The Extracellular RNA Communication Consortium: Establishing Foundational Knowledge and Technologies for Extracellular RNA Research.

The Extracellular RNA Communication Consortium (ERCC) was launched to accelerate progress in the new field of extracellular RNA (exRNA) biology and to establish whether exRNAs and their carriers, including extracellular vesicles (EVs), can mediate intercellular communication and be utilized for clinical applications. Phase 1 of the ERCC focused on exRNA/EV biogenesis and function, discovery of exRNA biomarkers, development of exRNA/EV-based therapeutics, and construction of a robust set of reference exRNA profiles for a variety of biofluids. Here, we present progress by ERCC investigators in these areas, and we discuss collaborative projects directed at development of robust methods for EV/exRNA isolation and analysis and tools for sharing and computational analysis of exRNA profiling data.

Protein N-glycans in Healthy and Sclerotic Glomeruli in Diabetic Kidney Disease.

BACKGROUND: Diabetes is expected to directly impact renal glycosylation, yet to date, there has not been a comprehensive evaluation of alterations in N-glycan composition in the glomeruli of patients with diabetic kidney disease (DKD). METHODS: We used untargeted mass spectrometry imaging to identify N-glycan structures in healthy and sclerotic glomeruli in FFPE sections from needle biopsies of five patients with DKD and three healthy kidney samples. Regional proteomics was performed on glomeruli from additional biopsies from the same patients to compare the abundances of enzymes involved in glycosylation. Secondary analysis of single nuclei transcriptomics (snRNAseq) data was used to inform on transcript levels of glycosylation machinery in different cell types and states. RESULTS: We detected 120 N-glycans, and among them identified twelve of these protein post-translated modifications that were significantly increased in glomeruli. All glomeruli-specific N-glycans contained an N-acetyllactosamine (LacNAc) epitope. Five N-glycan structures were highly discriminant between sclerotic and healthy glomeruli. Sclerotic glomeruli had an additional set of glycans lacking fucose linked to their core, and they did not show tetra-antennary structures that are common in healthy glomeruli. Orthogonal omics analyses revealed lower protein abundance and lower gene expression involved in synthesizing fucosylated and branched N-glycans in sclerotic podocytes. In snRNAseq and regional proteomics analyses, we observed that genes and/or proteins involved in sialylation and LacNAc synthesis were also downregulated in DKD glomeruli, but this alteration remained undetectable by our spatial N-glycomics assay. CONCLUSIONS: Integrative spatial glycomics, proteomics, and transcriptomics revealed protein N-glycosylation characteristic of sclerotic glomeruli in DKD.

Unbiased kidney-centric molecular categorization of chronic kidney disease as a step towards precision medicine.

Current classification of chronic kidney disease (CKD) into stages using indirect systemic measures (estimated glomerular filtration rate (eGFR) and albuminuria) is agnostic to the heterogeneity of underlying molecular processes in the kidney thereby limiting precision medicine approaches. To generate a novel CKD categorization that directly reflects within kidney disease drivers we analyzed publicly available transcriptomic data from kidney biopsy tissue. A Self-Organizing Maps unsupervised artificial neural network machine-learning algorithm was used to stratify a total of 369 patients with CKD and 46 living kidney donors as healthy controls. Unbiased stratification of the discovery cohort resulted in identification of four novel molecular categories of disease termed CKD-Blue, CKD-Gold, CKD-Olive, CKD-Plum that were replicated in independent CKD and diabetic kidney disease datasets and can be further tested on any external data at kidneyclass.org. Each molecular category spanned across CKD stages and histopathological diagnoses and represented transcriptional activation of distinct biological pathways. Disease progression rates were highly significantly different between the molecular categories. CKD-Gold displayed rapid progression, with significant eGFR-adjusted Cox regression hazard ratio of 5.6 [1.01-31.3] for kidney failure and hazard ratio of 4.7 [1.3-16.5] for composite of kidney failure or a 40% or more eGFR decline. Urine proteomics revealed distinct patterns between the molecular categories, and a 25-protein signature was identified to distinguish CKD-Gold from other molecular categories. Thus, patient stratification based on kidney tissue omics offers a gateway to non-invasive biomarker-driven categorization and the potential for future clinical implementation, as a key step towards precision medicine in CKD.

Micro-dissection and integration of long and short reads to create a robust catalog of kidney compartment-specific isoforms.

Studying isoform expression at the microscopic level has always been a challenging task. A classical example is kidney, where glomerular and tubulo-interstitial compartments carry out drastically different physiological functions and thus presumably their isoform expression also differs. We aim at developing an experimental and computational pipeline for identifying isoforms at microscopic structure-level. We microdissected glomerular and tubulo-interstitial compartments from healthy human kidney tissues from two cohorts. The two compartments were separately sequenced with the PacBio RS II platform. These transcripts were then validated using transcripts of the same samples by the traditional Illumina RNA-Seq protocol, distinct Illumina RNA-Seq short reads from European Renal cDNA Bank (ERCB) samples, and annotated GENCODE transcript list, thus identifying novel transcripts. We identified 14,739 and 14,259 annotated transcripts, and 17,268 and 13,118 potentially novel transcripts in the glomerular and tubulo-interstitial compartments, respectively. Of note, relying solely on either short or long reads would have resulted in many erroneous identifications. We identified distinct pathways involved in glomerular and tubulo-interstitial compartments at the isoform level, creating an important experimental and computational resource for the kidney research community.

Tobacco exposure in adults and children with proteinuric glomerulopathies: a NEPTUNE cohort study.

BACKGROUND: Tobacco exposure has been recognized as a risk factor for cardiovascular disease (CVD) and progression of kidney disease. Patients with proteinuric glomerulopathies are at increased risk for cardiovascular morbidity and mortality. Multiple studies have linked tobacco exposure to CVD and chronic kidney disease, but the relationships between smoking and proteinuric glomerulopathies in adults and children have not been previously explored. METHODS: Data from the Nephrotic Syndrome Study Network (NEPTUNE), a multi-center prospective observational study of participants with proteinuric glomerulopathies, was analyzed. 371 adults and 192 children enrolled in NEPTUNE were included in the analysis. Self-reported tobacco exposure was classified as non-smoker, active smoker, former smoker, or exclusive passive smoker. Baseline serum cotinine levels were measured in a sub-cohort of 178 participants. RESULTS: The prevalence of active smokers, former smokers and exclusive passive smoking among adults at baseline was 14.6%, 29.1% and 4.9%, respectively. Passive smoke exposure was 16.7% among children. Active smoking (reference non-smoking) was significantly associated with greater total cholesterol among adults (ß 17.91 95% CI 0.06, 35.76, p = 0.049) while passive smoking (reference non-smoking) was significantly associated with greater proteinuria over time among children (ß 1.23 95% CI 0.13, 2.33, p = 0.03). Higher cotinine levels were associated with higher baseline eGFR (r = 0.17, p = 0.03). CONCLUSION: Tobacco exposure is associated with greater risk for CVD and worse kidney disease outcomes in adults and children with proteinuric glomerulopathies. Preventive strategies to reduce tobacco exposure may help protect against future cardiovascular and kidney morbidity and mortality in patients with proteinuric glomerulopathies.

The Michigan O'Brien Kidney Research Center: transforming translational kidney research through systems biology.

Research on kidney diseases is being transformed by the rapid expansion and innovations in omics technologies. The analysis, integration, and interpretation of big data, however, have been an impediment to the growing interest in applying these technologies to understand kidney function and failure. Targeting this urgent need, the University of Michigan O'Brien Kidney Translational Core Center (MKTC) and its Administrative Core established the Applied Systems Biology Core. The Core provides need-based support for the global kidney community centered on enabling incorporation of systems biology approaches by creating web-based, user-friendly analytic and visualization tools, like Nephroseq and Nephrocell, guiding with experimental design, and processing, analysis, and integration of large data sets. The enrichment core supports systems biology education and dissemination through workshops, seminars, and individualized training sessions. Meanwhile, the Pilot and Feasibility Program of the MKTC provides pilot funding to both early-career and established investigators new to the field, to integrate a systems biology approach into their research projects. The relevance and value of the portfolio of training and services offered by MKTC are reflected in the expanding community of young investigators, collaborators, and users accessing resources and engaging in systems biology-based kidney research, thereby motivating MKTC to persevere in its mission to serve the kidney research community by enabling access to state-of-the-art data sets, tools, technologies, expertise, and learning opportunities for transformative basic, translational, and clinical studies that will usher in solutions to improve the lives of people impacted by kidney disease.

Phospholipase Cε insufficiency causes ascending aortic aneurysm and dissection.

Phospholipase Cε (PLCε) is a phospholipase C isoform with a wide range of physiological functions. It has been implicated in aortic valve disorders, but its role in frequently associated aortic disease remains unclear. To determine the role of PLCε in thoracic aortic aneurysm and dissection (TAAD) we used PLCε-deficient mice, which develop aortic valve insufficiency and exhibit aortic dilation of the ascending thoracic aorta and arch without histopathological evidence of injury. Fourteen days of infusion of Plce1+/+ and Plce1-/- mice with angiotensin II (ANG II), which induces aortic dilation and dissection, led to sudden death secondary to ascending aortic dissection in 43% of Plce1-/- versus 5% of Plce1+/+ mice (P < 0.05). Medial degeneration and TAAD were detected in 80% of Plce1-/- compared with 10% of Plce1+/+ mice (P < 0.05) after 4 days of ANG II. Treatment with ANG II markedly increased PLCε expression within the ascending aortic adventitia. Total RNA sequencing demonstrated marked upregulation of inflammatory and fibrotic pathways mediated by interleukin-1ß, interleukin-6, and tumor necrosis factor-α. In silico analysis of whole exome sequences of 258 patients with type A dissection identified 5 patients with nonsynonymous PLCE1 variants. Our data suggest that PLCε deficiency plays a role in the development of TAAD and aortic insufficiency.NEW & NOTEWORTHY We describe a novel phenotype by which PLCε deficiency predisposes to aortic valve insufficiency and ascending aortic aneurysm, dissection, and sudden death in the setting of ANG II-mediated hypertension. We demonstrate PLCE1 variants in patients with type A aortic dissection and aortic insufficiency, suggesting that PLCE1 may also play a role in human aortic disease. This finding is of very high significance because it has not been previously demonstrated that PLCε directly mediates aortic dissection.

Continuous Renal Replacement Therapy among Patients with COVID-19 and Acute Kidney Injury.

BACKGROUND AND OBJECTIVES: Acute kidney injury (AKI) is a common complication among patients with COVID-19 and acute respiratory distress syndrome. Reports suggest that COVID-19 confers a pro-thrombotic state, which presents challenges in maintaining hemofilter patency and delivering continuous renal replacement therapy (CRRT). We present our initial experience with CRRT in critically ill patients with COVID-19, emphasizing circuit patency and the association between fluid balance during CRRT and respiratory parameters. DESIGN, SETTING, PARTICIPANTS, AND MEASUREMENTS: Retrospective chart review of 32 consecutive patients with COVID-19 and AKI managed with continuous venovenous hemodiafiltration with regional citrate anticoagulation (CVVHDF-RCA) according to the University of Michigan protocol. Primary outcome was mean CRRT circuit life per patient during the first 7 days of CRRT. We used simple linear regression to assess the relationship between patient characteristics and filter life. We also explored the relationship between fluid balance on CRRT and respiratory parameters using repeated measures modeling. RESULTS: Patients' mean age was 54.8 years and majority were Black (75%). Comorbidities included hypertension (90.6%), obesity (70.9%) diabetes (56.2%), and chronic kidney disease (40.6%). Median CRRT circuit life was 53.5 [interquartile range 39.1-77.6] hours. There was no association between circuit life and inflammatory or pro-thrombotic laboratory values (ferritin p = 0.92, C-reactive protein p = 0.29, D-dimer p = 0.24), or with systemic anticoagulation (p = 0.37). Net daily fluid removal during the first 7 days of CRRT was not associated with daily (closest recorded values to 20:00) PaO2/FIO2 ratio (p = 0.21) or positive end-expiratory pressure requirements (p = 0.47). CONCLUSIONS: We achieved adequate CRRT circuit life in COVID-19 patients using an established CVVHDF-RCA protocol. During the first 7 days of CRRT therapy, cumulative fluid balance was not associated with improvements in respiratory parameters, even after accounting for baseline fluid balance.

Comprehensive Search for Novel Circulating miRNAs and Axon Guidance Pathway Proteins Associated with Risk of ESKD in Diabetes.

BACKGROUND: Mechanisms underlying the pro gression of diabetic kidney disease to ESKD are not fully understood. METHODS: We performed global microRNA (miRNA) analysis on plasma from two cohorts consisting of 375 individuals with type 1 and type 2 diabetes with late diabetic kidney disease, and targeted proteomics analysis on plasma from four cohorts consisting of 746 individuals with late and early diabetic kidney disease. We examined structural lesions in kidney biopsy specimens from the 105 individuals with early diabetic kidney disease. Human umbilical vein endothelial cells were used to assess the effects of miRNA mimics or inhibitors on regulation of candidate proteins. RESULTS: In the late diabetic kidney disease cohorts, we identified 17 circulating miRNAs, represented by four exemplars (miR-1287-5p, miR-197-5p, miR-339-5p, and miR-328-3p), that were strongly associated with 10-year risk of ESKD. These miRNAs targeted proteins in the axon guidance pathway. Circulating levels of six of these proteins-most notably, EFNA4 and EPHA2-were strongly associated with 10-year risk of ESKD in all cohorts. Furthermore, circulating levels of these proteins correlated with severity of structural lesions in kidney biopsy specimens. In contrast, expression levels of genes encoding these proteins had no apparent effects on the lesions. In in vitro experiments, mimics of miR-1287-5p and miR-197-5p and inhibitors of miR-339-5p and miR-328-3p upregulated concentrations of EPHA2 in either cell lysate, supernatant, or both. CONCLUSIONS: This study reveals novel mechanisms involved in progression to ESKD and points to the importance of systemic factors in the development of diabetic kidney disease. Some circulating miRNAs and axon guidance pathway proteins represent potential targets for new therapies to prevent and treat this condition.

Epigenetic regulation of arginine vasopressin receptor 2 expression by PAX2 and Pax transcription interacting protein.

Renal arginine vasopressin receptor 2 (AVPR2) plays a crucial role in osmoregulation. Engagement of ligand with AVPR2 results in aquaporin 2 movement to the apical membrane and water reabsorption from the urinary filtrate. Despite this essential role, little is known about transcriptional regulation of Avpr2. Here, we identify novel roles for PAX2, a transcription factor crucial for kidney development, and its adaptor protein, Pax transcription interacting protein (PTIP), for epigenetic regulation of Avpr2 and thus body water balance. Chromatin immunoprecipitation (ChIP) from murine inner medulla cells (IMCD-3) identified the minimal DNA-binding region of PAX2 on the Avpr2 promoter. Regulation of Avpr2 by PAX2 was confirmed using a heterologous DNA expression system. PAX2 recruits the adaptor protein PTIP and its associated histone methyltransferase (HMT) complex to Avpr2 promoter, imposing epigenetic marks on this region and throughout the coding sequence that modulate Avpr2 gene transcription. Reduction of PAX2 or PTIP protein levels by siRNA prevented histone lysine methylation and expression of Avpr2. ChIP using mouse or human kidneys determined that PAX2 is highly enriched in the AVPR2 promoter alongside PTIP and HMT proteins, leading to high levels of histone H3 lysine trimethylation within the promoter and throughout the gene. In conclusion, PAX2 provides locus specificity for PTIP, allowing the HMT complex to impart epigenetic changes at the Avpr2 locus and regulate Avpr2 transcription. These finding have major implications for understanding regulation of body water balance.NEW & NOTEWORTHY The transcription factor PAX2 plays an indispensable role in kidney development. In the adult kidney, we identified the first described protein this protein regulates. PAX2 and its interacting partner Pax transcription interacting protein recruit a histone methyltransferase complex to the promoter and epigentically regulate the expression of arginine vasopressin receptor 2, a protein that plays a crucial role in osmoregulation in the distal tubule.

Hypertension induces glomerulosclerosis in phospholipase C-ε1 deficiency.

Loss-of-function mutations in phospholipase C-ε1 (PLCE1) have been detected in patients with nephrotic syndrome, but other family members with the same mutation were asymptomatic, suggesting additional stressor are required to cause the full phenotype. Consistent with these observations, we determined that global Plce1-deficient mice have histologically normal glomeruli and no albuminuria at baseline. Angiotensin II (ANG II) is known to induce glomerular damage in genetically susceptible individuals. Therefore, we tested whether ANG II enhances glomerular damage in Plce1-deficient mice. ANG II increased blood pressure equally in Plce1-deficient and wild-type littermates. Additionally, it led to 20-fold increased albuminuria and significantly more sclerotic glomeruli in Plce1-deficient mice compared with wild-type littermates. Furthermore, Plce1-deficient mice demonstrated diffuse mesangial expansion, podocyte loss, and focal podocyte foot process effacement. To determine whether these effects are mediated by hypertension and hyperfiltration, rather than directly through ANG II, we raised blood pressure to a similar level using DOCA + salt + uninephrectomy and norepinephrine. This caused a fivefold increase in albuminuria in Plce1-deficient mice and a significant increase in the number of sclerotic glomeruli. Consistent with previous findings in mice, we detected strong PLCE1 transcript expression in podocytes using single cell sequencing of human kidney tissue. In hemagglutinin-tagged Plce1 transgenic mice, Plce1 was detected in podocytes and also in glomerular arterioles using immunohistochemistry. Our data demonstrate that Plce1 deficiency in mice predisposes to glomerular damage secondary to hypertensive insults.

Absence of miR-146a in Podocytes Increases Risk of Diabetic Glomerulopathy via Up-regulation of ErbB4 and Notch-1.

Podocyte injury is an early event in diabetic kidney disease and is a hallmark of glomerulopathy. MicroRNA-146a (miR-146a) is highly expressed in many cell types under homeostatic conditions, and plays an important anti-inflammatory role in myeloid cells. However, its role in podocytes is unclear. Here, we show that miR-146a expression levels decrease in the glomeruli of patients with type 2 diabetes (T2D), which correlates with increased albuminuria and glomerular damage. miR-146a levels are also significantly reduced in the glomeruli of albuminuric BTBR ob/ob mice, indicating its significant role in maintaining podocyte health. miR-146a-deficient mice (miR-146a-/-) showed accelerated development of glomerulopathy and albuminuria upon streptozotocin (STZ)-induced hyperglycemia. The miR-146a targets, Notch-1 and ErbB4, were also significantly up-regulated in the glomeruli of diabetic patients and mice, suggesting induction of the downstream TGFß signaling. Treatment with a pan-ErbB kinase inhibitor erlotinib with nanomolar activity against ErbB4 significantly suppressed diabetic glomerular injury and albuminuria in both WT and miR-146a-/- animals. Treatment of podocytes in vitro with TGF-ß1 resulted in increased expression of Notch-1, ErbB4, pErbB4, and pEGFR, the heterodimerization partner of ErbB4, suggesting increased ErbB4/EGFR signaling. TGF-ß1 also increased levels of inflammatory cytokine monocyte chemoattractant protein-1 (MCP-1) and MCP-1 induced protein-1 (MCPIP1), a suppressor of miR-146a, suggesting an autocrine loop. Inhibition of ErbB4/EGFR with erlotinib co-treatment of podocytes suppressed this signaling. Our findings suggest a novel role for miR-146a in protecting against diabetic glomerulopathy and podocyte injury. They also point to ErbB4/EGFR as a novel, druggable target for therapeutic intervention, especially because several pan-ErbB inhibitors are clinically available.

FSGS as an Adaptive Response to Growth-Induced Podocyte Stress.

Glomerular sclerotic lesions develop when the glomerular filtration surface area exceeds the availability of podocyte foot process coverage, but the mechanisms involved are incompletely characterized. We evaluated potential mechanisms using a transgenic (podocin promoter-AA-4E-BP1) rat in which podocyte capacity for hypertrophy in response to growth factor/nutrient signaling is impaired. FSGS lesions resembling human FSGS developed spontaneously by 7 months of age, and could be induced earlier by accelerating kidney hypertrophy by nephrectomy. Early segmental glomerular lesions occurred in the absence of a detectable reduction in average podocyte number per glomerulus and resulted from the loss of podocytes in individual glomerular capillary loops. Parietal epithelial cell division, accumulation on Bowman's capsule, and tuft invasion occurred at these sites. Three different interventions that prevented kidney growth and glomerular enlargement (calorie intake reduction, inhibition of mammalian target of rapamycin complex, and inhibition of angiotensin-converting enzyme) protected against FSGS lesion development, even when initiated late in the process. Ki67 nuclear staining and unbiased transcriptomic analysis identified increased glomerular (but not podocyte) cell cycling as necessary for FSGS lesion development. The rat FSGS-associated transcriptomic signature correlated with human glomerular transcriptomes associated with disease progression, compatible with similar processes occurring in man. We conclude that FSGS lesion development resulted from glomerular growth that exceeded the capacity of podocytes to adapt and adequately cover some parts of the filtration surface. Modest modulation of the growth side of this equation significantly ameliorated FSGS progression, suggesting that glomerular growth is an underappreciated therapeutic target for preservation of renal function.

Defining cell-type specificity at the transcriptional level in human disease.

Cell-lineage-specific transcripts are essential for differentiated tissue function, implicated in hereditary organ failure, and mediate acquired chronic diseases. However, experimental identification of cell-lineage-specific genes in a genome-scale manner is infeasible for most solid human tissues. We developed the first genome-scale method to identify genes with cell-lineage-specific expression, even in lineages not separable by experimental microdissection. Our machine-learning-based approach leverages high-throughput data from tissue homogenates in a novel iterative statistical framework. We applied this method to chronic kidney disease and identified transcripts specific to podocytes, key cells in the glomerular filter responsible for hereditary and most acquired glomerular kidney disease. In a systematic evaluation of our predictions by immunohistochemistry, our in silico approach was significantly more accurate (65% accuracy in human) than predictions based on direct measurement of in vivo fluorescence-tagged murine podocytes (23%). Our method identified genes implicated as causal in hereditary glomerular disease and involved in molecular pathways of acquired and chronic renal diseases. Furthermore, based on expression analysis of human kidney disease biopsies, we demonstrated that expression of the podocyte genes identified by our approach is significantly related to the degree of renal impairment in patients. Our approach is broadly applicable to define lineage specificity in both cell physiology and human disease contexts. We provide a user-friendly website that enables researchers to apply this method to any cell-lineage or tissue of interest. Identified cell-lineage-specific transcripts are expected to play essential tissue-specific roles in organogenesis and disease and can provide starting points for the development of organ-specific diagnostics and therapies.

Cognitive Impairment and Dementia in Older Adults With Chronic Kidney Disease: A Review.

Individuals with chronic kidney disease (CKD), especially older adults, are at more risk of experiencing cognitive impairment, possibly leading to mild cognitive impairment and/or dementia. Studies report associations between CKD and cognitive impairment; although unclear, there seems to be a graded association between stage of CKD and affected cognitive domains, with executive function being affected earlier in the process than episodic memory and global ability. In CKD, dysexecutive mild cognitive impairment and vascular dementia are also more prominent than other subtypes. Explanations are directed toward traditional and nontraditional vascular factors, which may also explain or mediate the association between CKD and type of cognitive impairment. Future research is urged to focus on the longitudinal association between specific domains of cognitive function, including executive function and memory and CKD; to develop screening tools fit for every CKD stage in elderly individuals, and lastly, to use imaging methods that may help clarify the underlying mechanisms connecting the kidney and the brain.

Glomerular Aging and Focal Global Glomerulosclerosis: A Podometric Perspective.

Kidney aging is associated with an increasing proportion of globally scarred glomeruli, decreasing renal function, and exponentially increasing ESRD prevalence. In model systems, podocyte depletion causes glomerulosclerosis, suggesting age-associated glomerulosclerosis could be caused by a similar mechanism. We measured podocyte number, size, density, and glomerular volume in 89 normal kidney samples from living and deceased kidney donors and normal poles of nephrectomies. Podocyte nuclear density decreased with age due to a combination of decreased podocyte number per glomerulus and increased glomerular volume. Compensatory podocyte cell hypertrophy prevented a change in the proportion of tuft volume occupied by podocytes. Young kidneys had high podocyte reserve (podocyte density >300 per 10(6) µm(3)), but by 70-80 years of age, average podocyte nuclear density decreased to, <100 per 10(6) µm(3), with corresponding podocyte hypertrophy. In older age podocyte detachment rate (urine podocin mRNA-to-creatinine ratio) was higher than at younger ages and podocytes were stressed (increased urine podocin-to-nephrin mRNA ratio). Moreover, in older kidneys, proteinaceous material accumulated in the Bowman space of glomeruli with low podocyte density. In a subset of these glomeruli, mass podocyte detachment events occurred in association with podocytes becoming binucleate (mitotic podocyte catastrophe) and subsequent wrinkling of glomerular capillaries, tuft collapse, and periglomerular fibrosis. In kidneys of young patients with underlying glomerular diseases similar pathologic events were identified in association with focal global glomerulosclerosis. Podocyte density reduction with age may therefore directly lead to focal global glomerulosclerosis, and all progressive glomerular diseases can be considered superimposed accelerators of this underlying process.

MicroRNA-21 in glomerular injury.

TGF-ß(1) is a pleotropic growth factor that mediates glomerulosclerosis and podocyte apoptosis, hallmarks of glomerular diseases. The expression of microRNA-21 (miR-21) is regulated by TGF-ß(1), and miR-21 inhibits apoptosis in cancer cells. TGF-ß(1)-transgenic mice exhibit accelerated podocyte loss and glomerulosclerosis. We determined that miR-21 expression increases rapidly in cultured murine podocytes after exposure to TGF-ß(1) and is higher in kidneys of TGF-ß(1)-transgenic mice than wild-type mice. miR-21-deficient TGF-ß(1)-transgenic mice showed increased proteinuria and glomerular extracellular matrix deposition and fewer podocytes per glomerular tuft compared with miR-21 wild-type TGF-ß(1)-transgenic littermates. Similarly, miR-21 expression was increased in streptozotocin-induced diabetic mice, and loss of miR-21 in these mice was associated with increased albuminuria, podocyte depletion, and mesangial expansion. In cultured podocytes, inhibition of miR-21 was accompanied by increases in the rate of cell death, TGF-ß/Smad3-signaling activity, and expression of known proapoptotic miR-21 target genes p53, Pdcd4, Smad7, Tgfbr2, and Timp3. In American-Indian patients with diabetic nephropathy (n=48), albumin-to-creatinine ratio was positively associated with miR-21 expression in glomerular fractions (r=0.6; P<0.001) but not tubulointerstitial fractions (P=0.80). These findings suggest that miR-21 ameliorates TGF-ß(1) and hyperglycemia-induced glomerular injury through repression of proapoptotic signals, thereby inhibiting podocyte loss. This finding is in contrast to observations in murine models of tubulointerstitial kidney injury but consistent with findings in cancer models. The aggravation of glomerular disease in miR-21-deficient mice and the positive association with albumin-to-creatinine ratio in patients with diabetic nephropathy support miR-21 as a feedback inhibitor of TGF-ß signaling and functions.

miR-21 mediates hematopoietic suppression in MDS by activating TGF-ß signaling.

Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis that leads to peripheral cytopenias. We observed that SMAD7, a negative regulator of transforming growth factor-beta (TGF-ß) receptor-I kinase, is markedly reduced in MDS and leads to ineffective hematopoiesis by overactivation of TGF-ß signaling. To determine the cause of SMAD7 reduction in MDS, we analyzed the 3'UTR of the gene and determined that it contains a highly conserved putative binding site for microRNA-21. We observed significantly elevated levels of miR-21 in MDS marrow samples when compared with age-matched controls. miR-21 was shown to directly bind to the 3'UTR of SMAD7 and reduce its expression in hematopoietic cells. Next, we tested the role of miR-21 in regulating TGF-ß signaling in a TGF-ß-overexpressing transgenic mouse model that develops progressive anemia and dysplasia and thus serves as a model of human bone marrow failure. Treatment with a chemically modified miR-21 inhibitor led to significant increases in hematocrit and led to an increase in SMAD7 expression in vivo. Inhibition of miR-21 also led to an increase in erythroid colony formation from primary MDS bone marrow progenitors, demonstrating its ability in stimulating hematopoiesis in vitro. Taken together, these studies demonstrate the role of miR-21 in regulating overactivated TGF-ß signaling in MDS.

Strategy and rationale for urine collection protocols employed in the NEPTUNE study.

BACKGROUND: Glomerular diseases are potentially fatal, requiring aggressive interventions and close monitoring. Urine is a readily-accessible body fluid enriched in molecular signatures from the kidney and therefore particularly suited for routine clinical analysis as well as development of non-invasive biomarkers for glomerular diseases. METHODS: The Nephrotic Syndrome Study Network (NEPTUNE; ClinicalTrials.gov Identifier NCT01209000) is a North American multicenter collaborative consortium established to develop a translational research infrastructure for nephrotic syndrome. This includes standardized urine collections across all participating centers for the purpose of discovering non-invasive biomarkers for patients with nephrotic syndrome due to minimal change disease, focal segmental glomerulosclerosis, and membranous nephropathy. Here we describe the organization and methods of urine procurement and banking procedures in NEPTUNE. RESULTS: We discuss the rationale for urine collection and storage conditions, and demonstrate the performance of three experimental analytes (neutrophil gelatinase-associated lipocalin [NGAL], retinol binding globulin, and alpha-1 microglobulin) under these conditions with and without urine preservatives (thymol, toluene, and boric acid). We also demonstrate the quality of RNA and protein collected from the urine cellular pellet and exosomes. CONCLUSIONS: The urine collection protocol in NEPTUNE allows robust detection of a wide range of proteins and RNAs from urine supernatant and pellets collected longitudinally from each patient over 5 years. Combined with the detailed clinical and histopathologic data, this provides a unique resource for exploration and validation of new or accepted markers of glomerular diseases. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT01209000.

Genetic Analysis of Obesity-Induced Diabetic Nephropathy in BTBR Mice.

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease in the U.S. and has a significant impact on human suffering. Leptin-deficient BTBR (BTBRob/ob) mice develop hallmark features of obesity-induced DN, whereas leptin-deficient C57BL/6J (B6ob/ob) mice do not. To identify genetic loci that underlie this strain difference, we constructed an F2 intercross between BTBRob/ob and B6ob/ob mice. We isolated kidneys from 460 F2 mice and histologically scored them for percent mesangial matrix and glomerular volume (∼50 glomeruli per mouse), yielding ∼45,000 distinct measures in total. The same histological measurements were made in kidneys from B6 and BTBR mice, either lean or obese (Lepob/ob), at 4 and 10 weeks of age, allowing us to assess the contribution of strain, age, and obesity to glomerular pathology. All F2 mice were genotyped for ∼5,000 single nucleotide polymorphisms (SNPs), ∼2,000 of which were polymorphic between B6 and BTBR, enabling us to identify a quantitative trait locus (QTL) on chromosome 7, with a peak at ∼30 Mbp, for percent mesangial matrix, glomerular volume, and mesangial volume. The podocyte-specific gene nephrin (Nphs1) is physically located at the QTL and contains high-impact SNPs in BTBR, including several missense variants within the extracellular immunoglobulin-like domains.