In plants, methylation catalyzed by HEN1 (small RNA methyl transferase) prevents microRNAs (miRNAs) from degradation triggered by uridylation. How methylation antagonizes uridylation of miRNAs in vivo is not well understood. In addition, 5′ RNA fragments (5′ fragments) produced by miRNA-mediated RNA cleavage can be uridylated in plants and animals. However, the biological significance of this modification is unknown, and enzymes uridylating 5′ fragments remain to be identified. Here, we report that in Arabidopsis, HEN1 suppressor 1 (HESO1, a miRNA nucleotidyl transferase) uridylates 5′ fragments to trigger their degradation. We also show that Argonaute 1 (AGO1), the effector protein of miRNAs, interacts with HESO1 through its Piwi/Argonaute/Zwille and PIWI domains, which bind the 3′ end of miRNA and cleave the target mRNAs, respectively. Furthermore, HESO1 is able to uridylate AGO1-bound miRNAs in vitro. miRNA uridylation in vivo requires a functional AGO1 in hen1, in which miRNA methylation is impaired, demonstrating that HESO1 can recognize its substrates in the AGO1 complex. On the basis of these results, we propose that methylation is required to protect miRNAs from AGO1-associated HESO1 activity that normally uridylates 5′ fragments. S mall interfering RNAs (siRNAs) and microRNAs (miRNAs), ∼20-25 nucleotides (nt) in size, are important regulators of gene expression. miRNAs and siRNAs are derived from imperfect hairpin transcripts and perfect long double-stranded RNAs, respectively (1, 2). miRNAs and siRNAs are then associated with Argonaute (AGO) proteins to repress gene expression through target cleavage and/or translational inhibition (3). The cleavage of target mRNAs usually occurs at a position opposite the tenth and eleventh nucleotides of miRNAs, resulting in a 5′ RNA fragment (5′ fragment) and a 3′ fragment (4). In Arabidopsis, the major effector protein for miRNA-mediated gene silencing is AGO1, which possesses the endonuclease activity required for target cleavage (5-7). In Drosophila, the exosome removes the 5′ fragments through its 3′-to-5′ exoribonuclease activity (8). How 5′ fragments are degraded in higher plants remains unknown. It has been shown that the 5′ fragments are subject to untemplated uridine addition at their 3′ termini (uridylation) in both animals and plants (9). However, the biological significance of this modification remains unknown because of a lack of knowledge of the enzymes targeting 5′ fragments for uridylation.Uridylation plays important roles in regulating miRNA biogenesis. In animals, TUT4, a terminal uridyl transferase, is recruited by Lin-28 (an RNA binding protein) to the let-7 precursor (prelet-7), resulting in uridylation of prelet-7 (10, 11). This modification impairs the stability of prelet-7, resulting in reduced levels of let-7. In addition, monouridylation has been shown to be required for the processing of some miRNA precursors (12). Deep sequencing analysis reveals that precursor uridylation is a widespread phenomenon occurring in many miRNA families in ani...

The objective of this study was to compare the ratio of renal oxygen availability (RO2) to glomerular filtration rate (GFR), a measure of relative renal hypoxia, in adolescents with and without type 1 diabetes (T1D) and relate the ratio to albuminuria, renal plasma flow (RPF), fat mass, and insulin sensitivity (M/I). RO2 was estimated by blood oxygen level–dependent MRI; fat mass was estimated by DXA; GFR and RPF were estimated by iohexol and p-aminohippurate clearance; albuminuria was estimated by urine albumin-to-creatinine ratio (UACR); and M/I was estimated from steady-state glucose infusion rate/insulin (mg/kg/min) by hyperglycemic clamp in 50 adolescents with T1D (age 16.1 ± 3.0 years, HbA1c 8.6 ± 1.2%) and 20 control patients of similar BMI (age 16.1 ± 2.9 years, HbA1c 5.2 ± 0.2%). The RO2:GFR (ms/mL/min) was calculated as RO2 (T2*, ms) divided by GFR (mL/min). Whole-kidney RO2:GFR was 25% lower in adolescents with T1D versus control patients (P < 0.0001). In adolescents with T1D, lower whole-kidney RO2:GFR was associated with higher UACR (r = −0.31, P = 0.03), RPF (r = −0.52, P = 0.0009), and fat mass (r = −0.33, P = 0.02). Lower medullary RO2:GFR was associated with lower M/I (r = 0.31, P = 0.03). In conclusion, adolescents with T1D exhibited relative renal hypoxia that was associated with albuminuria and with increased RPF, fat mass, and insulin resistance. These data suggest a potential role of renal hypoxia in the development of diabetic kidney disease.

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Kidney fibrosis constitutes the shared final pathway of nearly all chronic nephropathies, but biomarkers for the non-invasive assessment of kidney fibrosis are currently not available. To address this, we characterize five candidate biomarkers of kidney fibrosis: Cadherin-11 (CDH11), Sparc-related modular calcium binding protein-2 (SMOC2), Pigment epithelium-derived factor (PEDF), Matrix-Gla protein, and Thrombospondin-2. Gene expression profiles in single-cell and single-nucleus RNA-sequencing (sc/snRNA-seq) datasets from rodent models of fibrosis and human chronic kidney disease (CKD) were explored, and Luminex-based assays for each biomarker were developed. Plasma and urine biomarker levels were measured using independent prospective cohorts of CKD: the Boston Kidney Biopsy Cohort, a cohort of individuals with biopsyconfirmed semiquantitative assessment of kidney fibrosis, and the Seattle Kidney Study, a cohort of patients with common forms of CKD. Ordinal logistic regression and Cox proportional hazards regression models were used to test associations of biomarkers with interstitial fibrosis and tubular atrophy and progression to end-stage kidney disease and death, respectively. Sc/snRNA-seq data confirmed cell-specific expression of biomarker genes in fibroblasts. After multivariable adjustment, higher levels of plasma CDH11, SMOC2, and PEDF and urinary CDH11 and PEDF were significantly associated with increasing severity of interstitial fibrosis and tubular atrophy in the Boston Kidney Biopsy Cohort. In both cohorts, higher levels of plasma and urinary SMOC2 and urinary CDH11 were independently associated with progression to end-stage kidney disease. Higher levels of urinary PEDF associated with end-stage kidney disease in the Seattle Kidney Study, with a similar signal in the Boston Kidney Biopsy Cohort, although the latter narrowly missed statistical significance. Thus, we identified CDH11, SMOC2, and PEDF as promising non-invasive biomarkers of kidney fibrosis.

Objective Glomerular injury is a recognized complication of diabetic ketoacidosis (DKA), yet the tubular lesions are poorly understood. The aim of this prospective study was to evaluate the presence and reversibility of tubular injury during DKA in children with type 1 diabetes (T1D). Research Design and Methods Blood and urine samples were collected from 40 children with DKA (52% boys, mean age 11 ± 4 years, venous pH 7.2 ± 0.1, glucose 451 ± 163 mg/dL) at three timepoints: 0–8 and 12–24 h after starting insulin, and 3 months after discharge. Mixed‐effects models evaluated the changes in tubular injury markers over time (neutrophil gelatinase‐associated lipocalin [NGAL], kidney injury molecule 1 [KIM‐1], and interleukin 18 [IL‐18]). We also evaluated the relationships among the tubular injury biomarkers, copeptin, a vasopressin surrogate, and serum uric acid (SUA). Results Serum NGAL, KIM‐1, and IL‐18 were highest at 0–8 h (306.5 ± 45.9 ng/mL, 128.9 ± 10.1 pg/mL, and 564.3 ± 39.2 pg/mL, respectively) and significantly decreased over 3 months (p = 0.03, p = 0.01, and p < 0.001, respectively). There were strong relationships among increases in copeptin and SUA and rises in tubular injury biomarkers. At 0–8 h, participants with acute kidney injury (AKI) [17%] showed significantly higher concentrations of tubular injury markers, copeptin, and SUA. Conclusions DKA was characterized by tubular injury, and the degree of injury associated with elevated copeptin and SUA. Tubular injury biomarkers, copeptin and SUA may be able to predict AKI in DKA.