement therapy or death. RESULTS At the end of the study, PAC therapy significantly reduced the risk for the decline in renal function compared to ACE-I monotherapy by 54%. Survival time was longer in PAC group (21.7 A+/- 2.2 and 35.1 A+/- 3.9 months, in ACE-I monotherapy and PAC therapy, p < 0.05). Cox regression analysis indicated that age, sex and blood pressure except urinary protein excretion did not relate to the risk reduction by PAC therapy. CONCLUSION PAC therapy was proved to reduce the progression of end-stage renal failure.

Low-responders to angiotensin II receptor blockers and genetic polymorphism in angiotensin-converting enzyme

AIMS The existence of low-responders to angiotensin II receptor blockers (ARBs) in terms of the preservation of renal function is reported here. We investigated the relationship between the responsiveness to ARBs and insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene. METHODS The effects of ARBs on proteinuria and the progression of chronic renal failure were examined in 113 patients with chronic kidney disease for 34 months before and 27 months after the addition of ARBs. RESULTS Although a decrease in blood pressure was seen in the II, DI and DD patient subgroups of the ACE gene, the decrease in proteinuria and the amelioration of loss of renal function were observed in the II and DI but not in the DD patients. Kaplan-Meier analysis was employed with a decrease of the reciprocal of serum creatinine of more than 0.2, the induction of renal replacement therapy or death as endpoints. The analysis comparing the periods before and after the addition of ARBs revealed the extension of time to an end-point by the addition of ARBs in all groups together (II + DI + DD), in Group II, and Group DI but not in the DD patient Group. CONCLUSIONS These data suggest that DD patients with ACE gene demonstrate diminished response to ARBs in terms of renoprotection and that ACE gene polymorphism needs to be taken into account when using ARBs as a means of renoprotective therapy.

Effects of Atrial Natriuretic Peptide on Bicarbonate Transport in Long- and Short-Looped Medullary Thick Ascending Limbs of Rats

Atrial natriuretic peptide (ANP) is known to influence NaCl transport in the medullary thick ascending limbs (MAL), where the largest NaCl reabsorption occurs among distal nephron segments in response to arginine vasopressin (AVP). In the present study, we investigated the effect of ANP on bicarbonate (HCO3 −) transport in the MAL using an isolated tubule perfusion technique. The HCO3 − concentration was measured using free-flow ultramicro-fluorometer. We first observed basal HCO3 − reabsorption in both long- and short-looped MALs (lMALs, and sMALs, respectively). AVP inhibited HCO3 − reabsorption in both lMALs and sMALs, whereas ANP did not change HCO3 − transport. However, in the presence of AVP, ANP restored the HCO3 − reabsorption inhibited by AVP both in lMAL and sMAL. The effects of ANP on HCO3 − transport was mimicked by cyclic GMP. The mRNA expression level of the vasopressin V2 receptor in lMALs was significantly higher than in sMALs, whereas expression of the V1a receptor was unchanged. In summary, AVP inhibits HCO3 − transport, and ANP counteracts the action of AVP on HCO3 − transport both in lMALs and sMALs.

The collagen-derived compound collagen tripeptide induces collagen expression and extends lifespan via a conserved p38 mitogen-activated protein kinase cascade

The skin consists mostly of extracellular matrix (ECM) composed mainly of collagen, which provides a protective barrier from the environment. The skin continuously experiences harmful stress and damage. As aging progresses, the expression of various genes declines, and physiological functional deterioration occurs. The reduction of collagen accompanying aging impairs the barrier function of the skin and weakens protection from stressors. In the nematode Caenorhabditis elegans, ECM proteins turn over during aging. Older worms of longevity mutants exhibit increased collagen expression, whereas knockdown of collagen genes shortens lifespan. However, it is unclear whether the progression of aging can be delayed by increasing collagen production via an external stimulus. In this study, we examined the effects of collagen tripeptide (CTP), a collagen-derived compound, on lifespan and aging. Our results showed that CTP upregulated collagen genes via the p38 mitogen-activated protein kinase (MAPK)/SKN-1 pathway. Moreover, CTP extended lifespan and delayed aging through p38 MAPK/SKN-1 pathway. In addition, CTP also induced collagen expression via the p38 MAPK pathway in mammals. Our findings supported that external stimuli such as CTP could promote ECM youthfulness using a conserved signaling pathway, thereby contributing to suppression of aging.

Fludrocortisone stimulates erythropoietin production in the intercalated cells of the collecting ducts

Erythropoietin has been thought to be secreted to plasma soon after the production because of the difficulty of Western blot analysis and immunohistochemistry. We established the new methods of Western blot analysis and immunohistochemistry. Using the new methods, we investigated the effects of aldosterone and fludrocortisone, an analogue of aldosterone on erythropoietin mRNA and protein production by the kidneys. Aldosterone stimulated Epo and HIF2α mRNA expressions in tubule suspensions and microdissected medullary thick ascending limbs and outer medullary collecting ducts. Western blot analysis showed a recombinant erythropoietin at 34-45 kDa and kidney erythropoietin at 36-40 and 42 kDa, both of which shifted to 22 kDa by deglycosylation. Erythropoietin protein expression was observed in the nephrons but not in the interstitial cells in control condition. Fludrocortisone stimulated erythropoietin mRNA and protein expressions in the distal nephrons, particularly in the intercalated cells of the collecting ducts. These data show that erythropoietin is produced by the nephrons by the regulation of renin-angiotensin-aldosterone system and not by the renal interstitial cells in control condition.

δ-Tocopherol promotes thermogenic gene expression via PGC-1α upregulation in 3T3-L1 cells

Activation of thermogenic adipocytes (brown and beige) has been considered an attractive target for weight loss and treatment of metabolic disease. Peroxisome proliferator-activated receptor γ co-activator-1 α (PGC1-α) is a master regulator of thermogenic gene expression in thermogenic adipocytes. We previously reported that α-tocopherol upregulated PGC-1α gene expression and promoted thermogenic adipocyte differentiation in mammalian adipocytes. In this study, we investigated the effects of the vitamin E analogs (α-, γ- and δ-tocopherol) on PGC-1α and uncoupling protein 1 (UCP1) gene expression in 3T3-L1 cells. The expression of PGC-1α and UCP1 increased significantly with the addition of δ-tocopherol. In δ-tocopherol-treated cells, nuclear translocation of PGC-1α increased, as did p38 mitogen-activated protein kinase (MAPK) expression and phosphorylation. Our results suggest that p38 MAPK activation by δ-tocopherol contributes to PGC-1α activation and UCP1 induction.

PS 04-04 COMPUTATIONAL ANALYSIS OF THE EFFECTS OF ALDOSTERONE AND METABOLIC ACIDOSIS ON GENE EXPRESSIONS IN RENAL TUBULES

Objective: Renin-angiotensin-aldosterone system (RAAS) blockers are the first-line agent to treat patients with chronic kidney disease (CKD). Although RAAS blockers slow CKD progression, they sometimes cause metabolic acidosis as an adverse effect, which possibly worsens CKD. We investigated metabolic acidosis-induced, but aldosterone-independent, kidney injury by discovering gene expressions in renal tubules. Design and Method: We searched publications and picked up known gene transcripts that are induced in epithelial cells in the kidney either by administration of aldosterone or acid loading. To discover aldosterone-stimulated gene transcripts, we examined transcriptional start site-sequencing using a rat intercalated cell line (IN-IC cells), and then added newly found aldosterone-stimulated gene transcripts to the gene lists. Using DAVID Bioinformatics Database, we examined Gene Ontology (GO) analysis for the gene transcripts. Using oPOSSUM, we analyzed promoter regions of the genes and estimated enriched consensus motifs in the promoters of the genes. Results: Among publications and our experiments, 64 and 105 genes were picked up for aldosterone- and acid-stimulated genes, respectively. Only 5 genes were overlapped between two stimulations. GO analysis indicated 5 common GO terms in molecular function and biological process between two stimulations such as regulation of blood pressure, ATPase activity, and ion transport. Acid-stimulated genes indicated specific GO terms such as oxidative stress and protein catabolic process. The promoter analysis raised 27 and 21 consensus motifs for aldosterone- and acid-induced genes, respectively (Z-score > 5.0). There were 4 motifs overlapped between two stimulations, which include the hormone response element, a target of mineralocorticoid receptor (MR). Specific motifs for acid-induced genes include the motifs for Ets and NFAT TF families, and they locate on the promoter region of ATP6AP2 (Prorenin receptor). Conclusions: The results suggest that metabolic acidosis induced by RAAS blockers could stimulate many aldosterone-independent genes in renal tubules, which may affect blood pressure and CKD progression.