Glomerular Markers Research Articles

Evaluation of an algorithm based on quantitative assays of glomerular parameters (albuminuria and urinary IgG) for proteinuria typing

The first orientation test for proteinuria typing is electrophoresis. However, this technique has several drawbacks, such as delayed turnaround time and subjective readings. Some laboratories therefore use quantitative assays of glomerular markers combined with tubular markers. However, the cost of reagents and the instability of certain markers are significant drawbacks for some peripheral laboratories. The aim of this study is to evaluate the implementation of an algorithm based on parameters that can be used by all laboratories for proteinuria typing within a timeframe compatible with the urgency of the situation. Albuminuria and urinary IgG were determined on 161 urines. ROC curves were produced, using urine electrophoresis read by an expert center as the reference method. The decision thresholds used are: glomerular proteinuria is defined by a Albumin+IgGproteinsratio greater than 75.4% (100% specificity), and tubular or overload proteinuria is defined by by a Albuminproteinsratio less than 37.3% (100% sensitivity). Agreement between the results of the algorithm selected and the reference method used in our study was 88%, with a kappa value of 0.807 (95% CI [0.729 to 0.885]). The algorithm's performance suggests that it can find its place in the diagnostic strategy for clinically significant proteinuria, despite its limited indications. It is up to each biologist to assess the value of this algorithm in relation to the recruitment, habits and needs of clinicians.

Human Nephrogenesis can Persist Beyond 40 Postnatal Days in Preterm Infants

IntroductionHuman nephrogenesis is typically completed by 36 weeks (wks) gestation, but impacted by preterm birth. Early studies suggested that nephrogenesis persisted for ≤ 40 postnatal days in preterm infants. However, the postmenstrual age (PMA) of the preterm infants who survived > 40 days was uncertain. In this study, we sought to re-examine postnatal kidney development in preterm infants surviving > 40 days. MethodsHuman kidney samples were obtained from an institutional biobank. Samples were considered controls if survival was ≤ 4 days after birth with PMA of 30 to ≤ 36 wks. Kidneys from preterm neonates with postnatal survival > 40 days and PMA of 30 to ≤ 36 wks were compared to controls. We counted glomerular generations, measured nephrogenic zone widths, and performed immunofluorescence (IF) with SIX1 and RET. We compared kidney weights and quantified the cross-sectional area of proximal (LTL, SL22A2), distal (SLC12A3, KCNJ10), and glomerular (nephrin) markers using IF. ResultsSeven preterm infants surviving > 40 days and 8 controls were analyzed. 4/7 preterm infants had histologic and molecular evidence of nephrogenesis. Cessation of nephrogenesis in preterm infants occurred two weeks earlier than PMA-matched controls with attenuated expression of both SIX1 and RET. We found increased kidney weight: body weight ratio, increased distal tubular cross-sectional staining in the superficial nephrons, and distal tubular hypertrophy/hyperplasia in the preterm infant kidneys. ConclusionOur study supports that nephrogenesis in preterm infants persists longer than previously thought with evidence of early nephron stress, placing importance on the neonatal environment.

Urinary transferrin and proinflammatory markers predict the earliest diabetic nephropathy onset

Aim This study aimed to determine the earliest markers of diabetic nephropathy (DN) onset with discriminative potentials from controlled diabetes (CD). Methods Sixty male Wistar rats were allocated into three groups (20/group), the two diabetic groups CD and DN received 45 and 65 mg/kg STZ in 0.1 mole/L citrate buffer, respectively, while the control group received only the vehicle. Serum/urinary levels of glomerular, tubular, oxidative and proinflammatory markers were weekly monitored. Results Each diabetic group showed a different pattern of inflammatory, oxidative and signs of nephropathy along the study period, but none had a discriminative power until the fourth week. At this time point, levels of urinary transferrin, serum/urinary IL-6 and TNF-α as well as urinary IL-18 were significantly higher in DN group compared to CD (p = 0.0217, <0.0001, 0.0005, 0.0004, 0.0006, 0.0019, respectively). Predictive thresholds of these markers were calculated by receiver operating characteristic (ROC) curve that showed area under curve (AUC) of 0.9375 for transferrin with cut-off value of 35.2 mg/dL and 1.000 for serum/urinary IL-6 and TNF-α and urinary IL-18 with cut-of values 224.1, 82.11, 6.596, 125.9 and 21.86 pg/mL, respectively. Conclusion Urinary transferrin and the inflammatory endpoints proposed in this study might represent promising biomarkers for the early DN onset.

Long-Term Preservation of Renal Function in Septic Shock Burn Patients Requiring Renal Replacement Therapy for Acute Kidney Injury.

Background. The real impact of septic shock-associated acute kidney injury (AKI) on the long-term renal outcome is still debated, and little is known about AKI-burn patients. In a cohort of burn survivors treated by continuous renal replacement therapy (CRRT) and sorbent technology (CPFA-CRRT), we investigated the long-term outcome of glomerular and tubular function. Methods. Out of 211 burn patients undergoing CRRT from 2001 to 2017, 45 survived, 40 completed the clinical follow-up (cumulative observation period 4067 months, median 84 months, IR 44-173), and 30 were alive on 31 December 2020. Besides creatinine and urine albumin, in the 19 patients treated with CPFA-CRRT, we determined the normalized GFR by 99mTc-DTPA (NRI-GFR) and studied glomerular and tubular urine protein markers. Results. At the follow-up endpoint, the median plasma creatinine and urine albumin were 0.99 (0.72–1.19) and 0.0 mg/dL (0.0–0.0), respectively. NRI-GFR was 103.0 mL/min (93.4–115). Four patients were diabetic, and 22/30 presented at least one risk factor for chronic disease (hypertension, dyslipidemia, and overweight). Proteinuria decreased over time, from 0.47 g/day (0.42–0.52) at 6 months to 0.134 g/day (0.09–0.17) at follow-up endpoint. Proteinuria positively correlated with the peak of plasma creatinine (r 0.6953, p 0.006) and the number of CRRT days (r 0.5650, p 0.035) during AKI course, and negatively with NRI–GFR (r −0.5545, p 0.049). In seven patients, urine protein profile showed a significant increase of glomerular marker albumin and glomerular/tubular index. Conclusions. Burn patients who experienced septic shock and AKI treated with CRRT had a long-term expectation of preserved renal function. However, these patients were more predisposed to microalbuminuria, diabetes, and the presence of risk factors for intercurrent comorbidities and chronic renal disease.

Association of Urine Biomarkers of Kidney Tubule Injury and Dysfunction With Frailty Index and Cognitive Function in Persons With CKD in SPRINT

The associations of the glomerular markers of kidney disease, estimated glomerular filtration rate (eGFR) and albuminuria, with frailty and cognition are well established. However, the relationship of kidney tubule injury and dysfunction with frailty and cognition is unknown. Observational cross-sectional study. 2,253 participants with eGFR<60mL/min/1.73m2 in the Systolic Blood Pressure Intervention Trial (SPRINT). Eight urine biomarkers: interleukin 18 (IL-18), kidney injury molecule 1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), chitinase-3-like protein 1 (YKL-40), monocyte chemoattractant protein 1 (MCP-1), α1-microglobulin (A1M), β2-microglobulin (B2M), and uromodulin (Umod). Frailty was measured using a previously validated frailty index (FI), categorized as fit (FI≤0.10), less fit (0.10<FI≤0.21), and frail (FI>0.21). Cognitive function was assessed using the Montreal Cognitive Assessment (MoCA). Associations between kidney tubule biomarkers with categorical FI were evaluated using multinomial logistic regression with the fit group as the reference. Cognitive function was evaluated using linear regression. Models were adjusted for demographic, behavioral, and clinical variables including eGFR and urine albumin. Three of the 8 urine biomarkers of tubule injury and dysfunction were independently associated with FI. Each 2-fold higher level of urine KIM-1, a marker of tubule injury, was associated with a 1.22 (95% CI, 1.01-1.49) greater odds of being in the frail group. MCP-1, a marker of tubulointerstitial fibrosis, was associated with a 1.30 (95% CI, 1.04-1.64) greater odds of being in the frail group, and A1M, a marker of tubule reabsorptive capacity, was associated with a 1.48 (95% CI, 1.11-1.96) greater odds of being in the frail group. These associations were independent of confounders including eGFR and urine albumin, and were stronger than those of urine albumin with FI (1.15 [95% CI, 0.99-1.34]). Higher urine B2M, another marker of tubule reabsorptive capacity, was associated with worse cognitive scores at baseline (β:-0.09 [95% CI,-0.17 to-0.01]). Urine albumin was not associated with cognitive function. Cross-sectional design, and FI may not be generalizable in other populations. Urine biomarkers of tubule injury, fibrosis, and proximal tubule reabsorptive capacity are variably associated with FI and worse cognition, independent of glomerular markers of kidney health. Future studies are needed to validate these results among other patient populations.

Formation and optimization of three-dimensional organoids generated from urine-derived stem cells for renal function in vitro

BackgroundOrganoids play an important role in basic research, drug screening, and regenerative medicine. Here, we aimed to develop a novel kind of three-dimensional (3D) organoids generated from urine-derived stem cells (USCs) and to explore whether kidney-specific extracellular matrix (kECM) could enable such organoids for renal function in vitro.MethodsUSCs were isolated from human urine samples and cultured with kECM extraction to generate 3D organoids in vitro. Eight densities from 1000 to 8000 cells per organoids were prepared, and both ATP assay and Live/Dead staining were used to determine the optimal USC density in forming organoids and kECM additive concentration. The morphology and histology of as-made organoids were evaluated by hematoxylin and eosin (H.E.) staining, immunofluorescence staining and whole mount staining. Additionally, RT-qPCR was implemented to detect renal-related gene expression. Drug toxicity test was conducted to evaluate the potential application for drug screening. The renal organoids generated from whole adult kidney cells were used as a positive control in multiple assessments.ResultsThe optimized cell density to generate ideal USC-derived organoids (USC-organoids) was 5000 cells/well, which was set as applying density in the following experiments. Besides, the optimal concentration of kECM was revealed to be 10%. On this condition, Live/Dead staining showed that USC-organoids were well self-organized without significant cell death. Moreover, H.E. staining showed that compact and viable organoids were generated without obvious necrosis inside organoids, which were very close to renal organoids morphologically. Furthermore, specific proximal tubule marker Aquaporin-1 (AQP1), kidney endocrine product erythropoietin (EPO), kidney glomerular markers Podocin and Synaptopodin were detected positively in USC-organoids with kECM. Nephrotoxicity testing showed that aspirin, penicillin G, and cisplatin could exert drug-induced toxicity on USC-organoids with kECM.ConclusionsUSC-organoids could be developed from USCs via an optimal procedure. Combining culture with kECM, USC-organoid properties including morphology, histology, and specific gene expression were identified to be similar with real renal organoids. Additionally, USC-organoids posed kECM in vitro showed the potential to be a drug screening tool which might take the place of renal organoids to some extent in the future.

2149-PUB: The Changes in the Panel of Circulating Cytokines Associated with Chronic Kidney Disease in Patients with Type 2 Diabetes

Aim: To estimate the differences in the panel of circulating cytokines mediating low-grade inflammation and fibrosis in patients with type 2 diabetes (T2D) and chronic kidney disease.Methods: We examined 130 patients with T2D duration ≥10 years. Among them, 65 individuals had reduced estimated glomerular filtration rate (eGFR; 15-59 ml/min/1.73 m2) and 64 patients had increased albuminuria. Thirty healthy subjects were acted as control. Serum IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17A, bFGF, eotaxin, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, MIP-1β, PDGF-BB, RANTES, TNF-α, and VEGF were assessed by multiplex analysis. Urinary nephrin and podocin, as podocyte injury markers, and WFDC-2, a marker of tubulointerstitial fibrosis, were assessed by ELISA. Results: Patients with T2D and reduced eGFR had increased levels of IL-1ra, IL-8, IL-13 and decreased IL-7, IL-9 and IL-15 concentrations when compared to those with preserved renal function and control (all p&amp;lt;0.05). Excretion of WFDC-2, but not podocyte markers, was increased in these patients (p≤0.02). The elevation of albuminuria was associated with enhanced excretion of nephrin and podocin (p&amp;lt;0.0001), but no differences were found in the levels of all measured cytokines between patients with normal and elevated albuminuria. The levels of IL-2, IL-4, IL-5, IL-17A, bFGF, G-CSF, IP-10 and MIP-1α correlated negatively with eGFR. No correlations were found between measured cytokines and glomerular markers. There was positive correlation between WFDC-2 and MIP-1α. The levels of IL-10 and IL-15, two anti-inflammatory cytokines, correlated negatively with WFDC-2. Conclusion: The obtained results demonstrate that decreased eGFR and increased urinary excretion of WFDC-2, a marker of tubulointerstitial fibrosis, are associated with a predominance of pro-inflammatory and fibrogenic molecules in the panel of circulating cytokines in T2D subjects. Disclosure A.I. Korbut: None. O. Fazullina: None. V. Klimontov: None.

Neutrophil Gelatinase-Associated Lipocalin and Retinol-Binding Protein-4 as Biomarkers for Diabetic Kidney Disease

Aims: This study was designed to evaluate the conflicting association between 2 tubular protein markers including neutrophil gelatinase-associated lipocalin (NGAL) and retinol-binding protein-4 (RBP-4) with albuminuria and glomerular filtration rate (GFR) and calculate the accuracy of the role of NGAL and RBP-4 in diagnosis of diabetic nephropathy (DN) in patients with type2 diabetes. Methods: This is a cross-sectional study that included 133 patients with type 2 diabetes. There were 3 diabetic study groups with normoalbuminuria, moderately increased albuminuria, severely increased albuminuria, and non-diabetic control group without any renal disease. We analyzed the difference of urinary NGAL (uNGAL) and RBP-4 between nondiabetics and diabetics, as well as within the diabetic group. We also assessed the association between albuminuria and NGAL and RBP-4. Results: The urinary levels of NGAL and RBP-4 were higher in patients with type 2 diabetes compared to nondiabetics as well as in albuminuric diabetics compared to nonalbuminuric patients with diabetes (p value <0.001). These 2 proteins were higher in patients with severely increased albuminuria compared to patients with moderately increased albuminuria, even after adjustment for other metabolic factors (all p < 0.01). Moreover, areas under the curve of NGAL and RBP-4 for the diagnosis of chronic kidney disease were 80.6 and 74.6%, respectively. Conclusion: uNGAL and RBP-4 are potential markers of tubular damage that may increase before the onset of glomerular markers such as albuminuria and GFR in patients with type 2 diabetes. Therefore, these markers can be used as complementary measurements to albuminuria and GFR in the earlier diagnosis of DN.

P6533Proenkephalin as a new glomerular filtration marker for rule-out of sustained kidney injury after cardiac catheterization: main results from the prospective PANCAKE study

Abstract Introduction Use of contrast media is necessary for diagnostic imaging and PCI. However, contrast-induced kidney injury has been identified as the most frequent cause of hospital-acquired acute kidney injury and is associated with poor prognosis. Currently, contrast-induced kidney injury cannot be diagnosed on the day of cardiac catheterization or on the following day, when the majority of patients who undergo elective cardiac catheterization are discharged from the hospital in the real-world setting. Recently, proenkephalin (penKid) was introduced as a new glomerular filtration marker, which is capable of identifying normal subjects at high risk of future decline in renal function. The aim of this study was to investigate whether the change in penKid level on the day following cardiac catheterization can predict kidney injury before hospital discharge and thus allows for early detection of affected patients. Methods A total of 214 consecutive patients who underwent routine cardiac catherization were recruited, and blood was drawn at three time-points: immediately before catherization, 12–24 hours after catheterization and 4–8 weeks after discharge. Creatinine was measured for endpoint definition, while the markers urea, CRP (C-reactive protein), NGAL (neutrophil gelatinase-associated lipocalin), KIM-1 (kidney injury marker-1), cystatin C, suPAR (soluble urokinase-type plasminogen activator receptor), and penKid were measured as biomarkers of interest. The main outcome measure was sustained kidney injury (SKI), which was defined as an increase above 20% in serum creatinine between time-points 1 and 3. The main test was whether the change in biomarkers between baseline and immediately before discharge (time-points 1 and 2) can predict the development of sustained kidney injury. Results While only 5.6% of subjects developed acute kidney injury as defined according to KDIGO guidelines (delta serum creatinine between time-points 1 and 2), sustained kidney injury at mid-term follow-up (4–8 weeks) was found in 28.7% of the subjects. None of the baseline biomarkers, including creatinine, reliably predicted SKI (AUC in ROC analyses between 0.50 and 0.60). In rule-out analyses, stable values of penKid (not increasing from before catherization to discharge next day) reliably ruled out SKI at a specificity of 96.0% (90.1–98.9), while specificity was lower for the other candidate biomarkers [CRP: 63.4% (53.2–72.7); NGAL: 55.3% (44.1–66.1); KIM-1: 63.9% (53.5–73.4); cystatin C: 93.0% (86.1–97.1); suPAR: 52.0% (41.8–62.1)]. Using penKid, only 7 patients were categorized as false-positive, while all other patients would have been discharged safely. Study Flow PANCAKE-Study Conclusion Change in penKid levels between cardiac catheterization and discharge reliably rules out sustained kidney injury after contrast administration. PenKid thereby holds promise as an early biomarker for contrast-induced kidney injury and should be evaluated in pilot interventional trials.

491-P: Obesity-Induced Kidney Damage Is Attenuated by Genetic Deletion of miR-379 in Mice

Obesity is significantly associated with diabetes and related complications like kidney disease. Evidence shows dysregulation of certain non-coding RNAs including microRNAs can accelerate diabetic kidney disease (DKD). Recently, we found that miR-379 and its host transcript long non-coding RNA, lncMGC, were regulated by endoplasmic reticulum (ER) stress in glomerular cells, and lncMGC could promote features of DKD in mice. In this study, we examined the in vivo role of miR-379 in obesity-induced kidney disease by testing miR-379-knockout (KO) mice generated by CRISPR-Cas9 editing. Male (M) and female (F) mice were fed with control diet, or high fat diet (HFD) for 24 weeks to induce obesity in wild type (WT) and miR-379KO mice. Kidney function was examined by measuring urinary albumin and creatinine. Expression of miR-379 target genes (Edem3, Fis1), Leptin receptor (LepR) and profibrotic genes (Tgf-β1, Ctgf, Col4a1, Col1a2) was measured in isolated glomerular cells. Cortical sections were stained for histopathology. Blood glucose levels were similar in miR379KO-HFD and WT-HFD. miR379KO-HFD-F mice exhibited significantly lower body weight and body fat composition versus WT-HFD-F. Hyperinsulinemia observed in WT-HFD-F mice was attenuated in miR379KO-HFD-F. LepR expression was significantly decreased in WT-HFD-F but not miR379KO-HFD-F mice relative to controls. Increased expression of profibrotic genes, glomerular hypertrophy and interstitial fibrosis observed in WT-HFD-F were significantly attenuated in miR379KO-HFD-F mice. Similar to females, male miR-379KO-HFD-M mice showed significant reductions in glomerular hypertrophy, fibrosis and ER stress markers vs. WT-HFD-M. However, unlike females, miR379KO-HFD-M mice did not display reduced body weight compared to WT-HFD-M. These results suggest miR-379 deficiency protects mice from HFD-induced obesity and kidney injury, with some gender differences. miR-379 inhibition may be a novel therapy for HFD-induced metabolic disorders. Disclosure M. Abdollahi: None. M. Kato: None. R. Tunduguru: None. L.L. Lanting: None. R. Natarajan: None. Funding National Institutes of Health

Study of the effect of different iron-chelating agents on early renal glomerular and tubular function markers in children with beta-thalassemia

Background Advances in the management of patients of beta-thalassemia major (BTM) and the advent of effective chelators have led to the discovery of many renal complications. Mechanisms of renal impairment in BTM are still not fully investigated. Chronic anemia and hypoxia, iron overload and its complications, and direct nephrotoxic effects of some chelators are the commonly used explanations. Objectives In this cross-sectional study, the aim was to investigate the prevalence of renal impairment in patients with BTM using some of both conventional and early markers of glomerular and tubular renal function and to investigate the relation between them and iron overload (s. ferritin) and the use of a specific chelator. Patients and methods The study included 60 transfusion-dependent patients with BTM without overt renal disease or risk factors, for example, diabetes mellitus, and 30 age-matched and sex-matched healthy children as a control group (group C). Patients were divided into two equal groups according to type of chelator: group A children receiving standard deferoxamine (DFO) and/or deferiprone and group B children receiving deferasirox (new treatment). Serum levels of ferritin, creatinine, and blood urea nitrogen; albumin/creatinine ratio (ACR) in urine; estimated glomerular filtration rate using Schwartz formula; urinary beta 2 microglobulin (UB2MG); and urinary calcium/creatinine ratio were measured in all patients and healthy control children. Results Serum creatinine was within the normal range in most of the patients, and no significant differences were found between the two thalassemic groups and the control group (P=0.473). ACR in urine and UB2MG were significantly higher in the two thalassemic groups, and the deferasirox group showed significantly higher levels than both DFO and/or deferiprone group and control group (P1=0.05 and P2=0.005, respectively, for ACR and P1=0.025 and P2=0.000, respectively, for UB2MG). UB2MG was abnormally high in 67 and 76% of the DFO and/or deferiprone group and deferasirox group, respectively, followed by microalbuminuria (16 and 30%, respectively). Glomerular hyperfiltration was prevalent, but there were no significant differences in the median of the three groups (P=0.766). Hypercalciuria was also prevalent in the two thalassemic groups with abnormally elevated urinary calcium/creatinine ratio in 30% of both groups but without significant differences in the median of the three groups (P=0.123). Serum ferritin was significantly positively correlated with ACR (P=0.002) and with UB2MG (P=0.000). Conclusions ACR can be used as an early marker of glomerular function. Deferasirox use is probably safe in the usual doses in low-risk patients, keeping into consideration that high doses of deferasirox cause disruption of the renal function.

Human Pluripotent Stem Cell-Derived Kidney Model for Nephrotoxicity Studies.

Current in vitro models for identifying nephrotoxins are poorly predictive. We differentiated human pluripotent stem cells (hPSCs) into three-dimensional, multicellular structures containing proximal tubule cells (PTCs) and podocytes and evaluated them as a platform for predicting nephrotoxicity. The PTCs showed megalin-dependent, cubilin-mediated endocytosis of fluorescently labeled dextran and active gamma-glutamyl transpeptidase enzymes. Transporters from both the ATP-binding cassette (ABC) and the solute carrier (SLC) families were present at physiological levels in the differentiated cells, but important renal transporters such as organic anion transporter 1 (OAT1), OAT3, and organic cation transporter 2 (OCT2) were present only at lower levels. Radioactive uptake studies confirmed the functional activity of organic cation transporter, novel, type 2 (OCTN2), organic anion transporter polypeptide 4C1 (OATP4C1), and OCTs/multidrug and toxin extrusion proteins (MATEs). When treated with 10 pharmacologic agents as a test of the platform, the known nephrotoxic compounds were distinguished from the more benign compounds by an increase in tubular (PTC, kidney injury molecule 1 (KIM-1), and heme oxygenase 1 (HO-1)) and glomerular (nephrin [NPHS1]/Wilms tumor protein [WT1]) markers associated with nephrotoxicity, and we were able to distinguish the type of nephrotoxin by examining the relative levels of these markers. Given the functions demonstrated and with improved expression of key renal transporters, this hPSC-derived in vitro kidney model shows promise as a platform for detection of mechanistically different nephrotoxins.

Febrile Proteinuria in Hospitalized Children: Characterization of Urinary Proteins.

Background: Transient proteinuria during febrile illness is a common phenomenon. Recent studies have re-examined the pathophysiology of proteinuria and new urinary markers to characterize it, including B7-1 (CD80), which is expressed also in glomerular podocytes and influences the glomerular barrier.Aim: To investigate the pattern of proteinuria in febrile non-renal diseases, including B7-1.Methods: We prospectively analyzed urine samples of 44 febrile children and 28 afebrile controls for different protein components: albumin (glomerular marker), β2-microglobulin (tubular marker), uromodulin (Tamm Horsfall protein-THP, a renal endogenous protein) and B7-1. Febrile illness was characterized as focal bacterial vs. viral. Exclusion criteria were underlying renal disease, steroid treatment or urinary tract infection.Results: Elevated urine albumin (64.5 ± 10.3 vs. 17.8 ± 4 mg/g, mean ± S.E.M., p = 0.0009) and β2-microglobulin (1.44 ± 0.34 vs. 0.182 ± 0.03 mg/g, mean ± S.E.M., p = 0.005] and decreased uromodulin (10.5 ± 1 vs. 26.7 ± 2.2 Arbitrary units, mean ± S.E.M., p = 0.0001) excretion were found during febrile illness vs. controls. Urine B7-1 was also increased in the febrile group (0.27 ± 0.05 vs. 0.07 ± 0.01 ng/ml, mean ± S.E.M., p = 0.001), and was the only marker which was significantly higher in bacterial vs. viral disease.Conclusions: Febrile proteinuria is not generalized: while proteins of both glomerular and tubular origin increase, uromodulin decreases. Urine B7-1 is increased during fever, more significantly in bacterial infections. Thus, urinary B7-1 may be used as an additional marker to differentiate between febrile states of bacterial vs. viral origin.

Renal Evaluation in Common Variable Immunodeficiency.

Introduction Common variable immunodeficiency (CVID) comprises a heterogeneous group of disorders characterized by impaired antibody production. Kidney involvement in CVID is described in isolated and sporadic case reports. The objective of this study was to study the renal function pattern in CVID patients through glomerular and tubular function tests. Methods Study of 12 patients with CVID diagnosis and 12 healthy control individuals. Glomerular filtration rate (GFR), fractional excretion of sodium (FENa+) and potassium (FEK+), urinary concentration, and acidification capacity were measured. In addition, microalbuminuria and urinary monocyte chemoattractant protein-1 (MCP-1) were evaluated as markers of selectivity of the glomerular barrier and inflammation, respectively. Results In relation to glomerular markers, all CVID patients had normal GFR (>90 mL/min/1.73 m2), and microalbuminuria and urinary MCP-1 levels were also similar to those of controls. Interestingly, CVID patients had reduced urinary concentration capacity, as demonstrated by lower U/POsm ratio, when compared to controls. Also, while all control subjects achieved a urinary pH less than 5.3, no CVID patients showed a decrease in urinary pH to such levels in response to acid loading with CaCl2, characterizing impaired urinary acidification capacity. Conclusion Patients showed a trend towards an elevated prevalence of tubular dysfunction, mainly related to urinary acidification and concentration capacities.

Potential serum biomarkers for early detection of diabetic nephropathy

AimDiabetic nephropathy (DN) is considered as one of the diabetic complications affecting up to 40% of patients with type 1 or type 2 diabetes. In clinical practice, the frequently used markers of renal disease and progression are serum creatinine, estimated glomerular filtration rate (eGFR) and albuminuria. The aim of this study is to determine new biomarkers in human serum which are promising for early detection of DN. MethodsThis study included 50 patients with type 2 diabetes mellitus (T2DM) and 25 clinically healthy individuals. The patients were divided into two groups; group I included 25 T2DM patients with normoalbuminuria, and group II consisted of 25 T2DM patients with microalbuminuria. In all groups, neutrophil gelatinase-associated lipocalin (NGAL), β-trace protein (βTP) and microRNA- 130b (miR-130b) were estimated. ResultsThe serum levels of NGAL and βTP were significantly elevated in T2DM patients with microalbuminuria (group II) compared with T2DM patients with normoalbuminuria (group I) and control subjects but there was no significant difference between group I and control subjects. Serum miR-130b level was significantly decreased in patients with T2DM (groups I and II) compared with healthy control subjects, with a higher decrease in their levels in group II compared with group I. ConclusionOur results suggest that serum NGAL and βTP as tubular and glomerular markers respectively, together with serum miR-130b may be independent and reliable biomarkers for early detection of DN in patients with T2DM.

Patient-specific evolution of renal function in chronic heart failure patients dynamically predicts clinical outcome in the Bio-SHiFT study

Renal dysfunction is an important component of chronic heart failure (CHF), but its single assessment does not sufficiently reflect clinically silent progression of CHF prior to adverse clinical outcome. Therefore, we aimed to investigate temporal evolutions of glomerular and tubular markers in 263 stable patients with CHF, and to determine if their patient-specific evolutions during this clinically silent period can dynamically predict clinical outcome. We determined the risk of clinical outcome (composite endpoint of Heart Failure hospitalization, cardiac death, Left Ventricular Assist Device placement, and heart transplantation) in relation to marker levels, slopes and areas under their trajectories. In each patient, the trajectories were estimated using repeatedly measured glomerular markers: creatinine/estimated glomerular filtration rate (eGFR), cystatin C (CysC), and tubular markers: urinary N-acetyl-beta-D-glucosaminidase (NAG) and kidney injury molecule (KIM)-1, plasma and urinary neutrophil gelatinase-associated lipocalin (NGAL). During 2.2 years of follow-up, we collected on average 8 urine and 9 plasma samples per patient. All glomerular markers predicted the endpoint (univariable hazard ratio [95% confidence interval] per 20% increase: creatinine: 1.18[1.07-1.31], CysC: 2.41[1.81-3.41], and per 20% eGFR decrease: 1.13[1.05-1.23]). Tubular markers, NAG, and KIM-1 also predicted the endpoint (NAG: 1.06[1.01-1.11] and KIM-1: 1.08[1.04-1.11]). Larger slopes were the strongest predictors (creatinine: 1.57[1.39-1.84], CysC: 1.76[1.52-2.09], eGFR: 1.59[1.37-1.90], NAG: 1.26[1.11-1.44], and KIM-1: 1.64[1.38-2.05]). Associations persisted after multivariable adjustment for clinical characteristics. Thus, during clinically silent progression of CHF, glomerular and tubular functions deteriorate, but not simultaneously. Hence, patient-specific evolutions of these renal markers dynamically predict clinical outcome in patients with CHF.

The renal tubular damage marker urinary N-acetyl-\u03b2-d-glucosaminidase may be more closely associated with early detection of atherosclerosis than the glomerular damage marker albuminuria in patients with type 2 diabetes

BackgroundTo determine the association between urinary N-acetyl-β-d-glucosaminidase (NAG), a marker of renal tubulopathy, and carotid intima-media thickness (IMT) and plaques in patients with type 2 diabetes mellitus (T2D) and to compare the predictive value of NAG versus albuminuria, a marker of renal glomerulopathy.MethodsA total of 343 participants were enrolled in this retrospective cross-sectional study. We recruited participants with T2D who were tested for blood glucose parameters, urinary NAG, and urinary albumin-to-creatinine ratio (ACR) and had been checked for carotid ultrasonography.ResultsWe classified participants into a below-median urinary NAG group (Group I; n = 172) or an above-median group (Group II; n = 171). Mean, maximum, and mean of maximum carotid IMT and the proportion of patients with carotid plaques were significantly higher in Group II compared with Group I. In multiple linear regression analyses, high urinary NAG (Group II) was significantly associated with carotid IMT, independently of urinary ACR and other confounding factors. In terms of carotid plaques, both urinary NAG and ACR were significantly higher in participants with carotid plaques than in those without carotid plaques. After adjustment for confounding factors, both urinary NAG and ACR were significantly associated with the presence of carotid plaques.ConclusionsElevated urinary NAG, a marker of renal tubular damage, was related to increased carotid IMT and the presence of carotid plaques in patients with T2D. Urinary NAG may be a more sensitive biomarker than urinary albumin for early detection of atherosclerosis.

Stabilization of endogenous Nrf2 by minocycline protects against Nlrp3-inflammasome induced diabetic nephropathy.

While a plethora of studies support a therapeutic benefit of Nrf2 activation and ROS inhibition in diabetic nephropathy (dNP), the Nrf2 activator bardoxolone failed in clinical studies in type 2 diabetic patients due to cardiovascular side effects. Hence, alternative approaches to target Nrf2 are required. Intriguingly, the tetracycline antibiotic minocycline, which has been in clinical use for decades, has been shown to convey anti-inflammatory effects in diabetic patients and nephroprotection in rodent models of dNP. However, the mechanism underlying the nephroprotection remains unknown. Here we show that minocycline protects against dNP in mouse models of type 1 and type 2 diabetes, while caspase -3,-6,-7,-8 and -10 inhibition is insufficient, indicating a function of minocycline independent of apoptosis inhibition. Minocycline stabilizes endogenous Nrf2 in kidneys of db/db mice, thus dampening ROS-induced inflammasome activation in the kidney. Indeed, minocycline exerts antioxidant effects in vitro and in vivo, reducing glomerular markers of oxidative stress. Minocycline reduces ubiquitination of the redox-sensitive transcription factor Nrf2 and increases its protein levels. Accordingly, minocycline mediated Nlrp3 inflammasome inhibition and amelioration of dNP are abolished in diabetic Nrf2−/− mice. Taken together, we uncover a new function of minocycline, which stabilizes the redox-sensitive transcription factor Nrf2, thus protecting from dNP.

Driving iPSC-derived progenitor fate using in vitro synthetized kidney extracellular matrices assembled under macromolecular crowding

Event Abstract Back to Event Driving iPSC-derived progenitor fate using in vitro synthetized kidney extracellular matrices assembled under macromolecular crowding Valentina Magno1, Bella Roßbach2, Krithika Hariharan2, Jens Friedrichs1, Andreas Kurtz2 and Carsten Werner1, 3 1 Leibniz Institute of Polymer Research Dresden, Max Bergmann Center of Biomaterials, Germany 2 Charité – Universitätsmedizin Berlin, Berlin-Brandenburg Center for Regenerative Therapies, Germany 3 Center for Regenerative Therapies Dresden, Technische Universität Dresden, Germany Introduction: Cell-derived extracellular matrices (ECMs) have shown in the latest years to have broad application in the field of tissue engineering and regenerative medicine. The recent introduction of macromolecular crowding (MMC) for the modulation of in vitro ECM production allowed, furthermore, to better mimic physiological microenvironments[1]. In this work, we aim to explore the effect of MMC on the secretion of ECM by primary mouse mesangial cells and to study the impact of decellularized ECMs produced under standard and MMC conditions on the growth and differentiation of human induced pluripotent stem cell (iPSC)-derived intermediate mesoderm (IM) progenitors[2]. Methods: Covalently attached mesangial cell-derived ECMs were produced on a maleic anhydride co-polymer platform[3]. The ECM secretion was carried out for 8 days either under standard (Std) or MMC culture conditions by supplementing the medium with the synthetic polysaccharide Ficoll. During the ECM deposition phase, cell viability was assessed using a PrestoBlue assay. Cell layers were decellularised using an hypotonic solution and DNA remnants were removed by DNase treatment. The impact of MMC on ECM composition and morphology was investigated by colorimetric assays, immunohistochemistry, atomic force microscopy and scanning electron microscopy. IM progenitors were seeded in parallel on the mesangial cell-derived ECMs as well as on Geltrex and collagen IV as control substrates, with or without the supplementation of the ROCK inhibitor Y-27632. Adhesion and proliferation of IM progenitors were analyzed respectively using a Crystal Violet and a PrestoBlue assay. Furthermore, the expression of relevant kidney markers was examined via real time PCR. Results and Discussion: PrestoBlue assay showed that MMC did not affect the viability of the mesangial cells during ECM deposition. MMC was found to influence the composition, the mechanical properties and the supramolecular assembly of the mesangial cell-derived ECM. IM progenitors showed significantly higher cell viability and adhesion on both types of ECM (Std and MMC) compared to Geltrex and collagen IV. Particularly, in presence of the ROCK inhibitor, a significantly higher adhesion was found on MMC-ECM compared to both Std-ECM and control substrates. Higher proliferation rates were observed on both types of ECM vs. the controls, with the MMC-ECM outperforming Std-ECM. A significant increase in the expression of some mesangial markers and other glomerular markers was found for IM progenitors cultivated on MMC-ECM vs. Std-ECM, with even less mesangial-like characteristics found on the control substrates. Conclusion: Our results indicate that matrix based signals support the differentiation of the IM progenitors toward the mesangial lineage without the addition of soluble cues. In the future, the combination of cell-derived ECM and MMC may be utilized to study kidney progenitor differentiation and to develop bioengineered kidney tissues. A systematic study on different crowder categories and concentrations may also allow to control progenitor differentiation to obtain a reliable cell source for regenerative approaches. This work was supported by EU Marie Curie NephroTools FP7 Initial Training Network (ITN)

The proepicardium keeps a potential for glomerular marker expression which supports its evolutionary origin from the pronephros.

The proepicardium is the embryonic primordium of the epicardium. This transient structure is essential for cardiac development giving rise to the epicardium and supplying the heart with vascular and cardiac connective tissue progenitors. However, their nature and evolutionary origin are poorly-known. We have suggested elsewhere (Pombal et al. Evol. Dev. 10: 210-216, 2008; Cano et al., J. Dev. Biol. 1: 3-19, 2013) that the proepicardium is an evolutionary derivative of the primordium of an ancient external pronephric glomerulus, devoid of its original excretory function. In this study, we describe for the first time expression of two podocyte markers in the chick proepicardium (glepp1 and synaptopodin) and we have shown how these podocyte markers as well as the intermediate mesoderm marker Pax2 are strongly upregulated when the proepicardium is cultured with nephrogenic inducers. Retinoic acid treatment also induced in the proepicardium expression of Hoxb4, a gene which confers to intermediate mesoderm competence to respond to nephrogenic signals. Thus, a latent nephrogenic potential persists in the proepicardium and also that its original glomerular fate can be partially rescued. The transcription factor Wt1, essential for kidney and epicardial development, plays opposite roles in both tissues, inducing epithelial-mesenchymal transition in the proepicardium and promoting epithelialization in the kidneys (Essafi et al., Dev. Cell 21: 559-574, 2011). Consistently with this antithetical function of Wt1, we have observed an upregulation of podocalyxin in the epicardium of mouse embryos with conditional deletion of the Wt1 gene, while this protein is transcriptionally activated by Wt1 in podocytes.