bluuebunny/crossref_metadata_2025 huggingface dataset:
#815 Maternal and fetal outcomes in kidney transplant recipients: a single-center observational study
Authors: Artan Ayse Serra , Mirioglu Safak , Sivrikoz Tuğba Saraç , Ünal Elif , Hocaoglu Rabia Hacer , Suleymanova Vafa , Doksan Müge , Oto Ozgur Akin , Ozturk Savas , Yazici Halil , Turkmen Aydin
Published: May 2024

Background and Aims

Pregnancy poses a high risk for adverse maternal and fetal outcomes in kidney transplant recipients (KTRs), and data on long-term allograft functions compared to healthy population are still limited. Therefore, we aimed to conduct a comparative analysis of maternal and fetal outcomes in KTRs with age-matched non-transplanted controls.

Method

In this retrospective single-center study, KTRs who experienced at least one pregnancy after transplantation between 1984 and 2016 were evaluated (study group). In order to create an age-matched control group, healthy women who had one pregnancy each and received prenatal care were included. Maternal and fetal outcomes were gestational age, preterm birth, newborn mortality, admittance to neonatal intensive care unit (NICU), Apgar scores, birth weight, and obstetric complications (preeclampsia, peripartum hemorrhage, duration of maternal hospitalization) in all pregnancies, and composite kidney outcome of KTRs which was defined as progression to graft failure necessitating dialysis or re-transplantation or doubling of serum creatinine at the end of follow-up.

Results

In 53 KTRs, 68 pregnancies occurred. Mean age at birth was 31.6 ± 5.2 for KTRs and 30.5 ± 5.8 for controls (p = 0.288). Preeclampsia (29.4% vs. 2.9%, p < 0.001) and preterm birth (57.4% vs. 32.4%, p = 0.003) were significantly higher in KTRs. KTR pregnancies had lower mean birth weight (2354 ± 814 vs. 2856 ± 729 mg, p = 0.001) and longer durations of maternal hospitalization (3 vs. 2 days, p = 0.001), as well. However, neonatal mortality, admittance to NICU, and peripartum hemorrhage rates and Apgar scores were similar (Table 1). Baseline serum creatinine and eGFR levels of KTRs were 1 (0.8-1.2) mg/dl and 76.6 (59.9-91.5) ml/min/1.73 m2, respectively. Follow-up for a median of 105 months after the index birth showed higher serum creatinine levels at postpartum visits (p < 0.001) and last follow-up (p = 0.001) compared to baseline, with a tendency for increased proteinuria during pregnancies (Table 2). Six KTRs (11.3%) experienced composite kidney outcomes, including 5 patients with graft failure and 1 with doubling of serum creatinine.

Conclusion

KTRs exhibit comparable neonatal mortality and NICU rates but higher rates of preeclampsia and preterm birth. Notably, graft functions worsen significantly during postpartum follow-up.
#2599 Adam17 inhibition moderates interstitial fibrosis and macrophage infiltration in TD1 mouse model
Authors: Riera Marta , Palau Vanesa , Galdon Eric , Martyn Claudia , Cervera Cristina , Silva David , Amador Ana , Romeo Maria Jose Soler , Crespo Marta , Barrios-Barrera Clara
Published: May 2024

Background and Aims

ADAM17 participates in the release into circulation of inflammatory and fibrotic molecules, such as TNF-α actively involved in the progression of Diabetic Nephropathy. We studied the effect of specific deletion of ADAM17 in the endothelium and renal tubule in a T1 diabetic mouse model demonstrating its participation in the progression of kidney damage. As ADAM17 is also expressed in other cell types we wanted to study the effect of the complete deletion of Adam17 in type 1 diabetic mouse.

Method

We studied total inducible (by TAM) Adam17 knockout male mice (ADAM17KO) and their control littermates. Animals were rendered diabetic by STZ injection (DB group). Blood glucose, mesangial index (PAS staining), number of podocytes and positive area of α-SMA (by immunohistochemistry) were determined. We also studied the protein expression of the cytokine MCP-1 in renal cortex by Western Blot.

Results

The mesangial index value observed in the 20wk-DB group was compared with the diabetic ADAM17KO group. In the latter, the value significantly decreased. Podocyte loss detected in DB group was not observed in the KO-DB group. For interstitial fibrosis, the intensity and location of α-SMA was significantly lower in the ADAM17KO group. In addition, complete deletion of Adam17 partly prevented the infiltration of macrophages assessed by the expression of MCP-1 in the protein extract of the renal cortex.

Conclusion

Complete inhibition of Adam17 expression in T1DM mice prevents fibrosis progression and protects the glomerulus from hypertrophy. At the same time, it reduces macrophage infiltration, limiting the inflammatory response induced by diabetes. We demonstrate the influence of Adam17 on macrophages to reduce the expression of TNF-a partly responsible for the progression of diabetic nephropathy.
#1448 Regulation of renal phosphate excretion independent of FGF23 and PTH
Authors: Leifheit-Nestler Maren , Walter Stefanie , Walles Franziska , Richter Beatrice , Haffner Dieter
Published: May 2024

Background and Aims

The phosphaturic hormones fibroblast growth factor 23 (FGF23) and parathyroid hormone (PTH) promote renal phosphate excretion through FGFR1/Klotho and PTH1R-mediated ERK1/2 activation, respectively, leading to inhibition of the sodium-phosphate cotransporters NPT2a (SLC34A1) and NPT2c (SLC34A3) in proximal tubule (PT) cells. In bone cells, high extracellular phosphate can directly activate ERK1/2 via the sodium-phosphate cotransporters PiT-1 (SLC20A1) and PiT-2 (SLC20A2). To date, it is unclear to what extent phosphate can also regulate its own excretion in the kidney independently of FGF23 and PTH.

Method

To further investigate the role of phosphate in the regulation of renal phosphate homeostasis, male C57BL/6 mice were placed on a 0.8% control phosphate diet (CPD) or a 2% high phosphate diet (HPD) for 6 months. After four months, one HPD group was additionally treated with a calcimimetic (etelcalcetide) to lower FGF23 and PTH. In vitro, PT cells were stimulated with phosphate, FGF23 or PTH ± the phosphate transporter inhibitor foscarnet.

Results

HPD resulted in increased plasma concentrations of FGF23 and PTH, which were associated with reduced tubular phosphate reabsorption and increased fractional phosphate excretion. RNAseq analyses from isolated PT cells showed a reduction of Fgfr1, Kl, Slc34a1 and Slc34a3 and an induction of Slc20a2 in the HPD group compared to CPD. qPCR and immunoblot analyses of whole kidney tissue confirmed the reduction of the FGF23 co-receptor Klotho in the HPD group, while Fgfr1 was not significantly altered. Protein expression of NPT2a was decreased in isolated brush border membrane vesicles from HPD-fed mice, and immunofluorescence staining showed internalization of NPT2a from the apical brush border membrane. Treatment with etelcalcetide resulted in a reduction of circulating FGF23 and PTH, whereas urinary phosphate excretion in HPD mice remained elevated. In cultured PT cells, high phosphate induced phosphorylation of ERK1/2 and mRNA expression of Slc20a2, while Slc34a1 was suppressed. Phosphate-mediated PiT-2/ERK1/2 activation could be blocked by simultaneous treatment with foscarnet.

Conclusion

Our data suggest that high phosphate leads to internalization of NPT2a via direct activation of the PiT-2/ERK1/2 signaling cascade, independent of FGF23 and PTH, resulting in increased renal phosphate excretion.
#111 RNAseq of microdissected collecting ducts revealing the early signaling mediating the loss of aquaporin 2 after K+ deprivation
Authors: Sung Chih-Chien , Chen Min-Hsiu , Lin Shih-Hua
Published: May 2024

Background and Aims

Potassium (K+) deficiency could cause a reduction in urinary concentrating ability, resulting in nephrogenic diabetes insipidus (NDI), but the detailed mechanism remains unclear. Recently, transcriptomic and proteomic data from acquired NDI models reveal that oxidative stress, apoptosis, and inflammatory signaling are associated with AQP2 loss. We aim to explore the early signaling after K+ deprivation in collecting ducts.

Method

Immunoblotting of kidney lysate were performed at 0, 12, 24, and 48 hours after K+ deprivation in rats. Serum and urine biochemistry were also recorded. Based on immunoblotting, cortical collecting ducts (CCDs) were microdissected from rats at 6 hrs after K+ deprivation versus time controls. Small samples RNA-Seq was carried out independently in K+ deprivation rats versus controls (n = 4).

Results

Immunoblotting of bulk kidney showed a decreased in AQP2 protein abundance at 12 hours of K+ deprivation diet, and urine osmolality was significantly decreased at 24 hours, confirming the animal model of K+ deprivation-induced NDI. Small samples RNA-Seq data of CCDs at 6 hrs after K+ deprivation showed Aqp2, Aqp3, and Atp1a1 were significantly downregulated. It also revealed that chemokine transcripts (Ccl20 and Ccl28) were increased significantly. We also carried out analysis of Gene Ontology Biological Process terms that are statistically over-represented in the list of 88 “Increased Transcripts” at 6 hrs of K+ deprivation in CCDs, and many of the terms are related to glutathione metabolic process, cell chemotaxis, cellular response to lipopolysaccharide, consistent with an inflammatory response.

Conclusion

Our small samples RNA-Seq from microdissected CCDs in rats showed early cellular signaling changes in activation of oxidative stress and inflammatory signaling causing loss of aquaporin-2 in K+ deficiency induced NDI.
#883 P-cresyl sulphate and Indoxyl sulphate: two uremic toxins stimulating colon cancer progression in patients with chronic renal failure
Authors: Simeoni Mariadelina , DI Paola Rossella , De Ananya , Izhar Raafiah , Abate Marianna , Zappavigna Silvia , Perna Alessandra , Capasso Anna , Capasso Giovambattista , Caraglia Michele
Published: May 2024

Background and Aims

Chronic kidney disease (CKD) leads to the accumulation and production of uremic toxins, such as Indoxyl Sulphate (IS) and P-cresyl sulphate (p-CS). These toxins have been found to activate various processes that promote tumor growth. The literature has reported a two-fold increase in the incidence of colon cancer (CRC) and a worse prognosis in CKD patients, although the specific mechanisms underlying this association have not been fully elucidated. Given the pro-oncogenic activities of p-CS and IS at both the systemic and intestinal levels, it is plausible that these toxins contribute to the heightened aggressiveness of CRC in CKD patients.

Method

In order to assess the impact of toxins detected in uremic patients, we conducted experiments using three different colorectal cancer (CRC) cell lines (HT29, Hct116, LoVo). We tested various concentrations of IS and p-CS and measured their effects on tumor cell proliferation through a colorimetric assay called WST-8. For further analysis of cell survival, we performed a clonogenic assay. To investigate the migratory abilities of tumor cells after toxins treatment, we conducted a wound healing assay. Additionally, we assessed the invasive capacities of the three CRC cell lines post treatment with IS and p-CS using an invasion assay. In order to identify the specific pathways affected by toxins exposure, we conducted Real Time PCR and Western blot analysis to evaluate the inflammatory pathway and the epithelial-mesenchymal transition (EMT), respectively.

Results

The results of this study are remarkable, as we observed a notable increase in cell growth after exposure of cultured cells to various concentrations of toxins, as compared to the control group. Furthermore, the concentrations of IS and p-CS induced a substantial enhancement in cell migration and invasion. In the case of HCT116 cells treated with uremic toxins, there was an up-regulation of Vimentin protein expression, while E-cadherin expression was down-regulated compared to the control group. These proteins are crucial in the process of Epithelial-Mesenchymal Transition (EMT). Additionally, we evaluated the expression of genes associated with the inflammatory process (TNF-alpha, INOS, and IL-6) in the HT-29 and LoVo cell lines. Interestingly, we observed an up-regulation of these genes. Of particular importance, in the LoVo cell line, IL-6 exhibited the most significant increase in expression.

Conclusion

Based on these preliminary results, it appears that IS and p-CS play a key role in the progression of CRC in patients with CKD.
#2928 Fabry disease may imbalance integrin α3 related to endothelial dysfunction
Authors: Santana Aline Aparecida , Gregorio Paulo Cézar , Da Cunha Regiane Stafim , de Almeida Guilherme Miniskiskosky , Azevedo Carolina Amaral Bueno , Franco Célia Regina Cavichiolo , Barreto Fellype , Stinghen Andréa E M
Published: May 2024

Background and Aims

Fabry Disease (FD) is a rare disease characterized by a mutation in the GLA gene and consequently by the functional anomaly of the α-Galactosidase A (α-Gal) enzyme. The lack of this functional enzyme leads to lysosomal accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3), in different cell types, causing several symptoms throughout the body, which aggravate during the patient's longevity. We already demonstrated that exposure of endothelial cells to chloroquine, to mimic FD effects, leads to lysosomal accumulation in human endothelial cells. In the present study, we analyzed the endothelial changes in ultrastructural morphology and cell adhesion under evaluation of Integrin α3 expression, an important integrin that stabilizes cell adhesion and is involved in several signaling pathways in endothelial cells.

Method

Human endothelial cells (EA.hy926, ATCC CRL-2922) were exposed to chloroquine diphosphate (0.5 µg/mL) for 72 hours. The ultrastructural morphology was analyzed through scanning electron microscopy (SEM) and the Integrin α3 expression was analyzed through Western Blot and RT-qPCR.

Results

Through SEM (Fig. 1C-F) it was observed that after the exposure to chloroquine diphosphate, there was a decrease in cell expansion and adhesion and an increase in vesicular content. Curiously, the analysis of the Integrin α3 gene (ITGA3) and protein expression (Fig. 1A-B) demonstrated a significant increase (P < 0.05) in cells that were exposed to chloroquine diphosphate compared to non-exposed cells (control cells).

Conclusion

The in vitro condition of the FD in human endothelial cells demonstrated an increase in Integrin α3; however, there was a decrease in cell adhesion and expansion. The evaluation of the Integrin α3 expression could be important for monitoring the endothelial dysfunction in FD, as it is intrinsically involved in cell adhesion, remodeling, and fibrosis. These important changes can lead to renal impairment, proteinuria, and decreased glomerular filtration rate.
#2416 Bicc1 and PKD: linking RNA metabolism to kidney disease
Authors: Constam Daniel B
Published: May 2024

Background and Aims

Mutations in PKD1 or PKD2 lead to autosomal dominant polycystic kidney disease (PKD) by allowing urine flow-sensing primary cilia to activate tissue growth signals. However, the responsible cilia-associated factors and a strategy to block them and cure PKD are elusive. Cilia also specify organ laterality. In this process, flow-stimulated cilia activate the cytosolic protein Bicaudal-C (Bicc1) to curb the translation and/or the stability of specific target mRNAs. To identify candidate regulatory factors, we investigated if Bicc1 ribonucleoprotein particle (RNP) formation involves known proteins linked to cilia and cystic kidney diseases.

Method

To address this question, we here used AlphaFold structure modeling of protein complexes, combined with in vitro reconstitution assays and live imaging of Bicc1 RNP assembly. To begin to validate these interactions, we tested by genetic approaches if specific interacting factors also regulate Bicc1 targets in cells and mice.

Results

We found that multivalent interactions with structured domains of the ankyrin repeat and SAM domain containing (ANKS) 3 and their allosteric regulation by the related ANKS6 protein inhibit or restore Bicc1 access to target mRNAs, respectively. Concomitantly, ANKS3 alone increased the solubility and fluidity of Bicc1, whereas co-recruitment of ANKS6 had the opposite effect, liberating Bicc1 to self-polymerize and to induce phase-transitioning of target mRNAs in gel-like meshworks.

Conclusion

Perturbations in Bicc1-ANKS3-ANKS6 RNP dynamics and altered translation of Bicc1 target mRNAs emerge as likely culprits regulating cyst formation downstream of ciliary defects. ANKS6 can be sequestered in cilia by inversin, and mutations in these proteins provoke kidney cysts in nephronophthisis patients. Therefore, besides systematically profiling Bicc1 RNPs, future studies should test if ANKS6 mediates their dynamic regulation by flow-stimulated cilia.
#3020 A novel copy number analysis identifies human patients with NPHP1 whole gene deletions in previously genetically unsolved cases
Authors: Sayer John , Olinger Eric , Wilson Ian
Published: May 2024

Background and Aims

Genome sequencing is rapidly becoming a routine approach to investigating inherited human diseases in clinical practice. Nevertheless, data generated from genome sequencing requires extensive bioinformatic analysis and filtering of variants to determine pathogenicity and remains challenging. Despite well-established workflows and software in place to process raw data, the analysis of genome sequencing data is still complex. Copy Number Variations (CNVs) are increasingly recognised to be pathogenic, especially where this leads to a whole gene being deleted. Software tools to decipher CNVs across genome sequencing datasets are being utilised but are not completely accurate.

Method

Using a bespoke CNV analysis of Genomics England 100,000 Genomes Project (100 kGP) WGS data from 11754 parent-child trios, we searched for homozygous deletions overlapping with annotated genes, across the 22 autosomes.

Results

Homozygous ∼150 kb whole gene deletion of NPHP1-MALL on chromosome 1, which aligned with a known region containing segmental duplications (SD), was the most frequent observation among all autosomal recessive (AR) OMIM-morbid genes. Clinical phenotypes were consistent with this genotype including nephronophthisis and retinal dystrophy. We extended our analysis to look for NPHP1 whole gene deletions in 18877 additional singletons within the 100 kGP. This reverse genetics approach identified additional patients, up to this point genetically unsolved, with whole gene deletions and matching phenotypes. The frequency of heterozygous NPHP1 whole gene deletion was assessed in a control cohort of 15440 cancer patients within 100 kGP and was found to be 0.35%.

Conclusion

In conclusion, NPHP1 was the most frequently observed AR OMIM-morbid gene knockout in our analysis. Homozygous whole gene deletions in NPHP1 may be easily missed using standard WGS pipelines which don't assess fully for CNVs and a targeted read-depth approach to identify CNVs in parent-patient trios and singletons is valuable in identifying these deletions.
#1699 The role of interleukin 12 cytokine family in autosomal dominant polycystic kidney disease physiopathology
Authors: Ene Corina-Daniela , Penescu Mircea , Nicolae Ilinca
Published: May 2024

Background and Aims

Autosomal dominant polycystic kidney disease (ADPKD) is an inherited, complex disease, characterized by cysts formation and phenotypic changes. ADPKD physiopathology is under research, the role of proinflammatory chemokines responsible for perpetuating the cycle of chronic inflammation leading to kidney damage being intensively studied. We aimed to explore the individualized contribution of IL-12 interleukin family members in ADPKD physiopathology.

Method

We developed a prospective, case control study, in Carol Davila Clinical Hospital of Nephrology, including a group of 66 ADPKD subjects (56.1 years old, men:women 35:32), and a group of 40 healthy subjects, similar as sex, mean age. ADPKD diagnosis was based on familial history, clinical exam and CT or MRI scan. We excluded from the study subjects that presented cysts in other organs except the kidney, with eGFR<60 mL/min/1.73 mp, with history of hematuria, cysts infection, urinary tract infection, renal lithiasis, with unstable blood pressure in the last 6 months.

Laboratory tests: serum levels of monomers IL-12 p40 and IL-20 p35 and heterodimers IL-12 p70, IL-23, IL 35, assessed by ELISA method.

Statistical analysis. All the results were analyzed using IBM SPSS Statistics 2015 and were presented as average value ± standard deviation. p < 0.05 was considered with statistical significance.

Results

In ADPKD patients, we detected altered production of IL-12 cytokine, with low production of IL-12 p35 and IL-35, respectively high production of IL-12 p70, IL-23 and IL-12 p40 versus control. IL-12 p35 and IL-35 had statistically significant lower levels in ADPKD patients with eGFR<90 ml/min/1.73 mp, when compared with ADPKD patients with eGFR>90 ml/min/1.73 mp (Table 1). IL-12 p70, IL-23 and IL-12 p40 had statistically significant higher levels in ADPKD patients with eGFR<90 ml/min/1.73 mp, when compared with ADPKD patients with eGFR>90 ml/min/1.73 mp (Table 1). IL-12p70, IL-23 and IL-12 p40 had a negative significant correlation with eGFR, while IL-12p35 and IL-35 had a positive significant correlation with eGFR. Though, IL-12p35 and IL-35 serum levels correlated negatively with IL-12p70, IL-23 and IL-12 p40 serum levels in ADPKD patients.

Conclusion

The results of the present study suggest that ADPKD is a cellular source of IL-12 family, cytokines produced by antigenic stimulation. IL-12 family programming is abnormal in ADPKD patients. We detected synergic and divergent action of IL-12 cytokine family members, relevant for ADPKD physiopathology.
#1493 Upregulated miR-205 as a biomarker of diet-induced obesity-related glomerulopathy
Authors: Navarro Maru , López-Martínez Marina , Armengol Maria Pilar , Pey Irina , Farré Xavi , Rodríguez-Rodríguez Paula , Ferrer Mireia , Porrini Esteban , Luis-Lima Sergio , Martin Laura Díaz , Ampudia Rosa
Published: May 2024

Background and Aims

Obesity is a modifiable risk factor for the development and progression of chronic kidney disease (CKD). Pathways through which obesity might cause renal disease are not completely understood, as not all obese subjects develop CKD. In terms of deepening the knowledge of the preliminary steps of obesity-related renal damage, our group has developed a diet-induced obese murine model with obesity related glomerulopathy (ORG). The objective of this study was to investigate ORG at a cellular, structural and transcriptomic level as a way to discover the triggering pathogenic phenomena of ORG and evaluate potential biomarkers.

Method

Thirty Wistar rats were randomized into two groups: control rats (n = 15), which were fed with standard diet (SD) and study rats (n = 15), which were fed with high-fat diet (HFD). After 10 weeks, weight, parameters of kidney function, renal histological features, transcriptomic changes, miRNA and mRNA isolation were compared.

Results

HFD gained significantly more weight (55.8%) than SD(29.2%), p = 0.001. Albuminuria was also significantly higher in HFD (10,384.04) compared with SD (5,845.45), p = 0.001. HFD rats showed typical lesions of early-stages of ORG, with a predominance of mesangial matrix increase (MMI) and podocyte hypertrophy (PH). All histologic lesions correlated with genes differentially expressed (DE) in kidneys of HFD group and, both MMI and PH, also correlated with specific miRNAs DE in the urine of HFD group. The functional analysis showed 4 miRNAs DE in the kidneys of HFD group that negatively regulates PTEN gene, which promotes podocyte endocytosis of lipids in ORG. The electronic microscope confirmed the spaces of lipid vacuoles in the podocytes of HFD rats. Between those 4 miRNAs DE, miR-205 was also found to be upregulated in the urine of HFD group.

Conclusion

Wistar rats fed with HFD for 10 weeks developed early-stages of ORG, with a specific targetome of miRNAs and gene expression. The upregulation of miR-205 in kidney and its isolation in urine is associated with lipid endocytosis of podocytes, which takes place in the early-stages of ORG and could become a plausible biomarker of early-stages of ORG and open new avenues for future therapeutics research.
#2858 Immunohistochemical study reveals increased GLUT1 expression in proximal tubules of patients with acute interstitial nephritis
Authors: Shabaka Amir , Garcia-Fernandez Eugenia , Yébenes Laura , Alonso Patricia , Mendoza Ana , Triska Eugenia Landaluce , León-Póo Mariana , Garcia-Bermejo Laura , Juarez Gema Maria Fernandez
Published: May 2024

Background and Aims

Interstitial inflammation in acute interstitial nephritis can advance into tubular epithelial cells and produce proximal tubular dysfunction, which may be clinically expressed as renal glycosuria. Previous studies have shown that the presence of renal glycosuria in acute kidney injury is a diagnostic clue for acute interstitial nephritis (AIN) with an excellent specificity. Sodium-independent glucose transporter 1 (GLUT1) is localized in the basolateral membrane of epithelial cells along the entire length of the proximal tubule, mainly in the S3 segment, and it is involved in the transcellular glucose transport. The aim of this study was to examine renal expression of GLUT1 in the proximal tubules of patients with biopsy-proven AIN.

Method

We identified patients with biopsy-proven AIN. After excluding patients with diabetes mellitus and patients who had received steroid treatment previous to the kidney biopsy, we selected 10 patients with biopsy-proven AIN, and we selected 10 patients with acute tubular necrosis (ATN) matched for age, proteinuria and glomerular filtration rate as the control group. An immunohistochemical study for GLUT1 was performed on the biopsies of patients with AIN and ATN. Staining intensity was graded as 0 (negative), 1+ (weak), 2+ (moderate), and 3+ (strong). GLUT1 expression was defined as focal when <50% of epithelial cells stained positive, and diffuse when >50% of the tissue showed strong staining.

Results

Renal glycosuria was present in 60% of the selected patients with AIN, with a median glycosuria of 150 mg/dl (IQR 0-162 mg/dl), while only 1 patient (10%) with ATN presented glycosuria (p = 0.037). All patients with AIN showed moderate or strong cytoplasmic GLUT1 staining in the epithelial cells of proximal tubules, which was significantly more intense (2+; 20% vs 11%, 3+; 80% vs 33.3%, p = 0.008) and diffuse (80% vs 22.2%, p = 0.002) than in patients with ATN. There was a tendency for positive correlation between the concentration of glycosuria and the extension of GLUT1 staining (r = 0.316, p = 0.078).

Conclusion

We found higher expression of GLUT1 in the cytoplasm of proximal tubular cells in patients with AIN in comparison to ATN. These findings suggest that proximal tubular injury plays a key role in the pathogenesis of AIN, leading to renal glycosuria in some cases. We hypothesize that the localization of strong GLUT1 staining in the cytoplasm of proximal tubular cells may indicate a misallocation of GLUT1 from plasma membrane where it is functional and a reversed cell polarity which might account for the observed renal glycosuria.
#1978 Proteomic analysis of exosomes derived from mesangial cells treated with galactose-deficient IgA1
Authors: Narasimhan Gayathri , Ebefors Kerstin , Boi Roberto , Johansson Alva , Nyström Jenny
Published: May 2024

Background and Aims

IgA nephropathy (IgAN) is an autoimmune disease characterized by IgA deposits that build up in the kidneys, inflame and damage glomeruli causing hematuria and proteinuria. It is a leading cause of chronic kidney disease, which often results in end-stage kidney disease. There is no disease specific treatment for IgAN. The glomerulus, in which IgA1 lacking galactose residues (gdIgA1) are deposited, consists of mesangial cells, podocytes, and endothelial cells. Crosstalk between the cell types allows the glomerulus to act as a unit and react to its dynamic environment. There is very little evidence on mesangial cell derived exosomes and their cargo to understand the pathological crosstalk signaling in the glomerulus of IgAN. We isolated exosomes from mesangial cells that were treated with patient-derived gdIgA1 and IgA1 complexes. Proteomic analysis was performed to identify the exosome cargo and delineate the signaling pathways leading to maladaptive paracrine crosstalk. From our study, we aim to understand the molecular targets for therapeutic strategies in IgAN.

Method

Human mesangial cells were treated with/without IgA1, gdIgA1 or IgG for 24 h. Cell culture supernatant was collected for exosome isolations. Exosomes were analyzed by size, morphology and number using TEM and NanoFCM. Exosome protein extracts were processed for LC-Tandem mass spectrometry analysis using filter-aided sample preparation protocol. Proteomic analysis was performed using Qlucore omics explorer and pathway analysis using Ingenuity Pathway Analysis (IPA). Exosome marker expressions such as CD63, CD81 and FLOT1 and proteome validation was performed using Western blotting. Statistical analysis was performed using GraphPad Prism and values <0.05 were considered significant.

Results

We confirmed the release of exosomes by mesangial cells in response to IgA1/gdIgA1 complexes. Exosomes were fewer in unstimulated cells as compared to IgA1/gdIgA1 treated cells. They showed round morphology with a dense core and uniform size distribution. Exosomes from treated cells showed a mean size of about 75 nm and concentration in the range of 1-2 × 10^10 particles/ml. While untreated cells showed a mean size of about 100 nm and concentration of about 1 × 10^9 particles/ml. We confirmed the protein expression of exosome markers such as CD63, CD81 and FLOT1.

For the first time, we demonstrated global proteomics on exosomes from mesangial cells followed by pathway analysis. We have identified 89 unique proteins and pathways in exosomes derived from gdIgA1 treated mesangial cells that could mediate pathological signaling in the glomerulus in comparison to exosomes derived from IgA1 treated mesangial cells.

Conclusion

Our study provides an understanding of the pathological crosstalk signaling through exosomes and key molecular targets for IgAN.
#2549 Real world data of dapagliflozin effect on proteinuria in primary glomerulopathies in Croatia
Authors: Brinar Ivana Vukovic , Laganovic Mario , Zagorec Nikola , Šimunov Bojana , Radić Josipa , Gulin Tonko , Altabas Karmela , Bubic Ivan , Šimundić Tihana , Ivandic Ema , Pranjić Ita Jelić , Horvatic Ivica
Published: May 2024

Background and Aims

SGLT2 inhibitors, namely dapagliflozin, proved to slow progression of kidney disease in randomized clinical trials. Our aim was to assess real world efficacy of dapagliflozin on proteinuria level in primary glomerulopathies (GN).

Method

adult patients with biopsy proven glomerulonephritis from all university hospitals in Croatia who started dapagliflozin as a standard of care were enrolled. Data were analyzed if patients had at least six months of follow up. None of the patients were on immunosuppression. Data are shown as N (%) or median with interquartile range (IQR) and accompanied p levels.

Results

Overall 118 patients, predominantly male 84 (71.8%) of average age of 52 years (min–max 20–76), were enrolled. Most of the patients had IgA nephropathy (IgAN) (N = 55;46.6%), followed by membranous nephropathy (N = 27;22.9%), FSGS (N = 19;16.1%), membranoproliferative GN (N = 8;6.8%), Alport syndrome (N = 8;6.8%). 95.8% of patients were on ACE inhibitors or angiotensin receptor blockers and 10.7% were on mineralocorticoid receptor inhibitors and RAAS blockage was not significantly upscaled during follow up. During the median follow up of 12.2 (7.5-18.6) months, proteinuria was significantly reduced from 1139 (534–2265) to 701 (305-1543) mg/dU; p < 0.001. When divided by glomerulonephritis type, statistically significant reduction of proteinuria was observed in patients with IgAN and Alport syndrome, while trend of reduction of proteinuria was observed in all types of GN. There was a slight reduction in eGFR levels (54 (43-78) vs. 52 (39–73) ml/min/1,73 m²; p < 0.001) and rise in creatinine values (122 (89–149) vs. 128 (91–159) μmol/l; p < 0.001). There was a significant reduction in systolic (130 (120-140) vs.125 (115-134) mmHg; p < 0.001) and diastolic blood pressure (80 (75-90) vs. 80 (70-85) mmHg; p < 0.001). Reduction of uric acid level was noticed (377 (313–460) vs.343 (294–422); p < 0.001), while there was no effect on LDL levels (2.6 (2.12–3.53) vs.2.3 (1.9–3.30); p = 0.073). Dapagliflozin was well tolerated, one patient discontinued the therapy due to intolerance, one due to urinary infection and two due to relapse of GN which needed immunosuppressive therapy. One patient was excluded from analysis due to non-adherence.

Conclusion

SGLT2 inhibitor dapagliflozin has significant effect on reduction of proteinuria in patients with primary glomerulonephritis. Our data support a growing body of evidence that SGLT2 inhibitors should be used as a standard of care in patients with glomerular kidney diseases, especially IgAN. Future research should address earlier use of SGLT2 inhibitors during the treatment of glomerulonephritis alongside immunosuppressive therapy as well as look into the effect of SGLT2 inhibitors on mesangial cells.
#1089 Urinary Protein Biomarkers for Assessing Disease Activity and Chronicity in IgA Nephropathy
Authors: Post Tomas , Rother Nils , Pieterse Elmar , Rood Ilse , van Emst Liesbeth , Joosten Leo , Hilbrands Luuk , Rodriguez Julio Saez , Barratt Jonathan , Duivenvoorden Raphaël
Published: May 2024

Background and Objectives

IgA nephropathy (IgAN) is a progressive kidney disease characterized by the mesangial deposition of galactose-deficient IgA1, leading to kidney damage and may eventually lead to end-stage kidney disease. Current treatment decisions for IgAN patients rely on the degree of proteinuria. However, this is a non-specific parameter that can be the result of inflammatory disease activity and/or chronic glomerular damage. In this study, we employed the Olink proximity extension assay to comprehensively analyze the urinary proteome of IgAN patients. Our goal was to identify biomarkers capable of distinguishing between IgAN disease activity and disease chronicity.

Method

Urine samples were collected from 41 IgAN patients and 41 age- and eGFR-matched controls. Patient characteristics are shown in Table 1. Using Olink Proteomics, we measured 276 proteins in urine, from the inflammation, organ damage, and cardiometabolic panels. Comparisons were made between IgAN patients and controls, as well as between those with mild (eGFR ≥ 60 ml/min/1.73 m2 and uPCR < 100 mg/mmol) and active IgAN (eGFR ≥ 60 ml/min/1.73 m2 and uPCR ≥ 100 mg/mmol) to identify biomarkers for disease activity. Additionally, we compared IgAN patients with eGFR ≥ 60 ml/min/1.73 m2 to those with eGFR ≤ 30 ml/min/1.73 m2 to identify biomarkers associated with disease chronicity. Mann-Whitney U tests were used for group comparisons. Correction for multiple testing was performed using the Benjamini-Hochberg procedure. Disease activity and chronicity scores were calculated by averaging the z-scores of the identified biomarkers.

Results

L-Selectin (SELL), leukocyte inhibitory factor (LIF), interleukin-6 (IL-6), and carbonic anhydrase 1 (CA1) were most prominently elevated in IgAN patients compared to controls (Fig. 1A). SELL and CA1 were also markedly elevated in the active versus mild IgAN patients, and were therefore selected as biomarkers of disease activity (Fig. 1B). Contactin 2 (CNTN2) and chemokine (C-C motif) ligand 25 (CCL25) were most prominently elevated in IgAN patients with eGFR ≤ 30 ml/min/1.73 m2, and were selected as biomarkers of disease chronicity (Fig. 1C). We plotted the disease activity and chronicity scores calculated from the identified biomarkers to distinguish the different IgAN disease stages (Fig. 1D).

Conclusion

This study identified urine biomarkers indicative of IgAN disease activity and chronicity. Calculating an activity and chronicity score using these biomarkers offers a new approach to distinguish between inflammatory disease activity and chronicity, providing an alternative to proteinuria for guiding treatment decisions in IgAN patients. Validation of these biomarkers, and the scores calculated from them, in a larger cohort of IgAN patients is currently underway.
#1843 Deletion of IL17-producing follicular helper T cells restrains the germinal center reaction in systemic lupus erythematosus
Authors: Podesta' Manuel A , Cavazzoni Cecilia B , Richardson Sierra , Zhang Hengcheng , Gempler Maya , Nelson Cody , Chandrakar Pragya , Lee Jeong-M I , Sage Peter T
Published: May 2024

Background and Aims

Systemic lupus erythematosus (SLE) is an autoimmune disorder in which autoantibody formation can lead to severe kidney pathology. Interleukin (IL)17-producing T cells have been suggested to play an important role in the pathogenesis of SLE, and IL-17 inhibition was shown to ameliorate SLE pathology in lupus-prone mice. Moreover, the frequency of circulating follicular helper T (Tfh) cells, a T cell subset that orchestrates the maturation of high-affinity antibodies in the germinal center of secondary lymphoid organs, is associated with disease activity in SLE. Increased frequencies of IL17-producing Tfh have been reported in SLE patients compared to controls, but their role in SLE pathogenesis is still unknown.

Method

By leveraging the B6.Sle1NZM2410/AegYaa strain, we generated lupus-prone mice in which IL17-producing Tfh cells could be either fate-mapped (SLE Tfh17-FM, Il17aCRERosaLoxSTOPLox-YFPSle1+/+Yaa+) or fate-mapped and selectively depleted by intraperitoneal diphtheria toxin (DT) administration (SLE Tfh17-DTR, Il17aCRERosaLoxSTOPLox-YFPCxcr5LoxSTOPLox-DTRSle1+/+Yaa+). Tfh17-FM and Tfh17-DTR mice (both Sle1−/−Yaa−) were used as controls.

Results

Tfh17 comprised approximately 0.4% of total splenic Tfh in SLE mice at 6 weeks of life, and DT administration caused potent and selective depletion of these cells in the SLE Tfh17-DTR strain (Fig. 1A). Early Tfh17 deletion from week 4 to week 9 did not modify the increased Tfh frequency observed at 20 weeks in SLE mice, but resulted in long-term reduction of splenic germinal center (GC) B cells and plasmacells (Fig. 1B-C). IL17 receptor A was overexpressed in non-GC B cells and plasmacells of SLE mice compared to controls, suggesting a possible increased susceptibility of these subsets to IL17 (Fig. 1D). SLE Tfh17-DTR mice treated with DT displayed a trend towards decreased anti-DNA levels compared to SLE Tfh17-FM (Fig. 1E), but both glomerular immunocomplex deposition and renal pathology were not significantly impacted (Fig. 1F-G).

Conclusion

The germinal center reaction is affected by selective depletion of IL17-producing Tfh cells in SLE mice, with reduced differentiation of GC B cells and plasmacells. The modest effect of Tfh17 depletion on autoantibody levels and lupus nephritis pathology suggests that other Tfh subsets may play a role in GC-derived autoantibody generation. In addition, extrafollicular sources may also contribute to autoantibody production in SLE.
#68 Novel clinical application of urinary angiotensin-converting enzyme assay in renal sarcoidosis: a retrospective observational study
Authors: Okamoto Koji , Chiba Yuki , Murakami Koji , Miyazaki Mariko , Makino Rui , Yoshida Mai , Nagasawa Tasuku , Sato Hiroshi , Tamada Tsutomu , Tanaka Tetsuhiro
Published: May 2024

Background and Aims

Renal manifestations of sarcoidosis are observed in approximately 1–5% and characterized mainly by granulomatous interstitial nephritis, which could lead to kidney failure. Angiotensin-converting enzyme (ACE) reflects the presence of granuloma; hence, serum ACE and tubular injury markers are measured in renal sarcoidosis (RS); however, they either have low sensitivity or low specificity. Although its practicality has not been established, the use of ACE levels in cerebrospinal fluid has been reported for neuro-sarcoidosis. Therefore, we hypothesized that urinary ACE could reflect granuloma presence in the kidneys and could be a disease-specific marker for RS.

Method

This was a single-center, retrospective observational study. We evaluated clinical characteristics and kidney biopsy findings from electronic medical record surveys and kidney biopsy specimens. The control groups included sarcoidosis without renal involvement (SAR-control) and tubulointerstitial nephritis without a sarcoidosis etiology (TIN-control). Serum and urinary ACE levels were measured, and urinary fractional excretion of ACE (FEACE) was investigated. Serum ACE assay and urinary ACE levels were measured using the colorimetric method with p-hydroxybenzoyl-glycyl-L-histidyl-L-leucine. A total of six patients (three with RS; one with sarcoidosis as controls; and two with TIN controls) with undetectable urinary ACE levels were excluded from the urinary ACE and FEACE analyses. Tubulointerstitial lesions were visually assessed for essentially normal (%), inflammatory cell infiltrates (%), tubular atrophy (%), and interstitial fibrosis (%) by several board-certified nephrologists, and analyzed the correlation of these lesions with each marker.

Results

This study included 18 patients with RS, 18 as SAR-control, and 10 as TIN-control. Serum ACE, urinary ACE, and FEACE levels in the RS group were significantly higher than those in the control groups. Receiver operating characteristic analysis identified FEACE and serum ACE levels as useful biomarkers for diagnosing RS among patients with both controls, respectively. In the RS group, FEACE was positively correlated with the degree of tubulointerstitial injury (r = 0.69, P = 0.0045), and the cutoff value of FEACE for diffuse tubulointerstitial injury (50.0% or more) was 0.39%, with a sensitivity and specificity of 100.0%. Furthermore, obvious positive correlations were observed among FEACE, inflammatory cell infiltrates (r = 0.53, P = 0.044), and interstitial fibrosis (r = 0.56, P = 0.029) in the RS group but not in the TIN-control.

Conclusion

Combination with serum and urinary ACE could help diagnose and assess disease severity among patients with RS.
#1639 Nephrotic syndrome with anti-nephrin antibodies—case report of minimal change glomerulonephritis
Authors: Knehtl Maša , Piko Nejc , Zorman Tadej , Ekart Robert , Bevc Sebastjan
Published: May 2024

Background and Aims

Minimal change disease (MCD) in adults is one of the causes of nephrotic syndrome. Recently, anti-nephrin antibodies localized in glomerular podocytes have been described in a certain subset of patients with MCD, supporting the autoimmune etiology of the disease.

Method

A 79-year-old man with arterial hypertension, hypothirosis and mild dementia presented with new-onset nephrotic syndrome. Peripheral and periocular edema, hypoalbuminemia (serum albumin 17 g/l), proteinuria 15 g/day and hypercholesterolemia (serum cholesterol 6.7 mmol/l) were noted. Blood pressure was 133/85 mmHg. Creatinine was 165 mcmol/l on admission and increased to 361 mcmol/l in the following days. Immunologic tests were negative, including antibodies against phospholipase A2-receptor (anti-PLA2R) and thrombospondin-7A (anti-THSD7A). Anti-HBc was positive, HBV DNA was below 15 IU/ml. Liver tests and abdominal ultrasound were normal, with no signs of liver disease.

Results

Renal biopsy revealed 18 glomeruli with global sclerosis of one glomerulus (1/18) and 5% interstitial fibrosis. Tubular damage was noted, which could be attributed to severe proteinuria. Immunofluorescence showed discrete IgG podocyte deposits with a uniform kappa/lambda distribution and a high probability of idiopathic minimal change disease due to anti-nephrin antibodies. Electron microscopy showed diffuse effacement of the podocyte foot processes without deposits. The serum taken after the biopsy and before the start of immunosuppressive therapy was positive for anti-nephrin antibodies.

Due to a possible pharmacokinetic interaction with tenofovir, which the patient was receiving because of his anti-HBc positivity, we decided not to use cyclosporine. In addition to standard therapy with angiotensin convertase inhibitors, calcium channel blockers and furosemide, the patient was treated with low-dose oral glucocorticoids and mycophenolic acid. In addition, he received rituximab. After a few weeks, the oral glucocorticoids and mycophenolic acid were discontinued. The nephrotic syndrome regressed completely and renal function improved.

Conclusion

Anti-nephrin antibodies have contributed to a subgroup of patients with MCD, they can be found in a renal biopsy and are also detected in the serum of these patients. Targeted anti-B-cell therapy should be introduced to improve the prognosis of patients.
#1505 Late-onset lupus nephritis: clinical-epidemiological, histological, prognostic and therapeutical implications: a single center experience
Authors: Rodríguez Jorge González , Jatem Elías , Abó Anabel , Ribas Miquel Pinyol , Martin-Conde Maria Luisa
Published: May 2024

Background and Aims

Systemic lupus erythematosus (SLE), a chronic autoimmune disease, and lupus nephritis (LN), are typically diagnosed in women in fertile age. Late-onset SLE is defined as a disease onset over 50 years. Our main objective was to analyze the clinical, histological, prognostic and therapeutical features related to LN in our late-onset SLE population.

Method

A single center and retrospective study was performed comparing clinical-demographic, histological, prognostic and therapeutical data of 45 LN biopsied patients from 1994 to 2023. We divided the study population according to the age of onset of LN into classic-onset (< 45 years) and late-onset (> 45 years).

Results

Late-onset LN patients showed a higher association with Sjögren's and antiphospholipid syndrome, more cardiovascular comorbidities at presentation, poorer renal function at diagnosis and higher SDI score. On histology they showed more non-proliferative LN classes (II followed by V). Late-onset LN responders needed more time to achieve response but had fewer relapses. Non responsive patients had more incidence of acute kidney injury (AKI) and more glomerulosclerosis and interstitial fibrosis-tubular atrophy (IFTA) at presentation, as well as more need for renal replacement therapy (RRT) at diagnosis and at follow-up. The principal independent variables associated with relapse were the younger age at SLE diagnosis and an initial partial renal response (PRR). Patients of both groups who experienced relapse received higher doses of intravenous cyclophosphamide and less mycophenolic acid (MFA) analogues. Patients who received induction with MFA analogues were more likely to achieve complete renal response (CRR) or non-renal response (NRR).

Conclusion

late-onset LN is often accompanied by poor renal function at presentation. It seems imperative to establish a tailored approach in this fragile population in order to avoid unnecessary immunosuppression.