Histologically resolved small RNA maps in primary focal segmental glomerulosclerosis indicate progressive changes within glomerular and tubulointerstitial regions.

Pathological heterogeneity is common in clinical tissue specimens and complicates the interpretation of molecular data obtained from the specimen. As a typical example, a kidney biopsy specimen often contains glomeruli and tubulointerstitial regions with different levels of histological injury, including some that are histologically normal. We reasoned that the molecular profiles of kidney tissue regions with specific histological injury scores could provide new insights into kidney injury progression. Therefore, we developed a strategy to perform small RNA deep sequencing analysis for individually scored glomerular and tubulointerstitial regions in formalin-fixed, paraffin-embedded kidney needle biopsies. This approach was applied to study focal segmental glomerulosclerosis (FSGS), the leading cause of nephrotic syndrome in adults. Large numbers of small RNAs, including microRNAs, 3'-tRFs, 5'-tRFs, and mitochondrial tRFs, were differentially expressed between histologically indistinguishable tissue regions from patients with FSGS and matched healthy controls. A majority of tRFs were upregulated in FSGS. Several small RNAs were differentially expressed between tissue regions with different histological scores in FSGS. Notably, with increasing levels of histological damage, miR-21-5p was upregulated progressively and miR-192-5p was downregulated progressively in glomerular and tubulointerstitial regions, respectively. This study marks the first genome scale molecular profiling conducted in histologically characterized glomerular and tubulointerstitial regions. Thus, substantial molecular changes in histologically normal kidney regions in FSGS might contribute to initiating tissue injury or represent compensatory mechanisms. In addition, several small RNAs might contribute to subsequent progression of glomerular and tubulointerstitial injury, and histologically mapping small RNA profiles may be applied to analyze tissue specimens in any disease.

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.

Tissue-Specific MicroRNA Expression Patterns in Four Types of Kidney Disease.

MicroRNAs contribute to the development of kidney disease. Previous analyses of microRNA expression in human kidneys, however, were limited by tissue heterogeneity or the inclusion of only one pathologic type. In this study, we used laser-capture microdissection to obtain glomeruli and proximal tubules from 98 human needle kidney biopsy specimens for microRNA expression analysis using deep sequencing. We analyzed specimens from patients with diabetic nephropathy (DN), FSGS, IgA nephropathy (IgAN), membranoproliferative GN (MPGN) (n=19-23 for each disease), and a control group (n=14). Compared with control glomeruli, DN, FSGS, IgAN, and MPGN glomeruli exhibited differential expression of 18, 12, two, and 17 known microRNAs, respectively. The expression of several microRNAs also differed between disease conditions. Specifically, compared with control or FSGS glomeruli, IgAN glomeruli exhibited downregulated expression of hsa-miR-3182. Furthermore, in combination, the expression levels of hsa-miR-146a-5p and hsa-miR-30a-5p distinguished DN from all other conditions except IgAN. Compared with control proximal tubules, DN, FSGS, IgAN, and MPGN proximal tubules had differential expression of 13, 14, eight, and eight microRNAs, respectively, but expression of microRNAs did not differ significantly between the disease conditions. The abundance of several microRNAs correlated with indexes of renal function. Finally, we validated the differential glomerular expression of select microRNAs in a second cohort of patients with DN (n=19) and FSGS (n=21). In conclusion, we identified tissue-specific microRNA expression patterns associated with several kidney pathologies. The identified microRNAs could be developed as biomarkers of kidney diseases and might be involved in disease mechanisms.

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.

Characteristics of microRNAs enriched in specific cell types and primary tissue types in solid organs.

Knowledge of miRNA expression and function in specific cell types in solid organs is limited because of difficulty in obtaining appropriate specimens. We used laser capture microdissection to obtain nine tissue regions from rats, including the nucleus of the solitary tract, hypoglossal motor nucleus, ventral respiratory column/pre-Bötzinger complex, and midline raphe nucleus from the brain stem, myocardium and coronary artery from the heart, and glomerulus, proximal convoluted tubule, and medullary thick ascending limb from the kidney. Each tissue region consists of or is enriched for a specific cell type. Differential patterns of miRNA expression obtained by deep sequencing of minute amounts of laser-captured cells were highly consistent with data obtained from real-time PCR analysis. miRNA expression patterns correctly clustered the specimens by tissue regions and then by primary tissue types (neural, muscular, or epithelial). The aggregate difference in miRNA profiles between tissue regions that contained the same primary tissue type was as large as one-half of the aggregate difference between primary tissue types. miRNAs differentially expressed between primary tissue types are more likely to be abundant miRNAs, while miRNAs differentially expressed between tissue regions containing the same primary tissue type were distributed evenly across the abundance spectrum. The tissue type-enriched miRNAs were more likely to target genes enriched for specific functional categories compared with either cell type-enriched miRNAs or randomly selected miRNAs. These data indicate that the role of miRNAs in determining characteristics of primary tissue types may be different than their role in regulating cell type-specific functions in solid organs.

Cell-free DNA donor fraction analysis in pediatric and adult heart transplant patients by multiplexed allele-specific quantitative PCR: Validation of a rapid and highly sensitive clinical test for stratification of rejection probability.

Lifelong noninvasive rejection monitoring in heart transplant patients is a critical clinical need historically poorly met in adults and unavailable for children and infants. Cell-free DNA (cfDNA) donor-specific fraction (DF), a direct marker of selective donor organ injury, is a promising analytical target. Methodological differences in sample processing and DF determination profoundly affect quality and sensitivity of cfDNA analyses, requiring specialized optimization for low cfDNA levels typical of transplant patients. Using next-generation sequencing, we previously correlated elevated DF with acute cellular and antibody-mediated rejection (ACR and AMR) in pediatric and adult heart transplant patients. However, next-generation sequencing is limited by cost, TAT, and sensitivity, leading us to clinically validate a rapid, highly sensitive, quantitative genotyping test, myTAIHEART®, addressing these limitations. To assure pre-analytical quality and consider interrelated cfDNA measures, plasma preparation was optimized and total cfDNA (TCF) concentration, DNA fragmentation, and DF quantification were validated in parallel for integration into myTAIHEART reporting. Analytical validations employed individual and reconstructed mixtures of human blood-derived genomic DNA (gDNA), cfDNA, and gDNA sheared to apoptotic length. Precision, linearity, and limits of blank/detection/quantification were established for TCF concentration, DNA fragmentation ratio, and DF determinations. For DF, multiplexed high-fidelity amplification followed by quantitative genotyping of 94 SNP targets was applied to 1168 samples to evaluate donor options in staged simulations, demonstrating DF call equivalency with/without donor genotype. Clinical validation studies using 158 matched endomyocardial biopsy-plasma pairs from 76 pediatric and adult heart transplant recipients selected a DF cutoff (0.32%) producing 100% NPV for ≥2R ACR. This supports the assay's conservative intended use of stratifying low versus increased probability of ≥2R ACR. myTAIHEART is clinically validated for heart transplant recipients ≥2 months old and ≥8 days post-transplant, expanding opportunity for noninvasive transplant rejection assessment to infants and children and to all recipients >1 week post-transplant.

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.

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.

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.

Endogenous microRNAs in human microvascular endothelial cells regulate mRNAs encoded by hypertension-related genes.

The goal of this study was to systematically identify endogenous microRNAs (miRNAs) in endothelial cells that regulate mRNAs encoded by genes relevant to hypertension. Small RNA deep sequencing was performed in cultured human microvascular endothelial cells. Of the 50 most abundant miRNAs identified, 30 had predicted target mRNAs encoded by genes with known involvement in hypertension or blood pressure regulation. The cells were transfected with anti-miR oligonucleotides to inhibit each of the 30 miRNAs and the mRNA abundance of predicted targets was examined. Of 95 miRNA-target pairs examined, the target mRNAs were significantly upregulated in 35 pairs and paradoxically downregulated in 8 pairs. The result indicated significant suppression of the abundance of mRNA encoded by ADM by endogenous miR-181a-5p, ATP2B1 by the miR-27 family, FURIN by miR-125a-5p, FGF5 by the let-7 family, GOSR2 by miR-27a-3p, JAG1 by miR-21-5p, SH2B3 by miR-30a-5p, miR-98, miR-181a-5p, and the miR-125 family, TBX3 by the miR-92 family, ADRA1B by miR-22-3p, ADRA2A by miR-30a-5p and miR-30e-5p, ADRA2B by miR-30e-5p, ADRB1 by the let-7 family and miR-98, EDNRB by the miR-92 family, and NOX4 by the miR-92 family, miR-100-5p, and miR-99b-5p (n=3-9; P<0.05 versus scrambled anti-miR). Treatment with anti-miR-21 decreased blood pressure in mice fed a 4% NaCl diet. Inhibition of the miRNAs targeting NOX4 mRNA increased H2O2 release from endothelial cells. The findings indicate widespread, tonic control of mRNAs encoded by genes relevant to blood pressure regulation by endothelial miRNAs and provide a novel and uniquely informative basis for studying the role of miRNAs in hypertension.