The deacylase sirtuin 5 reduces malonylation in nonmitochondrial metabolic pathways in diabetic kidney disease.

Early diabetic kidney disease (DKD) is marked by dramatic metabolic reprogramming due to nutrient excess, mitochondrial dysfunction, and increased renal energy requirements from hyperfiltration. We hypothesized that changes in metabolism in DKD may be regulated by Sirtuin 5 (SIRT5), a deacylase that removes posttranslational modifications derived from acyl-coenzyme A and has been demonstrated to regulate numerous metabolic pathways. We found decreased malonylation in the kidney cortex (∼80% proximal tubules) of type 2 diabetic BKS db/db mice, associated with increased SIRT5 expression. We performed a proteomics analysis of malonylated peptides and found that proteins with significantly decreased malonylated lysines in the db/db cortex were enriched in nonmitochondrial metabolic pathways: glycolysis and peroxisomal fatty acid oxidation. To confirm relevance of these findings in human disease, we analyzed diabetic kidney transcriptomic data from a cohort of Southwestern American Indians, which revealed a tubulointerstitial-specific increase in Sirt5 expression. These data were further corroborated by immunofluorescence data of SIRT5 from nondiabetic and DKD cohorts. Furthermore, overexpression of SIRT5 in cultured human proximal tubules demonstrated increased aerobic glycolysis. Conversely, we observed reduced glycolysis with decreased SIRT5 expression. These findings suggest that SIRT5 may lead to differential nutrient partitioning and utilization in DKD. Taken together, our findings highlight a previously unrecognized role for SIRT5 in metabolic reprogramming in DKD.

Tubular dysfunction impairs renal excretion of pseudouridine in diabetic kidney disease.

Plasma nucleosides-pseudouridine (PU) and N2N2-dimethyl guanosine (DMG) predict the progression of type 2 diabetic kidney disease (DKD) to end-stage renal disease, but the mechanisms underlying this relationship are not well understood. We used a well-characterized model of type 2 diabetes (db/db mice) and control nondiabetic mice (db/m mice) to characterize the production and excretion of PU and DMG levels using liquid chromatography-mass spectrometry. The fractional excretion of PU and DMG was decreased in db/db mice compared with control mice at 24 wk before any changes to renal function. We then examined the dynamic changes in nucleoside metabolism using in vivo metabolic flux analysis with the injection of labeled nucleoside precursors. Metabolic flux analysis revealed significant decreases in the ratio of urine-to-plasma labeling of PU and DMG in db/db mice compared with db/m mice, indicating significant tubular dysfunction in diabetic kidney disease. We observed that the gene and protein expression of the renal tubular transporters involved with nucleoside transport in diabetic kidneys in mice and humans was reduced. In conclusion, this study strongly suggests that tubular handling of nucleosides is altered in early DKD, in part explaining the association of PU and DMG with human DKD progression observed in previous studies.NEW & NOTEWORTHY Tubular dysfunction explains the association between the nucleosides pseudouridine and N2N2-dimethyl guanosine and diabetic kidney disease.

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.

Sodium glucose co-transporter 2 inhibition increases epidermal growth factor expression and improves outcomes in patients with type 2 diabetes.

Underlying molecular mechanisms of the kidney protective effects of sodium glucose co-transporter 2 (SGLT2) inhibitors are not fully elucidated. Therefore, we studied the association between urinary epidermal growth factor (uEGF), a mitogenic factor involved in kidney repair, and kidney outcomes in patients with type 2 diabetes (T2D). The underlying molecular mechanisms of the SGLT2 inhibitor canagliflozin on EGF using single-cell RNA sequencing from kidney tissue were examined. Urinary EGF-to-creatinine ratio (uEGF/Cr) was measured in 3521 CANagliflozin cardioVascular Assessment Study (CANVAS) participants at baseline and week 52. Associations of uEGF/Cr with kidney outcome were assessed using multivariable-adjusted Cox regression models. Single-cell RNA sequencing was performed using protocol kidney biopsy tissue from ten young patients with T2D on SGLT2i, six patients with T2D on standard care only, and six healthy controls (HCs). In CANVAS, each doubling in baseline uEGF/Cr was associated with a 12% (95% confidence interval 1-22) decreased risk of kidney outcome. uEGF/Cr decreased after 52 weeks with placebo and remained stable with canagliflozin (between-group difference +7.3% (2.0-12.8). In young persons with T2D, EGF mRNA was primarily expressed in the thick ascending loop of Henle. Expression in biopsies from T2D without SGLT2i was significantly lower compared to HCs, whereas treatment with SGLT2i increased EGF levels closer to the healthy state. In young persons with T2D without SGLT2i, endothelin-1 emerged as a key regulator of the EGF co-expression network. SGLT2i treatment was associated with a shift towards normal EGF expression. Thus, decreased uEGF represents increased risk of kidney disease progression in patients with T2D. Canagliflozin increased kidney tissue expression of EGF and was associated with a downstream signaling cascade linked to tubular repair and reversal of tubular injury.

Precision nephrology identified tumor necrosis factor activation variability in minimal change disease and focal segmental glomerulosclerosis.

The diagnosis of nephrotic syndrome relies on clinical presentation and descriptive patterns of injury on kidney biopsies, but not specific to underlying pathobiology. Consequently, there are variable rates of progression and response to therapy within diagnoses. Here, an unbiased transcriptomic-driven approach was used to identify molecular pathways which are shared by subgroups of patients with either minimal change disease (MCD) or focal segmental glomerulosclerosis (FSGS). Kidney tissue transcriptomic profile-based clustering identified three patient subgroups with shared molecular signatures across independent, North American, European, and African cohorts. One subgroup had significantly greater disease progression (Hazard Ratio 5.2) which persisted after adjusting for diagnosis and clinical measures (Hazard Ratio 3.8). Inclusion in this subgroup was retained even when clustering was limited to those with less than 25% interstitial fibrosis. The molecular profile of this subgroup was largely consistent with tumor necrosis factor (TNF) pathway activation. Two TNF pathway urine markers were identified, tissue inhibitor of metalloproteinases-1 (TIMP-1) and monocyte chemoattractant protein-1 (MCP-1), that could be used to predict an individual's TNF pathway activation score. Kidney organoids and single-nucleus RNA-sequencing of participant kidney biopsies, validated TNF-dependent increases in pathway activation score, transcript and protein levels of TIMP-1 and MCP-1, in resident kidney cells. Thus, molecular profiling identified a subgroup of patients with either MCD or FSGS who shared kidney TNF pathway activation and poor outcomes. A clinical trial testing targeted therapies in patients selected using urinary markers of TNF pathway activation is ongoing.

Intraindividual variations of urinary biomarkers in hospitalized children with glomerular diseases: a prospective observational study.

This study aimed to assess the intraindividual variations of urinary biomarkers in hospitalized children with glomerular diseases. Hospitalized children with glomerular diseases participated in the study. For each patient, an overnight (9:00 p.m.-7:00 a.m.) urine was collected, followed by a 24-h urine (classified into four distinct periods: morning 7:00 a.m.-12:00 p.m., afternoon 12:00 p.m.-4:00 p.m., evening 4:00 p.m.-9:00 p.m., and overnight 9:00 p.m.-7:00 a.m.). The concentrations of protein, albumin, N-acetyl-beta-D-glucosaminidase, and epidermal growth factor (EGF) were measured and normalized by three correction factors (creatinine, osmolality, or specific gravity, respectively). Additionally, the 2nd overnight urine sample was grouped into different aliquots according to centrifugation, additives, storage temperature, or delayed processing. Twenty (14 boys, 6 girls) children were enrolled, with an average age of 11.3 years. Among the three correction factors, creatinine-normalized biomarkers provided the best agreements among different periods over 24 h. There were significant diurnal variations during 24 h in the concentrations of urinary protein, albumin, N-acetyl-beta-D-glucosaminidase, and EGF (p = 0.001, p = 0.003, p = 0.003, and p = 0.003, respectively). Evening urine overestimated 24-h urinary protein and albumin, while overnight urine underestimated 24-h urinary albumin. Urinary EGF showed low variability within a day or between the 2 days (coefficients of variation 10.2% and 10.6%, respectively) and excellent agreements (intraclass correlation coefficients > 0.9) with 24-h urinary concentration. Furthermore, urinary EGF was not affected by centrifugation, additives, storage temperature, or delayed processing of urine samples (all p > 0.05).  Conclusion: Given the diurnal variations of urinary biomarkers, urine samples should be collected during the same time period in clinical practice if possible. The results also extend the evidence for urinary EGF as a relatively stable biomarker applied in the future clinical practice. What is Known: • Urinary biomarkers have been widely used or discussed in making diagnoses and therapy regimens and estimating the prognosis of pediatric glomerular diseases. It remains unclear whether their levels would be affected by the time of sample collection, processing methods, and storage conditions in hospitalized children with glomerular diseases. What is New: • The levels of both commonly used biomarkers and novel biomarkers exhibited diurnal variations in hospitalized children with glomerular diseases. • Our results extend the evidence for urinary EGF as a relatively stable biomarker applied in the future clinical practice.

A role for NPY-NPY2R signaling in albuminuric kidney disease.

Albuminuria is an independent risk factor for the progression to end-stage kidney failure, cardiovascular morbidity, and premature death. As such, discovering signaling pathways that modulate albuminuria is desirable. Here, we studied the transcriptomes of podocytes, key cells in the prevention of albuminuria, under diabetic conditions. We found that Neuropeptide Y (NPY) was significantly down-regulated in insulin-resistant vs. insulin-sensitive mouse podocytes and in human glomeruli of patients with early and late-stage diabetic nephropathy, as well as other nondiabetic glomerular diseases. This contrasts with the increased plasma and urinary levels of NPY that are observed in such conditions. Studying NPY-knockout mice, we found that NPY deficiency in vivo surprisingly reduced the level of albuminuria and podocyte injury in models of both diabetic and nondiabetic kidney disease. In vitro, podocyte NPY signaling occurred via the NPY2 receptor (NPY2R), stimulating PI3K, MAPK, and NFAT activation. Additional unbiased proteomic analysis revealed that glomerular NPY-NPY2R signaling predicted nephrotoxicity, modulated RNA processing, and inhibited cell migration. Furthermore, pharmacologically inhibiting the NPY2R in vivo significantly reduced albuminuria in adriamycin-treated glomerulosclerotic mice. Our findings suggest a pathogenic role of excessive NPY-NPY2R signaling in the glomerulus and that inhibiting NPY-NPY2R signaling in albuminuric kidney disease has therapeutic potential.

Molecular Characterization of Membranous Nephropathy.

BACKGROUND: Molecular characterization of nephropathies may facilitate pathophysiologic insight, development of targeted therapeutics, and transcriptome-based disease classification. Although membranous nephropathy (MN) is a common cause of adult-onset nephrotic syndrome, the molecular pathways of kidney damage in MN require further definition. METHODS: We applied a machine-learning framework to predict diagnosis on the basis of gene expression from the microdissected kidney tissue of participants in the Nephrotic Syndrome Study Network (NEPTUNE) cohort. We sought to identify differentially expressed genes between participants with MN versus those of other glomerulonephropathies across the NEPTUNE and European Renal cDNA Bank (ERCB) cohorts, to find MN-specific gene modules in a kidney-specific functional network, and to identify cell-type specificity of MN-specific genes using single-cell sequencing data from reference nephrectomy tissue. RESULTS: Glomerular gene expression alone accurately separated participants with MN from those with other nephrotic syndrome etiologies. The top predictive classifier genes from NEPTUNE participants were also differentially expressed in the ERCB participants with MN. We identified a signature of 158 genes that are significantly differentially expressed in MN across both cohorts, finding 120 of these in a validation cohort. This signature is enriched in targets of transcription factor NF-κB. Clustering these MN-specific genes in a kidney-specific functional network uncovered modules with functional enrichments, including in ion transport, cell projection morphogenesis, regulation of adhesion, and wounding response. Expression data from reference nephrectomy tissue indicated 43% of these genes are most highly expressed by podocytes. CONCLUSIONS: These results suggest that, relative to other glomerulonephropathies, MN has a distinctive molecular signature that includes upregulation of many podocyte-expressed genes, provides a molecular snapshot of MN, and facilitates insight into MN's underlying pathophysiology.

Genome-wide meta-analysis and omics integration identifies novel genes associated with diabetic kidney disease.

AIMS/HYPOTHESIS: Diabetic kidney disease (DKD) is the leading cause of kidney failure and has a substantial genetic component. Our aim was to identify novel genetic factors and genes contributing to DKD by performing meta-analysis of previous genome-wide association studies (GWAS) on DKD and by integrating the results with renal transcriptomics datasets. METHODS: We performed GWAS meta-analyses using ten phenotypic definitions of DKD, including nearly 27,000 individuals with diabetes. Meta-analysis results were integrated with estimated quantitative trait locus data from human glomerular (N=119) and tubular (N=121) samples to perform transcriptome-wide association study. We also performed gene aggregate tests to jointly test all available common genetic markers within a gene, and combined the results with various kidney omics datasets. RESULTS: The meta-analysis identified a novel intronic variant (rs72831309) in the TENM2 gene associated with a lower risk of the combined chronic kidney disease (eGFR<60 ml/min per 1.73 m2) and DKD (microalbuminuria or worse) phenotype (p=9.8×10-9; although not withstanding correction for multiple testing, p>9.3×10-9). Gene-level analysis identified ten genes associated with DKD (COL20A1, DCLK1, EIF4E, PTPRN-RESP18, GPR158, INIP-SNX30, LSM14A and MFF; p<2.7×10-6). Integration of GWAS with human glomerular and tubular expression data demonstrated higher tubular AKIRIN2 gene expression in individuals with vs without DKD (p=1.1×10-6). The lead SNPs within six loci significantly altered DNA methylation of a nearby CpG site in kidneys (p<1.5×10-11). Expression of lead genes in kidney tubules or glomeruli correlated with relevant pathological phenotypes (e.g. TENM2 expression correlated positively with eGFR [p=1.6×10-8] and negatively with tubulointerstitial fibrosis [p=2.0×10-9], tubular DCLK1 expression correlated positively with fibrosis [p=7.4×10-16], and SNX30 expression correlated positively with eGFR [p=5.8×10-14] and negatively with fibrosis [p<2.0×10-16]). CONCLUSIONS/INTERPRETATION: Altogether, the results point to novel genes contributing to the pathogenesis of DKD. DATA AVAILABILITY: The GWAS meta-analysis results can be accessed via the type 1 and type 2 diabetes (T1D and T2D, respectively) and Common Metabolic Diseases (CMD) Knowledge Portals, and downloaded on their respective download pages ( https://t1d.hugeamp.org/downloads.html ; https://t2d.hugeamp.org/downloads.html ; https://hugeamp.org/downloads.html ).

Glomerular endothelial cell-podocyte stresses and crosstalk in structurally normal kidney transplants.

Increased podocyte detachment begins immediately after kidney transplantation and is associated with long-term allograft failure. We hypothesized that cell-specific transcriptional changes in podocytes and glomerular endothelial cells after transplantation would offer mechanistic insights into the podocyte detachment process. To test this, we evaluated cell-specific transcriptional profiles of glomerular endothelial cells and podocytes from 14 patients of their first-year surveillance biopsies with normal histology from low immune risk recipients with no post-transplant complications and compared these to biopsies of 20 healthy living donor controls. Glomerular endothelial cells from these surveillance biopsies were enriched for genes related to fluid shear stress, angiogenesis, and interferon signaling. In podocytes, pathways were enriched for genes in response to growth factor signaling and actin cytoskeletal reorganization but also showed evidence of podocyte stress as indicated by reduced nephrin (adhesion protein) gene expression. In parallel, transcripts coding for proteins required to maintain podocyte adherence to the underlying glomerular basement membrane were downregulated, including the major glomerular podocyte integrin α3 and the actin cytoskeleton-related gene synaptopodin. The reduction in integrin α3 protein expression in surveillance biopsies was confirmed by immunoperoxidase staining. The combined growth and stress response of patient allografts post-transplantation paralleled similar changes in a rodent model of nephrectomy-induced glomerular hypertrophic stress that progress to develop proteinuria and glomerulosclerosis with shortened kidney life span. Thus, even among patients with apparently healthy allografts with no detectable histologic abnormality including alloimmune injury, transcriptomic changes reflecting cell stresses are already set in motion that could drive hypertrophy-associated glomerular disease progression.

Molecular programs associated with glomerular hyperfiltration in early diabetic kidney disease.

Hyperfiltration is a state of high glomerular filtration rate (GFR) observed in early diabetes that damages glomeruli, resulting in an iterative process of increasing filtration load on fewer and fewer remaining functional glomeruli. To delineate underlying cellular mechanisms of damage associated with hyperfiltration, transcriptional profiles of kidney biopsies from Pima Indians with type 2 diabetes with or without early-stage diabetic kidney disease were grouped into two hyperfiltration categories based on annual iothalamate GFR measurements. Twenty-six participants with a peak GFR measurement within two years of biopsy were categorized as the hyperfiltration group, and 26 in whom biopsy preceded peak GFR by over two years were considered pre-hyperfiltration. The hyperfiltration group had higher hemoglobin A1c, higher urine albumin-to-creatinine ratio, increased glomerular basement membrane width and lower podocyte density compared to the pre-hyperfiltration group. A glomerular 1240-gene transcriptional signature identified in the hyperfiltration group was enriched for endothelial stress response signaling genes, including endothelin-1, tec-kinase and transforming growth factor-ß1 pathways, with the majority of the transcripts mapped to endothelial and inflammatory cell clusters in kidney single cell transcriptional data. Thus, our analysis reveals molecular pathomechanisms associated with hyperfiltration in early diabetic kidney disease involving putative ligand-receptor pairs with downstream intracellular targets linked to cellular crosstalk between endothelial and mesangial cells.

Serum Level of Polyubiquitinated PTEN and Loss of Kidney Function in American Indians With Type 2 Diabetes.

RATIONALE & OBJECTIVE: Fibrosis is a major driver of chronic kidney disease, and epithelial-mesenchymal transition (EMT) may contribute to its development. A polyubiquitinated form of phosphatase and tensin homolog (PTENK27polyUb) promotes EMT in vitro. Thus, it is a potentially useful biomarker of progressive kidney fibrosis and may predict loss of kidney function. STUDY DESIGN: Observational cohort study. SETTING & PARTICIPANTS: Southwest United States, American Indians (154 women, 80 men) with or at high risk for diabetic kidney disease (DKD). PREDICTORS: Serum level of PTENK27polyUb. OUTCOME: ≥40% loss of glomerular filtration rate (GFR) or onset of kidney failure. Kidney structural measures in a subset of study participants who underwent research kidney biopsies (n = 77). ANALYTICAL APPROACH: Cox proportional hazards models adjusted for age, sex, diabetes duration, hemoglobin A1c (HbA1c), blood pressure, use of renin angiotensin system (RAS) blockers, measured GFR, and albuminuria. Spearman correlations for associations with structural measures. RESULTS: At baseline, the participants' mean age was 42.8 ± 10.5 (SD) years, diabetes duration 11.5 ± 7.1 years, mean arterial pressure 90.5 ± 9.5 mm Hg, HbA1c 9.3 ± 2.4%, GFR 152 ± 45 mL/min, and median urinary albumin-creatinine ratio 38 (interquartile range, 14-215) mg/g. RAS blockers were being used by 64 participants (27.4%). A higher PTENK27polyUb value was associated with a greater risk of ≥40% loss of GFR during a median follow-up period of 6.3 years (HR for quartile 4 [Q4] vs Q1, 3.95 [95% CI, 2.23-6.98], P < 0.001). Serum PTENK27polyUb was associated with an increased risk of kidney failure over a median follow-up period of 15.8 years (HR for Q4 vs Q1, 5.66 [95% CI, 1.99-16.13], P = 0.001). Baseline serum PTENK27polyUb in the biopsy subset correlated with structural measures including glomerular basement membrane width (ρ = 0.370, P < 0.001) and mesangial fractional volume (ρ = 0.392, P < 0.001). LIMITATIONS: Small study in single population. CONCLUSIONS: Higher serum PTENK27polyUb is associated with increased risk for GFR decline and kidney failure in American Indians with type 2 diabetes.

Gene expression profiles of diabetic kidney disease and neuropathy in eNOS knockout mice: Predictors of pathology and RAS blockade effects.

Diabetic kidney disease (DKD) and diabetic peripheral neuropathy (DPN) are two common diabetic complications. However, their pathogenesis remains elusive and current therapies are only modestly effective. We evaluated genome-wide expression to identify pathways involved in DKD and DPN progression in db/db eNOS-/- mice receiving renin-angiotensin-aldosterone system (RAS)-blocking drugs to mimic the current standard of care for DKD patients. Diabetes and eNOS deletion worsened DKD, which improved with RAS treatment. Diabetes also induced DPN, which was not affected by eNOS deletion or RAS blockade. Given the multiple factors affecting DKD and the graded differences in disease severity across mouse groups, an automatic data analysis method, SOM, or self-organizing map was used to elucidate glomerular transcriptional changes associated with DKD, whereas pairwise bioinformatic analysis was used for DPN. These analyses revealed that enhanced gene expression in several pro-inflammatory networks and reduced expression of development genes correlated with worsening DKD. Although RAS treatment ameliorated the nephropathy phenotype, it did not alter the more abnormal gene expression changes in kidney. Moreover, RAS exacerbated expression of genes related to inflammation and oxidant generation in peripheral nerves. The graded increase in inflammatory gene expression and decrease in development gene expression with DKD progression underline the potentially important role of these pathways in DKD pathogenesis. Since RAS blockers worsened this gene expression pattern in both DKD and DPN, it may partly explain the inadequate therapeutic efficacy of such blockers.

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.

IGFBP-1 expression is reduced in human type 2 diabetic glomeruli and modulates ß1-integrin/FAK signalling in human podocytes.

AIMS/HYPOTHESIS: Podocyte loss or injury is one of the earliest features observed in the pathogenesis of diabetic kidney disease (DKD), which is the leading cause of end-stage renal failure worldwide. Dysfunction in the IGF axis, including in IGF binding proteins (IGFBPs), is associated with DKD, particularly in the early stages of disease progression. The aim of this study was to investigate the potential roles of IGFBPs in the development of type 2 DKD, focusing on podocytes. METHODS: IGFBP expression was analysed in the Pima DKD cohort, alongside data from the Nephroseq database, and in ex vivo human glomeruli. Conditionally immortalised human podocytes and glomerular endothelial cells were studied in vitro, where IGFBP-1 expression was analysed using quantitative PCR and ELISAs. Cell responses to IGFBPs were investigated using migration, cell survival and adhesion assays; electrical cell-substrate impedance sensing; western blotting; and high-content automated imaging. RESULTS: Data from the Pima DKD cohort and from the Nephroseq database demonstrated a significant reduction in glomerular IGFBP-1 in the early stages of human type 2 DKD. In the glomerulus, IGFBP-1 was predominantly expressed in podocytes and controlled by phosphoinositide 3-kinase (PI3K)-forkhead box O1 (FoxO1) activity. In vitro, IGFBP-1 signalled to podocytes via ß1-integrins, resulting in increased phosphorylation of focal-adhesion kinase (FAK), increasing podocyte motility, adhesion, electrical resistance across the adhesive cell layer and cell viability. CONCLUSIONS/INTERPRETATION: This work identifies a novel role for IGFBP-1 in the regulation of podocyte function and that the glomerular expression of IGFBP-1 is reduced in the early stages of type 2 DKD, via reduced FoxO1 activity. Thus, we hypothesise that strategies to maintain glomerular IGFBP-1 levels may be beneficial in maintaining podocyte function early in DKD.

Annexin A1 alleviates kidney injury by promoting the resolution of inflammation in diabetic nephropathy.

Since failed resolution of inflammation is a major contributor to the progression of diabetic nephropathy, identifying endogenously generated molecules that promote the physiological resolution of inflammation may be a promising therapeutic approach for this disease. Annexin A1 (ANXA1), as an endogenous mediator, plays an important role in resolving inflammation. Whether ANXA1 could affect established diabetic nephropathy through modulating inflammatory states remains largely unknown. In the current study, we found that in patients with diabetic nephropathy, the levels of ANXA1 were upregulated in kidneys, and correlated with kidney function as well as kidney outcomes. Therefore, the role of endogenous ANXA1 in mouse models of diabetic nephropathy was further evaluated. ANXA1 deficiency exacerbated kidney injuries, exhibiting more severe albuminuria, mesangial matrix expansion, tubulointerstitial lesions, kidney inflammation and fibrosis in high fat diet/streptozotocin-induced-diabetic mice. Consistently, ANXA1 overexpression ameliorated kidney injuries in mice with diabetic nephropathy. Additionally, we found Ac2-26 (an ANXA1 mimetic peptide) had therapeutic potential for alleviating kidney injuries in db/db mice and diabetic Anxa1 knockout mice. Mechanistic studies demonstrated that intracellular ANXA1 bound to the transcription factor NF-κB p65 subunit, inhibiting its activation thereby modulating the inflammatory state. Thus, our data indicate that ANXA1 may be a promising therapeutic approach to treating and reversing diabetic nephropathy.

An eQTL Landscape of Kidney Tissue in Human Nephrotic Syndrome.

Expression quantitative trait loci (eQTL) studies illuminate the genetics of gene expression and, in disease research, can be particularly illuminating when using the tissues directly impacted by the condition. In nephrology, there is a paucity of eQTL studies of human kidney. Here, we used whole-genome sequencing (WGS) and microdissected glomerular (GLOM) and tubulointerstitial (TI) transcriptomes from 187 individuals with nephrotic syndrome (NS) to describe the eQTL landscape in these functionally distinct kidney structures. Using MatrixEQTL, we performed cis-eQTL analysis on GLOM (n = 136) and TI (n = 166). We used the Bayesian "Deterministic Approximation of Posteriors" (DAP) to fine-map these signals, eQTLBMA to discover GLOM- or TI-specific eQTLs, and single-cell RNA-seq data of control kidney tissue to identify the cell type specificity of significant eQTLs. We integrated eQTL data with an IgA Nephropathy (IgAN) GWAS to perform a transcriptome-wide association study (TWAS). We discovered 894 GLOM eQTLs and 1,767 TI eQTLs at FDR < 0.05. 14% and 19% of GLOM and TI eQTLs, respectively, had >1 independent signal associated with its expression. 12% and 26% of eQTLs were GLOM specific and TI specific, respectively. GLOM eQTLs were most significantly enriched in podocyte transcripts and TI eQTLs in proximal tubules. The IgAN TWAS identified significant GLOM and TI genes, primarily at the HLA region. In this study, we discovered GLOM and TI eQTLs, identified those that were tissue specific, deconvoluted them into cell-specific signals, and used them to characterize known GWAS alleles. These data are available for browsing and download via our eQTL browser, "nephQTL."

Renin-angiotensin system inhibition reverses the altered triacylglycerol metabolic network in diabetic kidney disease.

OBJECTIVE: Dyslipidemia is a significant risk factor for progression of diabetic kidney disease (DKD). Determining the changes in individual lipids and lipid networks across a spectrum of DKD severity may identify lipids that are pathogenic to DKD progression. METHODS: We performed untargeted lipidomic analysis of kidney cortex tissue from diabetic db/db and db/db eNOS-/- mice along with non-diabetic littermate controls. A subset of mice were treated with the renin-angiotensin system (RAS) inhibitors, lisinopril and losartan, which improves the DKD phenotype in the db/db eNOS-/- mouse model. RESULTS: Of the three independent variables in this study, diabetes had the largest impact on overall lipid levels in the kidney cortex, while eNOS expression and RAS inhibition had smaller impacts on kidney lipid levels. Kidney lipid network architecture, particularly of networks involving glycerolipids such as triacylglycerols, was substantially disrupted by worsening kidney disease in the db/db eNOS-/- mice compared to the db/db mice, a feature that was reversed with RAS inhibition. This was associated with decreased expression of the stearoyl-CoA desaturases, Scd1 and Scd2, with RAS inhibition. CONCLUSIONS: In addition to the known salutary effect of RAS inhibition on DKD progression, our results suggest a previously unrecognized role for RAS inhibition on the kidney triacylglycerol lipid metabolic network.

Transcriptome analysis of primary podocytes reveals novel calcium regulated regulatory networks.

Podocytes are pivotal in establishing the selective permeability of the glomerular filtration barrier. Recently, we showed that an increase of the intracellular calcium ion concentration [Ca2+ ] causes a rapid and transient actin reset (CaAR) measurable through live imaging microscopy using lifeact-mCherry as an actin dye in different cell types including the podocyte. This and other studies show the critical role [Ca2+ ] and the actin cytoskeleton play in podocyte homeostasis. To further investigate the role of [Ca2+ ] and the actin cytoskeleton in podocytes, we used a double fluorescent reporter mouse model to establish a primary podocyte culture system. We treated these podocytes temporarily with a Calcium Ionophore and facultatively with Latrunculin A, an inhibitor of actin polymerization. Unbiased genome wide transcriptional analysis identified a transcriptional response in podocytes to elevated [Ca2+ ] levels, affecting mRNA levels of PDGF-BB, RICTOR, and MIR17HG as mediators of Ca2+ -signaling. Comparison of the ex vivo transcriptional response from the primary podocyte culture with glomerular transcripts across a wide spectrum of CKD disease confirmed co-regulation of transcript sets, establishing the disease relevance of the model system. Our findings demonstrate novel [Ca2+ ] regulated gene networks in podocytes deepening our understanding of podocyte biology and disease.

Urinary excretion of epidermal growth factor and rapid loss of kidney function.

BACKGROUND: Lower urinary excretion of the kidney tubule-specific biomarker epidermal growth factor (uEGF) is associated with increased risk of renal function [glomerular filtration rate (GFR)] loss in diabetes and in patients with established chronic kidney disease (CKD). We investigated whether uEGF is associated with rapid GFR decline or incident CKD in the general population. METHODS: Subjects without CKD or diabetes were recruited from the general population in Tromso, Norway [Renal Iohexol Clearance Survey (RENIS); N = 1249] and Groningen, the Netherlands [Prevention of REnal and Vascular END-stage disease (PREVEND); N = 4534], with a median follow-up of 5.6 and 7.4 years, respectively. GFR was measured by iohexol clearance in the RENIS and estimated using the CKD Epidemiology Collaboration creatinine-cystatin C equation in the PREVEND study. Rapid GFR decline was defined as an annual GFR loss >3.0 mL/min/1.73 m2 and in sensitivity analyses as subjects with the 10% steepest GFR slope within each cohort. RESULTS: Lower baseline uEGF excretion was associated with rapid GFR loss in both cohorts {RENIS, odds ratio [OR] per 1 µg/mmol lower uEGF 1.42 [95% confidence interval (CI) 1.06-1.91], P = 0.02; PREVEND, OR 1.29 [95% CI 1.10-1.53], P < 0.01}, adjusted for baseline GFR, albumin:creatinine ratio and conventional CKD risk factors. Similar results were obtained using the outcome of the 10% steepest GFR slope in each cohort. Lower uEGF levels were associated with incident CKD in the combined analysis of both cohorts. CONCLUSIONS: Lower uEGF levels are associated with increased risk of rapid GFR loss and incident CKD in the general population. This finding, together with previous findings in CKD and high-risk populations, supports that uEGF may serve as a broadly applicable biomarker representing the tubular component of the current glomerulus-centric clinical risk assessment system.