Broad-acting therapeutic effects of miR-29b-chitosan on hypertension and diabetic complications.

MicroRNA miR-29 promotes endothelial function in human arterioles in part by targeting LYPLA1 and increasing nitric oxide production. In addition, miR-29 is a master inhibitor of extracellular matrix gene expression, which may attenuate fibrosis but could also weaken tissue structure. The goal of this study was to test whether miR-29 could be developed as an effective, broad-acting, and safe therapeutic. Substantial accumulation of miR-29b and effective knockdown of Lypla1 in several mouse tissues were achieved using a chitosan-packaged, chemically modified miR-29b mimic (miR-29b-CH-NP) injected systemically at 200 µg/kg body weight. miR-29b-CH-NP, injected once every 3 days, significantly attenuated angiotensin II-induced hypertension. In db/db mice, miR-29b-CH-NP treatment for 12 weeks decreased cardiac and renal fibrosis and urinary albuminuria. In uninephrectomized db/db mice, miR-29b-CH-NP treatment for 20 weeks significantly improved myocardial performance index and attenuated proteinuria. miR-29b-CH-NP did not worsen abdominal aortic aneurysm in ApoE knockout mice treated with angiotensin II. miR-29b-CH-NP caused aortic root fibrotic cap thinning in ApoE knockout mice fed a high-cholesterol and high-fat diet but did not worsen the necrotic zone or mortality. In conclusion, systemic delivery of low-dose miR-29b-CH-NP is an effective therapeutic for several forms of cardiovascular and renal disease in mice.

Endogenous miR-204 Protects the Kidney against Chronic Injury in Hypertension and Diabetes.

BACKGROUND: Aberrant microRNA (miRNA) expression affects biologic processes and downstream genes that are crucial to CKD initiation or progression. The miRNA miR-204-5p is highly expressed in the kidney but whether miR-204-5p plays any role in the development of chronic renal injury is unknown. METHODS: We used real-time PCR to determine levels of miR-204 in human kidney biopsies and animal models. We generated Mir204 knockout mice and used locked nucleic acid-modified anti-miR to knock down miR-204-5p in mice and rats. We used a number of physiologic, histologic, and molecular techniques to analyze the potential role of miR-204-5p in three models of renal injury. RESULTS: Kidneys of patients with hypertension, hypertensive nephrosclerosis, or diabetic nephropathy exhibited a significant decrease in miR-204-5p compared with controls. Dahl salt-sensitive rats displayed lower levels of renal miR-204-5p compared with partially protected congenic SS.13BN26 rats. Administering anti-miR-204-5p to SS.13BN26 rats exacerbated interlobular artery thickening and renal interstitial fibrosis. In a mouse model of hypertensive renal injury induced by uninephrectomy, angiotensin II, and a high-salt diet, Mir204 gene knockout significantly exacerbated albuminuria, renal interstitial fibrosis, and interlobular artery thickening, despite attenuation of hypertension. In diabetic db/db mice, administering anti-miR-204-5p exacerbated albuminuria and cortical fibrosis without influencing blood glucose levels. In all three models, inhibiting miR-204-5p or deleting Mir204 led to upregulation of protein tyrosine phosphatase SHP2, a target gene of miR-204-5p, and increased phosphorylation of signal transducer and activator of transcription 3, or STAT3, which is an injury-promoting effector of SHP2. CONCLUSIONS: These findings indicate that the highly expressed miR-204-5p plays a prominent role in safeguarding the kidneys against common causes of chronic renal injury.

Fumarase Overexpression Abolishes Hypertension Attributable to endothelial NO synthase Haploinsufficiency in Dahl Salt-Sensitive Rats.

Human blood pressure salt sensitivity is associated with changes in urinary metabolites related to fumarase (Fh) and nitric oxide (NO) metabolism, and fumarase promotes NO production through an arginine regeneration pathway. We examined the role of the fumarase-NO pathway in the development of hypertension using genetically engineered rat models. Dahl salt-sensitive (SS) rats with heterozygous mutation of eNOS (endothelial NO synthase or Nos3; SS-Nos3+/-) were bred with SS rats with a hemizygous Fh transgene. SS-Nos3+/- rats without the Fh transgene (SS-Nos3+/-/Fh0/0) developed substantial hypertension with a mean arterial pressure of 134.2±3.7 mm Hg on a 0.4% NaCl diet and 178.0±3.5 mm Hg after 14 days on a 4% NaCl diet. Mean arterial pressure decreased remarkably to 123.1±1.4 mm Hg on 0.4% NaCl, and 143.3±1.5 mm Hg on 4% NaCl in SS-Nos3+/- rats with a Fh transgene (SS-Nos3+/-/Fh0/1), and proteinuria, renal fibrosis, and tubular casts were attenuated in SS-Nos3+/-/Fh0/1 rats compared with SS-Nos3+/-/Fh0/0 rats. eNOS protein abundance decreased in rats with the Nos3 heterozygous mutation, which was not influenced by Fh overexpression in rats on the 0.4% NaCl diet. However, the decrease in NO metabolite in the renal outer medulla of SS-Nos3+/-/Fh0/0 rats on the 0.4% NaCl diet was reversed in SS-Nos3+/-/Fh0/1 rats, and levels of L-arginine, but not the other 12 amino acids analyzed, were significantly higher in SS-Nos3+/-/Fh0/1 rats than in SS-Nos3+/+/Fh0/0 rats. In conclusion, fumarase has potent effects in restoring NO production and blunting the development of hypertension attributable to eNOS haploinsufficiency.

MiR-192-5p in the Kidney Protects Against the Development of Hypertension.

MicroRNA miR-192-5p is one of the most abundant microRNAs in the kidney and targets the mRNA for ATP1B1 (ß1 subunit of Na+/K+-ATPase). Na+/K+-ATPase drives renal tubular reabsorption. We hypothesized that miR-192-5p in the kidney would protect against the development of hypertension. We found miR-192-5p levels were significantly lower in kidney biopsy specimens from patients with hypertension (n=8) or hypertensive nephrosclerosis (n=32) compared with levels in controls (n=10). Similarly, Dahl salt-sensitive (SS) rats showed a reduced abundance of miR-192-5p in the renal cortex compared with congenic SS.13BN26 rats that had reduced salt sensitivity (n=9; P<0.05). Treatment with anti-miR-192-5p delivered through renal artery injection in uninephrectomized SS.13BN26 rats exacerbated hypertension significantly. Mean arterial pressure on a 4% NaCl high-salt diet at day 14 post anti-miR-192-5p treatment was 16 mm Hg higher than in rats treated with scrambled anti-miR (n=8 and 6; P<0.05). Similarly, Mir192 knockout mice on the high-salt diet treated with Ang II (angiotensin II) for 14 days exhibited a mean arterial pressure 22 mm Hg higher than wild-type mice (n=9 and 5; P<0.05). Furthermore, protein levels of ATP1B1 were higher in Dahl SS rats than in SS.13BN26 rats. Na+/K+-ATPase activity increased in the renal cortex of SS.13BN26 rats 9 days posttreatment with anti-miR-192-5p compared with that of control anti-miR treated rats. Intrarenal knockdown of ATP1B1 attenuated hypertension in SS.13BN26 rats with intrarenal knockdown of miR-192-5p. In conclusion, miR-192-5p in the kidney protects against the development of hypertension, which is mediated, at least in part, by targeting Atp1b1.

Role of DNA De Novo (De)Methylation in the Kidney in Salt-Induced Hypertension.

Numerous adult diseases involving tissues consisting primarily of nondividing cells are associated with changes in DNA methylation. It suggests a pathophysiological role for de novo methylation or demethylation of DNA, which is catalyzed by DNA methyltransferase 3 and ten-eleven translocases. However, the contribution of DNA de novo (de)methylation to these diseases remains almost completely unproven. Broad changes in DNA methylation occurred within days in the renal outer medulla of Dahl SS rats fed a high-salt diet, a classic model of hypertension. Intrarenal administration of anti-DNA methyltransferase 3a/ten-eleven translocase 3 GapmeRs attenuated high salt-induced hypertension in SS rats. The high-salt diet induced differential expression of 1712 genes in the renal outer medulla. Remarkably, the differential expression of 76% of these genes was prevented by anti-DNA methyltransferase 3a/ten-eleven translocase 3 GapmeRs. The genes differentially expressed in response to the GapmeRs were involved in the regulation of metabolism and inflammation and were significantly enriched for genes showing differential methylation in response to the GapmeRs. These data indicate a significant role of DNA de novo (de)methylation in the kidney in the development of hypertension in SS rats. The findings should help to shift the paradigm of DNA methylation research in diseases involving nondividing cells from correlative analysis to functional and mechanistic studies.

Tissue-specific effects of targeted mutation of Mir29b1 in rats.

BACKGROUND: miR-29 is a master regulator of extracellular matrix genes, but conflicting data on its anti-fibrotic effect have been reported. miR-29 improves nitric oxide (NO) production in arterioles by targeting Lypla1. Mir29b1 targeted mutation exacerbates hypertension in a model derived from the Dahl salt-sensitive rat. We examined the effect of Mir29b1 mutation on tissue fibrosis and NO levels with a focus on kidney regions. METHODS: Mir29b1 targeted mutant rats on the genetic background of SS-Chr13BN rats were studied. Masson trichrome staining, molecular and biochemical assays, metabolic cage studies, and bioinformatic analysis of human genomic data were performed. FINDINGS: The abundance of miR-29b and the co-transcribed miR-29a was substantially lower in mutant rats. Tissue fibrosis was significantly increased in the renal outer medulla, but not in the renal cortex, heart or liver in mutant rats on a 0.4% NaCl diet. Lypla1 protein abundance was significantly higher and NO levels lower in the renal outer medulla, but not in the renal cortex. After 14 days of a 4% NaCl diet, 24 h urine volume and urinary sodium excretion was significantly lower in mutant rats, and tissue fibrosis became higher in the heart. NO levels were lower in the renal outer medulla and heart, but not in the renal cortex. Human miR-29 genes are located in proximity with blood pressure-associated single nucleotide polymorphisms. INTERPRETATION: The renal outer medulla might be particularly susceptible to the injurious effects of a miR-29 insufficiency, which might contribute to the development of hypertension in Mir29b1 mutant rats.

MicroRNA-214-3p in the Kidney Contributes to the Development of Hypertension.

BACKGROUND: In spite of extensive study, the mechanisms for salt sensitivity of BP in humans and rodent models remain poorly understood. Several microRNAs (miRNAs) have been associated with hypertension, but few have been shown to contribute to its development. METHODS: We examined miRNA expression profiles in human kidney biopsy samples and rat models using small RNA deep sequencing. To inhibit an miRNA specifically in the kidney in conscious, freely moving rats, we placed indwelling catheters to allow both renal interstitial administration of a specific anti-miR and measurement of BP. A rat with heterozygous disruption of the gene encoding endothelial nitric oxide synthase (eNOS) was developed. We used bioinformatic analysis to evaluate the relationship between 283 BP-associated human single-nucleotide polymorphisms (SNPs) and 1870 human miRNA precursors, as well as other molecular and cellular methods. RESULTS: Compared with salt-insensitive SS.13BN26 rats, Dahl salt-sensitive (SS) rats showed an upregulation of miR-214-3p, encoded by a gene in the SS.13BN26 congenic region. Kidney-specific inhibition of miR-214-3p significantly attenuated salt-induced hypertension and albuminuria in SS rats. miR-214-3p directly targeted eNOS. The effect of miR-214-3p inhibition on hypertension and albuminuria was abrogated in SS rats with heterozygous loss of eNOS. Human kidney biopsy specimens from patients with hypertension or hypertensive nephrosclerosis showed upregulation of miR-214-3p; the gene encoding miR-214-3p was one of several differentially expressed miRNA genes located in proximity to human BP-associated SNPs. CONCLUSIONS: Renal miR-214-3p plays a functional and potentially genetic role in the development of hypertension, which might be mediated in part by targeting eNOS.

Urinary Metabolites Associated with Blood Pressure on a Low- or High-Sodium Diet.

Dietary salt intake has significant effects on arterial blood pressure and the development of hypertension. Mechanisms underlying salt-dependent changes in blood pressure remain poorly understood, and it is difficult to assess blood pressure salt-sensitivity clinically. Methods: We examined urinary levels of metabolites in 103 participants of the Dietary Approaches to Stop Hypertension (DASH)-Sodium trial after nearly 30 days on a defined diet containing high sodium (targeting 150 mmol sodium intake per day) or low sodium (50 mmol per day). Targeted chromatography/mass spectrometry analysis was performed in 24 h urine samples for 47 amino metabolites and 10 metabolites related to the tricarboxylic acid cycle. The effect of an identified metabolite on blood pressure was examined in Dahl salt-sensitive rats. Results: Urinary metabolite levels improved the prediction of classification of blood pressure salt-sensitivity based on race, age and sex. Random forest and generalized linear mixed model analyses identified significant (false discovery rate <0.05) associations of 24 h excretions of ß-aminoisobutyric acid, cystine, citrulline, homocysteine and lysine with systolic blood pressure and cystine with diastolic blood pressure. The differences in homocysteine levels between low- and high-sodium intakes were significantly associated with the differences in diastolic blood pressure. These associations were significant with or without considering demographic factors. Treatment with ß-aminoisobutyric acid significantly attenuated high-salt-induced hypertension in Dahl salt-sensitive rats. Conclusion: These findings support the presence of new mechanisms of blood pressure regulation involving metabolic intermediaries, which could be developed as markers or therapeutic targets for salt-sensitive hypertension.

miR-29 contributes to normal endothelial function and can restore it in cardiometabolic disorders.

We investigated the role of microRNAs (miRNA) in endothelial dysfunction in the setting of cardiometabolic disorders represented by type 2 diabetes mellitus (T2DM). miR-29 was dysregulated in resistance arterioles obtained by biopsy in T2DM patients. Intraluminal delivery of miR-29a-3p or miR-29b-3p mimics restored normal endothelium-dependent vasodilation (EDVD) in T2DM arterioles that otherwise exhibited impaired EDVD Intraluminal delivery of anti-miR-29b-3p in arterioles from non-DM human subjects or rats or targeted mutation of Mir29b-1/a gene in rats led to impaired EDVD and exacerbation of hypertension in the rats. miR-29b-3p mimic increased, while anti-miR-29b-3p or Mir29b-1/a gene mutation decreased, nitric oxide levels in arterioles. The mutation of Mir29b-1/a gene led to preferential differential expression of genes related to nitric oxide including Lypla1. Lypla1 was a direct target of miR-29 and could abrogate the effect of miR-29 in promoting nitric oxide production. Treatment with Lypla1 siRNA improved EDVD in arterioles obtained from T2DM patients or Mir29b-1/a mutant rats or treated with anti-miR-29b-3p. These findings indicate miR-29 is required for normal endothelial function in humans and animal models and has therapeutic potential for cardiometabolic disorders.

Elevation of fumarase attenuates hypertension and can result from a nonsynonymous sequence variation or increased expression depending on rat strain.

The activity of fumarase, an enzyme in the tricarboxylic acid cycle, is lower in Dahl salt-sensitive SS rats compared with SS.13BN rats. SS.13BN rats have a Brown Norway (BN) allele of fumarase and exhibit attenuated hypertension. The SS allele of fumarase differs from the BN allele by a K481E sequence variation. It remains unknown whether higher fumarase activities can attenuate hypertension and whether the mechanism is relevant without the K481E variation. We developed SS-TgFh1 transgenic rats overexpressing fumarase on the background of the SS rat. Hypertension was attenuated in SS-TgFh1 rats. Mean arterial pressure in SS-TgFh1 rats was 20 mmHg lower than transgene-negative SS littermates after 12 days on a 4% NaCl diet. Fumarase overexpression decreased H2O2, while fumarase knockdown increased H2O2 Ectopically expressed BN form of fumarase had higher specific activity than the SS form. However, sequencing of more than a dozen rat strains indicated most rat strains including salt-insensitive Sprague-Dawley (SD) rats had the SS allele of fumarase. Despite that, total fumarase enzyme activity in the renal medulla was still higher in SD rats than in SS rats, which was associated with higher expression of fumarase in SD. H2O2 can suppress the expression of fumarase. Renal medullary interstitial administration of fumarase siRNA in SD rats resulted in higher blood pressure on the high-salt diet. These findings indicate elevation of total fumarase activity attenuates the development of hypertension and can result from a nonsynonymous sequence variation in some rat strains and higher expression in other rat strains.

Malate and Aspartate Increase L-Arginine and Nitric Oxide and Attenuate Hypertension.

Fumarase catalyzes the interconversion of fumarate and L-malate in the tricarboxylic acid cycle. The Dahl salt-sensitive (SS) rat, a model of salt-sensitive hypertension, exhibits fumarase insufficiencies. To investigate the mechanism mediating the effect of fumarase-related metabolites on hypertension, we considered the pathway in which L-malate can be converted to oxaloacetate, aspartate, argininosuccinate, and L-arginine, the substrate of nitric oxide (NO) synthase. The levels of aspartate, citrulline, L-arginine, and NO were significantly decreased in the kidneys of SS rats compared to salt-insensitive consomic SS.13BN rats. Knockdown of fumarase in human kidney cells and vascular endothelial cells resulted in decreased levels of malate, aspartate, L-arginine, and NO. Supplementation of aspartate or malate increased renal levels of L-arginine and NO and attenuated hypertension in SS rats. These findings reveal a multi-step metabolic pathway important for hypertension in which malate and aspartate may modulate blood pressure by altering levels of L-arginine and NO.

Renal Delivery of Anti-microRNA Oligonucleotides in Rats.

MicroRNAs are endogenous small, non-protein-coding RNA molecules that play an important role in the regulation of a wide variety of cellular functions and disease processes. A novel role for microRNAs in the development of hypertension and hypertensive tissue injury is emerging in recent studies. Development of hypertension involves multiple organ systems and cannot be modeled in vitro. Therefore, the ability to experimentally alter genes, gene products, or biological pathways, including microRNAs, in an organ-specific manner in intact animal models is particularly valuable to hypertension research. The kidney plays a central role in the long-term regulation of arterial blood pressure. In this chapter, we describe a detailed protocol for using a renal interstitial injection method to deliver anti-miR oligonucleotides to knock down microRNA specifically in the kidney in conscious rats.

Renal Tumor Necrosis Factor α Contributes to Hypertension in Dahl Salt-Sensitive Rats.

Tumor necrosis factor α (TNFα) is a major proinflammatory cytokine and its level is elevated in hypertensive states. Inflammation occurs in the kidneys during the development of hypertension. We hypothesized that TNFα specifically in the kidney contributes to the development of hypertension and renal injury in Dahl salt-sensitive (SS) rats, a widely used model of human salt-sensitive hypertension and renal injury. SS rats were chronically instrumented for renal interstitial infusion and blood pressure measurement in conscious, freely moving state. Gene expression was measured using real-time PCR and renal injury assessed with histological analysis. The abundance of TNFα in the renal medulla of SS rats, but not the salt-insensitive congenic SS.13(BN26) rats, was significantly increased when rats had been fed a high-salt diet for 7 days (n = 6 or 9, p < 0.01). The abundance of TNFα receptors in the renal medulla was significantly higher in SS rats than SS.13(BN26) rats. Renal interstitial administration of Etanercept, an inhibitor of TNFα, significantly attenuated the development of hypertension in SS rats on a high-salt diet (n = 7-8, p < 0.05). Glomerulosclerosis and interstitial fibrosis were also significantly ameliorated. These findings indicate intrarenal TNFα contributes to the development of hypertension and renal injury in SS rats.

Antithrombin III/SerpinC1 insufficiency exacerbates renal ischemia/reperfusion injury.

Antithrombin III, encoded by SerpinC1, is a major anti-coagulation molecule in vivo and has anti-inflammatory effects. We found that patients with low antithrombin III activities presented a higher risk of developing acute kidney injury after cardiac surgery. To study this further, we generated SerpinC1 heterozygous knockout rats and followed the development of acute kidney injury in a model of modest renal ischemia/reperfusion injury. Renal injury, assessed by serum creatinine and renal tubular injury scores after 24 h of reperfusion, was significantly exacerbated in SerpinC1(+/-) rats compared to wild-type littermates. Concomitantly, renal oxidative stress, tubular apoptosis, and macrophage infiltration following this injury were significantly aggravated in SerpinC1(+/-) rats. However, significant thrombosis was not found in the kidneys of any group of rats. Antithrombin III is reported to stimulate the production of prostaglandin I2, a known regulator of renal cortical blood flow, in addition to having anti-inflammatory effects and to protect against renal failure. Prostaglandin F1α, an assayable metabolite of prostaglandin I2, was increased in the kidneys of the wild-type rats at 3 h after reperfusion. The increase of prostaglandin F1α was significantly blunted in SerpinC1(+/-) rats, which preceded increased tubular injury and oxidative stress. Thus, our study found a novel role of SerpinC1 insufficiency in increasing the severity of renal ischemia/reperfusion injury.

Ultrastructure of mitochondria and the endoplasmic reticulum in renal tubules of Dahl salt-sensitive rats.

Metabolic and functional abnormalities in the kidney precede or coincide with the initiation of overt hypertension in the Dahl salt-sensitive (SS) rat. However, renal histological injury in SS rats is mild before the development of overt hypertension. We performed electron microscopy analysis in 7-wk-old SS rats and salt-insensitive consomic SS.13(BN) rats and Sprague-Dawley (SD) rats fed a 4% NaCl diet for 7 days. Long mitochondria (>2 µm) accounted for a significantly smaller fraction of mitochondria in medullary thick ascending limbs in SS rats (4% ± 1%) than in SS.13(BN) rats (8% ± 1%, P < 0.05 vs. SS rats) and SD rats (9% ± 1%, P < 0.01 vs. SS rats), consistent with previous findings of mitochondrial functional insufficiency in the medulla of SS rats. Long mitochondria in proximal tubules, however, were more abundant in SS rats than in SS.13(BN) and SD rats. The width of the endoplasmic reticulum, an index of endoplasmic reticulum stress, was significantly greater in medullary thick ascending limbs of SS rats (107 ± 1 nm) than in SS.13(BN) rats (95 ± 2 nm, P < 0.001 vs. SS rats) and SD rats (74 ± 3 nm, P < 0.01 vs. SS or SS.13(BN) rats). The tubules examined were indistinguishable between rat strains under light microscopy. These data indicate that ultrastructural abnormalities occur in the medullary thick ascending limbs of SS rats before the development of histological injury in renal tubules, providing a potential structural basis contributing to the subsequent development of overt hypertension.

Delayed ischemic preconditioning contributes to renal protection by upregulation of miR-21.

Delayed ischemic preconditioning effectively protects kidneys from ischemia-reperfusion injury but the mechanism underlying renal protection remains poorly understood. Here we examined the in vivo role of microRNA miR-21 in the renal protection conferred by delayed ischemic preconditioning in mice. A 15-min renal ischemic preconditioning significantly increased the expression of miR-21 by 4 h and substantially attenuated ischemia-reperfusion injury induced 4 days later. A locked nucleic acid-modified anti-miR-21 given at the time of ischemic preconditioning knocked down miR-21 and significantly exacerbated subsequent ischemia-reperfusion injury in the mouse kidney. Knockdown of miR-21 resulted in significant upregulation of programmed cell death protein 4, a proapoptotic target gene of miR-21, and substantially increased tubular cell apoptosis. Hypoxia-inducible factor-1α in the kidney was activated after ischemic preconditioning and blockade of its activity with a decoy abolished the upregulation of miR-21 in cultured human renal epithelial cells treated with the inducer cobalt chloride. In the absence of ischemic preconditioning, knockdown of miR-21 alone did not significantly affect ischemia-reperfusion injury in the mouse kidney. Thus, upregulation of miR-21 contributes to the protective effect of delayed ischemic preconditioning against subsequent renal ischemia-reperfusion injury.

MiR-382 targeting of kallikrein 5 contributes to renal inner medullary interstitial fibrosis.

Previously we have shown that microRNA miR-382 can facilitate loss of renal epithelial characteristics in cultured cells. This study examined the in vivo role of miR-382 in the development of renal interstitial fibrosis in a mouse model. Unilateral ureteral obstruction was used to induce renal interstitial fibrosis in mice. With 3 days of unilateral ureteral obstruction, expression of miR-382 in the obstructed kidney was increased severalfold compared with sham-operated controls. Intravenous delivery of locked nucleic acid-modified anti-miR-382 blocked the increase in miR-382 expression and significantly reduced inner medullary fibrosis. Expression of predicted miR-382 target kallikrein 5, a proteolytic enzyme capable of degrading several extracellular matrix proteins, was reduced with unilateral ureteral obstruction. Anti-miR-382 treatment prevented the reduction of kallikrein 5 in the inner medulla. Furthermore, the protective effect of the anti-miR-382 treatment against fibrosis was abolished by renal knockdown of kallikrein 5. Targeting of kallikrein 5 by miR-382 was confirmed by 3'-untranslated region luciferase assay. These data support a completely novel mechanism in which miR-382 targets kallikrein 5 and contributes to the development of renal inner medullary interstitial fibrosis. The study provided the first demonstration of an in vivo functional role of miR-382 in any species and any organ system.

Renal medullary microRNAs in Dahl salt-sensitive rats: miR-29b regulates several collagens and related genes.

MicroRNAs are endogenous repressors of gene expression. We examined microRNAs in the renal medulla of Dahl salt-sensitive rats and consomic SS-13(BN) rats. Salt-induced hypertension and renal injury in Dahl salt-sensitive rats, particularly medullary interstitial fibrosis, have been shown previously to be substantially attenuated in SS-13(BN) rats. Of 377 microRNAs examined, 5 were found to be differentially expressed between Dahl salt-sensitive rats and consomic SS-13(BN) rats receiving a high-salt diet. Real-time PCR analysis demonstrated that high-salt diets induced substantial upregulation of miR-29b in the renal medulla of SS-13(BN) rats but not in SS rats. miR-29b was predicted to regulate 20 collagen genes, matrix metalloproteinase 2 (Mmp2), integrin beta1 (Itgb1), and other genes related to the extracellular matrix. Expression of 9 collagen genes and Mmp2 was upregulated by a high-salt diet in the renal medulla of SS rats, but not in SS-13(BN) rats, an expression pattern opposite to miR-29b. Knockdown of miR-29b in the kidneys of SS-13(BN) rats resulted in upregulation of several collagen genes. miR-29b reduced expression levels of several collagen genes and Itgb1 in cultured rat renal medullary epithelial cells. Moreover, miR-29b suppressed the activity of luciferase when the reporter gene was linked to a 3'-untranslated segment of collagen genes Col1a1, Col3a1, Col4a1, Col5a1, Col5a2, Col5a3, Col7a1, Col8a1, Mmp2, or Itgb1 but not Col12a1. The result demonstrated broad effects of miR-29b on a large number of collagens and genes related to the extracellular matrix and suggested involvement of miR-29b in the protection from renal medullary injury in SS-13(BN) rats.

Novel role of fumarate metabolism in dahl-salt sensitive hypertension.

In a previous proteomic study, we found dramatic differences in fumarase in the kidney between Dahl salt-sensitive rats and salt-insensitive consomic SS-13(BN) rats. Fumarase catalyzes the conversion between fumarate and l-malate in the tricarboxylic acid cycle. Little is known about the pathophysiological significance of fumarate metabolism in cardiovascular and renal functions, including salt-induced hypertension. The fumarase gene is located on the chromosome substituted in the SS-13(BN) rat. Sequencing of fumarase cDNA indicated the presence of lysine at amino acid position 481 in Dahl salt-sensitive rats and glutamic acid in Brown Norway and SS-13(BN) rats. Total fumarase activity was significantly lower in the kidneys of Dahl salt-sensitive rats compared with SS-13(BN) rats, despite an apparent compensatory increase in fumarase abundance in Dahl salt-sensitive rats. Intravenous infusion of a fumarate precursor in SS-13(BN) rats resulted in a fumarate excess in the renal medulla comparable to that seen in Dahl salt-sensitive rats. The infusion significantly exacerbated salt-induced hypertension in SS-13(BN) rats (140+/-3 vs125+/-2 mm Hg in vehicle control at day 5 on a 4% NaCl diet; P<0.05). In addition, the fumarate infusion increased renal medullary tissue levels of H2O2. Treatment of cultured human renal epithelial cells with the fumarate precursor also increased cellular levels of H2O2. These data suggest a novel role for fumarate metabolism in salt-induced hypertension and renal medullary oxidative stress.

Glucocorticoid response elements and 11 beta-hydroxysteroid dehydrogenases in the regulation of endothelial nitric oxide synthase expression.

AIMS: Hypertensive and other effects of excess glucocorticoids might be in part mediated by the suppression of endothelial nitric oxide synthase (eNOS) expression. We studied the transcriptional and biochemical mechanisms that mediate or modulate the suppression of eNOS expression by glucocorticoids. METHODS AND RESULTS: We found that a mere three-fold increase in the concentration of the natural glucocorticoid cortisol (from 30 to 100 nmol/L) significantly decreased the expression level of eNOS in human endothelial cells. Deletion analysis of the eNOS promoter indicated that the segment within -119 bp upstream from the transcription start site was significantly involved in the effect of cortisol. Site-directed mutagenesis and chromatin immunoprecipitation analyses demonstrated the presence of a suppressive glucocorticoid response element (GRE) at -111 to -105 bp. 11 beta-hydroxysteroid dehydrogenases (11 beta-HSD) catalyse the interconversion of active and inactive glucocorticoids. The suppression of 11 beta-HSD2 using small interfering RNA markedly exacerbated the inhibition of eNOS by cortisol. The suppression of 11 beta-HSD1 abolished the inhibition of eNOS expression by cortisol. CONCLUSION: We identified the first GRE in the eNOS promoter region and demonstrated that endogenous 11 beta-HSD1 and 11 beta-HSD2 play significant and distinct roles in modulating the effect of glucocorticoids on eNOS expression.