scRNA Transcription Profile of Adult Zebrafish Podocytes Using a Novel Reporter Strain.

BACKGROUND/AIMS: The role of podocytes is well conserved across species from drosophila to teleosts, and mammals. Identifying the molecular markers that actively maintain the integrity of the podocyte will enable a greater understanding of the changes that lead to damage. METHODS: We generated transgenic zebrafish, expressing fluorescent reporters driven by the podocin promoter, for the visualization and isolation of podocytes. We have conducted single cell RNA sequencing (scRNA-seq) on isolated podocytes from a zebrafish reporter line. RESULTS: We demonstrated that the LifeAct-TagRFP-T fluorescent reporter faithfully replicated podocin expression in vivo. We were also able to show spontaneous GCaMP6s fluorescence using light sheet (single plane illumination) microscopy. We identified many podocyte transcripts, encoding proteins related to calcium-binding and actin filament assembly, in common with those expressed in human and mouse mature podocytes. CONCLUSION: We describe the establishment of novel transgenic zebrafish and their use to identify and isolate podocyte cells for the preparation of a scRNA-seq library from normal podocytes. The scRNA-seq data identifies distinct populations of cells and potential gene switching between clusters. These data provide a foundation for future comparative studies and for exploiting the zebrafish as a model for kidney development, disease, injury and repair.

Kidney Single-Cell Atlas Reveals Myeloid Heterogeneity in Progression and Regression of Kidney Disease.

BACKGROUND: Little is known about the roles of myeloid cell subsets in kidney injury and in the limited ability of the organ to repair itself. Characterizing these cells based only on surface markers using flow cytometry might not provide a full phenotypic picture. Defining these cells at the single-cell, transcriptomic level could reveal myeloid heterogeneity in the progression and regression of kidney disease. METHODS: Integrated droplet- and plate-based single-cell RNA sequencing were used in the murine, reversible, unilateral ureteric obstruction model to dissect the transcriptomic landscape at the single-cell level during renal injury and the resolution of fibrosis. Paired blood exchange tracked the fate of monocytes recruited to the injured kidney. RESULTS: A single-cell atlas of the kidney generated using transcriptomics revealed marked changes in the proportion and gene expression of renal cell types during injury and repair. Conventional flow cytometry markers would not have identified the 12 myeloid cell subsets. Monocytes recruited to the kidney early after injury rapidly adopt a proinflammatory, profibrotic phenotype that expresses Arg1, before transitioning to become Ccr2+ macrophages that accumulate in late injury. Conversely, a novel Mmp12+ macrophage subset acts during repair. CONCLUSIONS: Complementary technologies identified novel myeloid subtypes, based on transcriptomics in single cells, that represent therapeutic targets to inhibit progression or promote regression of kidney disease.

Cardiovascular disease biomarkers are associated with declining renal function in type 2 diabetes.

AIMS/HYPOTHESIS: We investigated whether biochemical cardiovascular risk factors and/or markers of subclinical cardiovascular disease were associated with the development of reduced renal function in people with type 2 diabetes. METHODS: A cohort of 1066 Scottish men and women aged 60-74 years with type 2 diabetes from the Edinburgh Type 2 Diabetes Study were followed up for a median of 6.7 years. New-onset reduced renal function was defined as two eGFRs <60 ml-1 min-1 (1.73 m)-2 at least 3 months apart with a > 25% decline from baseline eGFR. Ankle brachial pressure index (ABI), N-terminal pro-B-type natriuretic peptide (NT-proBNP) and high-sensitivity troponin T (hsTnT) were measured at baseline. Pulse wave velocity (PWV) and carotid intima media thickness were measured 1 year into follow-up. Data were analysed using Cox proportional hazards models. RESULTS: A total of 119 participants developed reduced renal function during follow-up. ABI, PWV, NT-proBNP and hsTnT were all associated with onset of decline in renal function following adjustment for age and sex. These associations were attenuated after adjustment for additional diabetes renal disease risk factors (systolic BP, baseline eGFR, albumin:creatinine ratio and smoking pack-years), with the exception of hsTnT which remained independently associated (HR 1.51 [95% CI 1.22, 1.87]). Inclusion of hsTnT in a predictive model improved the continuous net reclassification index by 0.165 (0.008, 0.286). CONCLUSIONS/INTERPRETATION: Our findings demonstrate an association between hsTnT, a marker of subclinical cardiac ischaemia, and subsequent renal function decline. Further research is required to establish the predictive value of hsTnT and response to intervention.

Urinary peptidomics in a rodent model of diabetic nephropathy highlights epidermal growth factor as a biomarker for renal deterioration in patients with type 2 diabetes.

Many diabetic patients suffer from declining renal function without developing albuminuria. To identify alternative biomarkers for diabetic nephropathy (DN) we performed urinary peptidomic analysis in a rodent model in which hyperglycemia and hypertension synergize to promote renal pathologic changes consistent with human DN. We identified 297 increased and 15 decreased peptides in the urine of rats with DN compared with controls, including peptides derived from proteins associated with DN and novel candidate biomarkers. We confirmed by ELISA that one of the parent proteins, urinary epidermal growth factor (uEGF), was more than 2-fold reduced in rats with DN in comparison with controls. To assess the clinical utility of uEGF we examined renal outcomes in 642 participants from the Edinburgh Type 2 Diabetes Study who were normoalbuminuric and had preserved renal function at baseline. After adjustment for established renal risk factors, a lower uEGF to creatinine ratio was associated with new-onset estimated glomerular filtration rate less than 60 ml/min per 1.73m(2) (odds ratio 0.48; 95% confidence interval, 0.26-0.90), rapid (over 5% per annum) decline in renal function (odds ratio 0.44; 95% confidence interval, 0.27-0.72) or the composite of both outcomes (odds ratio 0.38; 95% confidence interval, 0.24-0.62). Thus, the utility of a low uEGF to creatinine ratio as a biomarker of progressive decline in renal function in normoalbuminuric patients should be assessed in additional populations.

An Update on the Use of Animal Models in Diabetic Nephropathy Research.

In the current review, we discuss limitations and recent advances in animal models of diabetic nephropathy (DN). As in human disease, genetic factors may determine disease severity with the murine FVB and DBA/2J strains being more susceptible to DN than C57BL/6J mice. On the black and tan, brachyuric (BTBR) background, leptin deficient (ob/ob) mice develop many of the pathological features of human DN. Hypertension synergises with hyperglycemia to promote nephropathy in rodents. Moderately hypertensive endothelial nitric oxide synthase (eNOS(-/-)) deficient diabetic mice develop hyaline arteriosclerosis and nodular glomerulosclerosis and induction of renin-dependent hypertension in diabetic Cyp1a1mRen2 rats mimics moderately severe human DN. In addition, diabetic eNOS(-/-) mice and Cyp1a1mRen2 rats recapitulate many of the molecular pathways activated in the human diabetic kidney. However, no model exhibits all the features of human DN; therefore, researchers should consider biochemical, pathological, and transcriptomic data in selecting the most appropriate model to study their molecules and pathways of interest.

MicroRNA-214 antagonism protects against renal fibrosis.

Renal tubulointerstitial fibrosis is the common end point of progressive renal disease. MicroRNA (miR)-214 and miR-21 are upregulated in models of renal injury, but the function of miR-214 in this setting and the effect of its manipulation remain unknown. We assessed the effect of inhibiting miR-214 in an animal model of renal fibrosis. In mice, genetic deletion of miR-214 significantly attenuated interstitial fibrosis induced by unilateral ureteral obstruction (UUO). Treatment of wild-type mice with an anti-miR directed against miR-214 (anti-miR-214) before UUO resulted in similar antifibrotic effects, and in vivo biodistribution studies demonstrated that anti-miR-214 accumulated at the highest levels in the kidney. Notably, in vivo inhibition of canonical TGF-ß signaling did not alter the regulation of endogenous miR-214 or miR-21. Whereas miR-21 antagonism blocked Smad 2/3 activation, miR-214 antagonism did not, suggesting that miR-214 induces antifibrotic effects independent of Smad 2/3. Furthermore, TGF-ß blockade combined with miR-214 deletion afforded additional renal protection. These phenotypic effects of miR-214 depletion were mediated through broad regulation of the transcriptional response to injury, as evidenced by microarray analysis. In human kidney tissue, miR-214 was detected in cells of the glomerulus and tubules as well as in infiltrating immune cells in diseased tissue. These studies demonstrate that miR-214 functions to promote fibrosis in renal injury independent of TGF-ß signaling in vivo and that antagonism of miR-214 may represent a novel antifibrotic treatment in the kidney.

Recent advances in animal models of diabetic nephropathy.

Diabetic nephropathy (DN) is the single most common cause of end-stage kidney disease. Therefore, it is imperative that novel therapies are developed. Progress has been hindered, however, by the lack of robust animal models. In the current review we describe recent advances in the field, including the impact of background strain, hypertension and transcriptomic profiling. While the C57BL/6J strain is relatively resistant to DN, the FVB strain appears more susceptible and Ove26 and db/db mice on this background may be useful in modelling types 1 and 2 DN, respectively. Black and tan, brachyury (BTBR) mice deficient for the leptin receptor (ob/ob) develop many of the pathological features of human DN and, remarkably, treatment with exogenous leptin ameliorates hyperglycaemia, albuminuria and glomerulosclerosis. Hypertension plays a key role in the progression of human DN and exacerbates nephropathy in diabetic rodents. Endothelial nitric oxide synthase deficiency (eNOS(-/-)) results in moderate hypertension and the development of nodular glomerulosclerosis and hyaline arteriosclerosis in streptozotocin-induced diabetic C57BL/6J mice. In Cyp1a1mRen2 rats, renin-dependent hypertension synergises with streptozotocin-induced hyperglycaemia to produce a 500-fold increase in albuminuria, glomerulosclerosis and tubulointerstitial fibrosis. Renal transcriptional profiling suggests that many of the gene expression changes observed in human DN are replicated in eNOS(-/-) mice and Cyp1a1mRen2 rats. Despite these advances, no model faithfully recapitulates all the features of human DN and further refinements are required. In the interim, it is likely that researchers may use publically available transcriptomic data to select the most appropriate model to study their molecule or pathway of interest.

Balloon angioplasty, with and without stenting, versus medical therapy for hypertensive patients with renal artery stenosis.

BACKGROUND: Atherosclerotic renal artery stenosis is the most common cause of secondary hypertension. Balloon angioplasty with stenting is widely used for the treatment of hypertensive patients with renal artery stenosis but the effectiveness of this procedure in treating hypertension, improving renal function and preventing adverse cardiovascular and renal events remains uncertain. This is an update, to include the results of recent, important large trials, of a review first published in 2003. OBJECTIVES: To compare the effectiveness of balloon angioplasty (with and without stenting) with medical therapy for the treatment of atherosclerotic renal artery stenosis in patients with hypertension. The following outcomes were compared: blood pressure control, renal function, frequency of cardiovascular and renal adverse events, presence or absence of restenosis of the renal artery, side effects of medical therapy, numbers and defined daily doses of antihypertensive drugs. SEARCH METHODS: For this update the Cochrane Peripheral Vascular Diseases Group Trials Search Co-ordinator searched the Specialised Register (last searched May 2014) and CENTRAL (2014, Issue 4). Bibliographies were also reviewed and trial authors were contacted for more information. SELECTION CRITERIA: Randomised controlled trials (RCTs) comparing balloon angioplasty with medical therapy in hypertensive patients with haemodynamically significant renal artery stenosis (greater than 50% reduction in luminal diameter) and with a minimum follow-up of six months. DATA COLLECTION AND ANALYSIS: Data were extracted independently on trial design, participants, interventions and outcome measures. A formal meta-analysis was completed to assess the effect on blood pressure, renal function and cardiovascular and renal adverse events. Peto's odds ratios (ORs) and corresponding 95% confidence intervals (CI) for dichotomous outcomes and mean differences (MD) and corresponding 95% CIs for continuous variables were calculated. MAIN RESULTS: Eight RCTs involving 2222 participants with renal artery stenosis were included in the review. The overall quality of evidence included in this review was moderate. Limited pooling of results was possible due to the variable presentation of some of the trial outcomes. Meta-analysis of the four studies reporting change in diastolic blood pressure (BP) found a small improvement in diastolic BP in the angioplasty group (MD -2.00 mmHg; 95% CI -3.72 to -0.27) whilst the meta-analysis of the five studies reporting change in systolic BP did not find any evidence of significant improvement (MD -1.07 mmHg; 95% CI -3.45 to 1.30). There was no significant effect on renal function as measured by serum creatinine (MD -7.99 µmol/L; 95% CI -22.6 to 6.62). Meta-analysis of the three studies that reported the mean number of antihypertensive drugs found a small decrease in antihypertensive drug requirements for the angioplasty group (MD -0.18; 95% CI -0.34 to -0.03). Repeat angiography was only performed on a small number of participants in a single trial and it was therefore not possible to comment on restenosis of the renal artery following balloon angioplasty. Based on the results of the seven studies that reported cardiovascular and renal clinical outcomes there were no differences in cardiovascular (OR 0.91; 95% CI 0.75 to 1.11) or renal adverse events (OR 1.02; 95% CI 0.75 to 1.38) between the angioplasty and medical treatment groups. A small number of procedural complications of balloon angioplasty were reported (haematoma at the site of catheter insertion (6.5%), femoral artery pseudoaneurysm (0.7%), renal artery or kidney perforation or dissection (2.5%) as well as peri-procedural deaths (0.4%)). No side effects of medical therapy were reported. AUTHORS' CONCLUSIONS: The available data are insufficient to conclude that revascularisation in the form of balloon angioplasty, with or without stenting, is superior to medical therapy for the treatment of atherosclerotic renal artery stenosis in patients with hypertension. However, balloon angioplasty results in a small improvement in diastolic blood pressure and a small reduction in antihypertensive drug requirements. Balloon angioplasty appears safe and results in similar numbers of cardiovascular and renal adverse events to medical therapy.

Tight blood glycaemic and blood pressure control in experimental diabetic nephropathy reduces extracellular matrix production without regression of fibrosis.

AIMS: Regression of albuminuria and renal fibrosis occurs in patients with diabetic nephropathy (DN) following tight control of blood glucose and blood pressure, however the pathways that promote regression remain poorly understood and we wished to characterize these using a rodent model. METHODS: Diabetes was induced with streptozotocin in Cyp1a1mRen2 rats and hypertension was generated by inducing renin transgene expression with dietary indole-3-carbinol (I-3-C) for 28 weeks. At this point an 'injury cohort' was culled, while in a 'reversal cohort' glycaemia was tightly controlled using insulin implants and blood pressure normalized by withdrawing dietary I-3-C for a further 8 weeks. Pathways activated during and following reversal of diabetes and hypertension were assessed by microarray profiling. RESULTS: Tight control of blood glucose and blood pressure reduced albuminuria and renal hypertrophy, but had no impact on renal fibrosis. 85 genes were up-regulated specifically during the injury phase, including genes encoding multiple myofibroblast and extracellular matrix (ECM) proteins. Conversely, 314 genes remained persistently elevated during reversal including genes linked to innate/adaptive immunity, phagocytosis, lysosomal processing and degradative metalloproteinases (MMPs). Despite increased MMP gene expression, MMP activity was suppressed during both injury and reversal, in association with up-regulation of tissue inhibitor of metalloproteinase-1 (TIMP-1) protein. Physical separation of the TIMP-1/MMP complexes during zymography of tissue homogenate restored MMP activity. CONCLUSION: Normalization of blood glucose and pressure ameliorates albuminuria and inhibits excess ECM production, however persistent TIMP-1 expression hinders attempts at ECM remodelling. Therapies which counteract the action of TIMPs may accelerate scar resolution.

Hyperglycemia and renin-dependent hypertension synergize to model diabetic nephropathy.

Rodent models exhibit only the earliest features of human diabetic nephropathy, which limits our ability to investigate new therapies. Hypertension is a prerequisite for advanced diabetic nephropathy in humans, so its rarity in typical rodent models may partly explain their resistance to nephropathy. Here, we used the Cyp1a1mRen2 rat, in which the murine renin-2 gene is incorporated under the Cytochrome P4501a1 promoter. In this transgenic strain, administration of low-dose dietary indole-3-carbinol induces moderate hypertension. In the absence of hypertension, streptozotocin-induced diabetes resulted in a 14-fold increase in albuminuria but only mild changes in histology and gene expression despite 28 weeks of marked hyperglycemia. In the presence of induced hypertension, hyperglycemia resulted in a 500-fold increase in albuminuria, marked glomerulosclerosis and tubulointerstitial fibrosis, and induction of many of the same pathways that are upregulated in the tubulointerstitium in human diabetic nephropathy. In conclusion, although induction of diabetes alone in rodents has limited utility to model human diabetic nephropathy, renin-dependent hypertension and hyperglycemia synergize to recapitulate many of the clinical, histological, and gene expression changes observed in humans.

Exploration of Metabolomic Markers Associated With Declining Kidney Function in People With Type 2 Diabetes Mellitus.

Background: Metabolomics, the study of small molecules in biological systems, can provide valuable insights into kidney dysfunction in people with type 2 diabetes mellitus (T2DM), but prospective studies are scarce. We investigated the association between metabolites and kidney function decline in people with T2DM. Methods: The Edinburgh Type 2 Diabetes Study, a population-based cohort of 1066 men and women aged 60 to 75 years with T2DM. We measured 149 serum metabolites at baseline and investigated individual associations with baseline estimated glomerular filtration rate (eGFR), incident chronic kidney disease [CKD; eGFR <60 mL/min/(1.73 m)2], and decliner status (5% eGFR decline per year). Results: At baseline, mean eGFR was 77.5 mL/min/(1.73 m)2 (n = 1058), and 216 individuals had evidence of CKD. Of those without CKD, 155 developed CKD over a median 7-year follow-up. Eighty-eight metabolites were significantly associated with baseline eGFR (ß range -4.08 to 3.92; PFDR < 0.001). Very low density lipoproteins, triglycerides, amino acids (AAs), glycoprotein acetyls, and fatty acids showed inverse associations, while cholesterol and phospholipids in high-density lipoproteins exhibited positive associations. AA isoleucine, apolipoprotein A1, and total cholines were not only associated with baseline kidney measures (PFDR < 0.05) but also showed stable, nominally significant association with incident CKD and decline. Conclusion: Our study revealed widespread changes within the metabolomic profile of CKD, particularly in lipoproteins and their lipid compounds. We identified a smaller number of individual metabolites that are specifically associated with kidney function decline. Replication studies are needed to confirm the longitudinal findings and explore if metabolic signals at baseline can predict kidney decline.

Indian Hedgehog release from TNF-activated renal epithelia drives local and remote organ fibrosis.

Progressive fibrosis is a feature of aging and chronic tissue injury in multiple organs, including the kidney and heart. Glioma-associated oncogene 1 expressing (Gli1+) cells are a major source of activated fibroblasts in multiple organs, but the links between injury, inflammation, and Gli1+ cell expansion and tissue fibrosis remain incompletely understood. We demonstrated that leukocyte-derived tumor necrosis factor (TNF) promoted Gli1+ cell proliferation and cardiorenal fibrosis through induction and release of Indian Hedgehog (IHH) from renal epithelial cells. Using single-cell-resolution transcriptomic analysis, we identified an "inflammatory" proximal tubular epithelial (iPT) population contributing to TNF- and nuclear factor κB (NF-κB)-induced IHH production in vivo. TNF-induced Ubiquitin D (Ubd) expression was observed in human proximal tubular cells in vitro and during murine and human renal disease and aging. Studies using pharmacological and conditional genetic ablation of TNF-induced IHH signaling revealed that IHH activated canonical Hedgehog signaling in Gli1+ cells, which led to their activation, proliferation, and fibrosis within the injured and aging kidney and heart. These changes were inhibited in mice by Ihh deletion in Pax8-expressing cells or by pharmacological blockade of TNF, NF-κB, or Gli1 signaling. Increased amounts of circulating IHH were associated with loss of renal function and higher rates of cardiovascular disease in patients with chronic kidney disease. Thus, IHH connects leukocyte activation to Gli1+ cell expansion and represents a potential target for therapies to inhibit inflammation-induced fibrosis.

Measuring urinary tubular biomarkers in type 2 diabetes does not add prognostic value beyond established risk factors.

Tubulointerstitial disease plays an important role in the pathophysiology of diabetic kidney disease. To determine whether biomarkers of tubular injury could predict renal outcome and mortality in patients with type 2 diabetes, we measured urinary levels of kidney injury molecule-1 (KIM-1) and glycoprotein non-metastatic melanoma B (Gpnmb), both normalized to the urinary creatinine, in 978 individuals from the Edinburgh Type 2 Diabetes Study. At baseline, 238 patients had an estimated glomerular filtration rate (eGFR) below 60 ml/min/1.73 m2 while 147 and 15 patients had microalbuminuria or overt proteinuria, respectively. Both the urine KIM-1 and Gpnmb to creatinine ratios correlated with the urinary albumin to creatinine ratio, the duration of diabetes, and the stringency of glycemic control but not with blood pressure or baseline eGFR. Higher ratios of each marker were associated with a faster decline in kidney function during 4 years of follow-up; however, this was not independent of the urinary albumin to creatinine ratio. Higher KIM-1, but not Gpnmb ratios were associated with an increased risk of mortality, but this association was no longer significant after adjustment for other risk factors, in particular albuminuria. Thus, tubular injury in persons with type 2 diabetes may contribute to the decline in kidney function; however, measuring the urinary concentration of these two tubular biomarkers does not confer additional prognostic information beyond established risk factors.

Macrophages in the kidney in health, injury and repair.

Macrophages are a key component of the renal mononuclear phagocyte system, playing a major role in defense against infection, renal injury and repair. Yolk sac macrophage precursors seed the early embryonic kidney and are important for renal development. Later, renal macrophages are derived from hematopoietic stem cells and in adult life, there is a significant contribution from circulating monocytes, which is enhanced in response to infection or injury. Macrophages are highly plastic and can alter their phenotype in response to cues from parenchymal renal cells. Danger-associated molecules released from injured kidney cells may activate macrophages toward a pro-inflammatory phenotype, mediating further recruitment of inflammatory cells, exacerbating renal injury and activating renal fibroblasts to promote scarring. In acute kidney injury, once the injury stimulus has abated, macrophages may adopt a more reparative phenotype, dampening the immune response and promoting repair of renal tissue. However, in chronic kidney disease ongoing activation of pro-inflammatory monocytes and persistence of reparative macrophages leads to glomerulosclerosis and tubulointerstitial fibrosis, the hallmarks of end-stage kidney disease. Several strategies to inhibit the recruitment, activation and secretory products of pro-inflammatory macrophages have proven beneficial in pre-clinical models and are now undergoing clinical trials in patients with kidney disease. In addition, macrophages may be utilized in cell therapy as a "Trojan Horse" to deliver targeted therapies to the kidney. Single-cell RNA sequencing has identified a previously unappreciated spectrum of macrophage phenotypes, which may be selectively present in injury or repair, and ongoing functional analyses of these subsets may identify more specific targets for therapeutic intervention.

Sonoporation of Human Renal Proximal Tubular Epithelial Cells In Vitro to Enhance the Liberation of Intracellular miRNA Biomarkers.

Ultrasound has previously been demonstrated to non-invasively cause tissue disruption. Small animal studies have demonstrated that this effect can be enhanced by contrast microbubbles and has the potential to be clinically beneficial in techniques such as targeted drug delivery or enhancing liquid biopsies when a physical biopsy may be inappropriate. Cavitating microbubbles in close proximity to cells increases membrane permeability, allowing small intracellular molecules to leak into the extracellular space. This study sought to establish whether cavitating microbubbles could liberate cell-specific miRNAs, augmenting biomarker detection for non-invasive liquid biopsies. Insonating human polarized renal proximal tubular epithelial cells (RPTECs), in the presence of SonoVue microbubbles, revealed that cellular health could be maintained while achieving the release of miRNAs, miR-21, miR-30e, miR-192 and miR-194 (respectively, 10.9-fold, 7.17-fold, 5.95-fold and 5.36-fold). To examine the mechanism of release, RPTECs expressing enhanced green fluorescent protein were generated and the protein successfully liberated. Cell polarization, cellular phenotype and cell viability after sonoporation were measured by a number of techniques. Ultrastructural studies using electron microscopy showed gap-junction disruption and pore formation on cellular surfaces. These studies revealed that cell-specific miRNAs can be non-specifically liberated from RPTECs by sonoporation without a significant decrease in cell viability.

Frequency and Phenotype Associations of Rare Variants in 5 Monogenic Cerebral Small Vessel Disease Genes in 200,000 UK Biobank Participants.

Background and Objectives: Based on previous case reports and disease-based cohorts, a minority of patients with cerebral small vessel disease (cSVD) have a monogenic cause, with many also manifesting extracerebral phenotypes. We investigated the frequency, penetrance, and phenotype associations of putative pathogenic variants in cSVD genes in the UK Biobank (UKB), a large population-based study. Methods: We used a systematic review of previous literature and ClinVar to identify putative pathogenic rare variants in CTSA, TREX1, HTRA1, and COL4A1/2. We mapped phenotypes previously attributed to these variants (phenotypes-of-interest) to disease coding systems used in the UKB's linked health data from UK hospital admissions, death records, and primary care. Among 199,313 exome-sequenced UKB participants, we assessed the following: the proportion of participants carrying ≥1 variant(s); phenotype-of-interest penetrance; and the association between variant carrier status and phenotypes-of-interest using a binary (any phenotype present/absent) and phenotype burden (linear score of the number of phenotypes a participant possessed) approach. Results: Among UKB participants, 0.5% had ≥1 variant(s) in studied genes. Using hospital admission and death records, 4%-20% of variant carriers per gene had an associated phenotype. This increased to 7%-55% when including primary care records. Only COL4A1 variant carrier status was significantly associated with having ≥1 phenotype-of-interest and a higher phenotype score (OR = 1.29, p = 0.006). Discussion: While putative pathogenic rare variants in monogenic cSVD genes occur in 1:200 people in the UKB population, only approximately half of variant carriers have a relevant disease phenotype recorded in their linked health data. We could not replicate most previously reported gene-phenotype associations, suggesting lower penetrance rates, overestimated pathogenicity, and/or limited statistical power.

Single-cell analysis of senescent epithelia reveals targetable mechanisms promoting fibrosis.

Progressive fibrosis and maladaptive organ repair result in significant morbidity and millions of premature deaths annually. Senescent cells accumulate with aging and after injury and are implicated in organ fibrosis, but the mechanisms by which senescence influences repair are poorly understood. Using 2 murine models of injury and repair, we show that obstructive injury generated senescent epithelia, which persisted after resolution of the original injury, promoted ongoing fibrosis, and impeded adaptive repair. Depletion of senescent cells with ABT-263 reduced fibrosis in reversed ureteric obstruction and after renal ischemia/reperfusion injury. We validated these findings in humans, showing that senescence and fibrosis persisted after relieved renal obstruction. We next characterized senescent epithelia in murine renal injury using single-cell RNA-Seq. We extended our classification to human kidney and liver disease and identified conserved profibrotic proteins, which we validated in vitro and in human disease. We demonstrated that increased levels of protein disulfide isomerase family A member 3 (PDIA3) augmented TGF-ß-mediated fibroblast activation. Inhibition of PDIA3 in vivo significantly reduced kidney fibrosis during ongoing renal injury and as such represented a new potential therapeutic pathway. Analysis of the signaling pathways of senescent epithelia connected senescence to organ fibrosis, permitting rational design of antifibrotic therapies.

The induction of macrophage hemeoxygenase-1 is protective during acute kidney injury in aging mice.

Aging is thought to be associated with a higher susceptibility to renal ischemia-reperfusion injury (IRI). To study whether defective induction of hemeoxygenase-1 (HO-1, a protective and anti-inflammatory enzyme) might contribute to this, we found that while 12-month-old mice had similar baseline renal function and HO-1 expression, the induction of HO-1 usually seen in ischemia-reperfusion was reduced. This was also associated with worsened renal function and acute tubular necrosis in the aged compared with young mice. In the older mice, heme arginate (HA) induced HO-1 in the cortex and medulla, significantly improved renal function, and reduced tissue injury. Cellular HO-1 induction in the medulla in response to injury or HA treatment was found to be interstitial rather than epithelial, as evidenced by its colocalization with macrophage markers. In vitro, HA treatment of primary macrophages resulted in marked HO-1 induction without impairment of classical activation pathways. Macrophage depletion, caused by diphtheria toxin treatment of 12-month-old CD11b-DTR transgenic animals, resulted in the loss of interstitial HO-1-positive cells and reversal of the protective phenotype of HA treatment. Thus, failure of HO-1 induction following renal IRI worsens structural and functional injury in older mice and represents a therapeutic target in the elderly. Hence, HO-1-positive renal macrophages mediate HA-induced protection in IRI.

A National Registry Study of Patient and Renal Survival in Adult Nephrotic Syndrome.

INTRODUCTION: We aimed to determine the mortality rate, cause of death, and rate of end-stage kidney disease (ESKD) in adults with nephrotic syndrome (NS). METHODS: We conducted a national registry-based study, including all 522 adults who had a kidney biopsy for NS in Scotland in 2014-2017. We linked the Scottish Renal Registry to death certificate data. We performed survival and Cox proportional hazards analyses, accounting for competing risks of death and ESKD. We compared mortality rates with those in the age- and sex-matched general population. RESULTS: A total of 372 patients had primary NS; 150 had secondary NS. Over a median follow-up of 866 days, 110 patients (21%) died. In patients with primary NS, observed versus population 3-year mortality was 2.1% (95% CI 0.0%-4.6%) versus 0.9% (0.8%-1.0%) in patients aged <60 years and 24.9% (18.4%-30.8%) versus 9.4% (8.3%-10.5%) in those aged ≥60 years. In secondary NS, this discrepancy was 17.1% (5.6%-27.2%) versus 1.1% (0.9%-1.2%) in <60-year-olds and 49.4% (36.6%-59.7%) versus 8.1% (6.6%-9.6%) in ≥60-year-olds. In primary NS, cardiovascular causes accounted for 28% of deaths, compared with 18% in the general population. Eighty patients (15%) progressed to ESKD. Incidence of ESKD by 3 years was 8.4% (95% CI 4.9%-11.7%) in primary and 35.1% (24.3%-44.5%) in secondary NS. Early remission of proteinuria and the absence of early acute kidney injury (AKI) were associated with lower rates of death and ESKD. CONCLUSIONS: Adults with NS have high rates of death and ESKD. Cardiovascular causes account for excess mortality in primary NS.