Uromodulin Research Articles

Knowledge mapping of UMOD of English published work from 1985 to 2022: a bibliometric analysis

BackgroundUMOD is exclusively produced by renal epithelial cells. Recent genome-wide association studies (GWAS) suggested that common variants in UMOD gene are closely connected with the risk of CKD. However, a comprehensive and objective report on the current status of UMOD research is lacking. Therefore, we aim to conduct a bibliometric analysis to quantify and identify the status quo and trending issues of UMOD research in the past.MethodsWe collected data from the Web of Science Core Collection database and used the Online Analysis Platform of Literature Metrology, the Online Analysis Platform of Literature Metrology and Microsoft Excel 2019 to perform bibliometricanalysis and visualization.ResultsBased on the data from the WoSCC database from 1985 to 2022, a total of 353 UMOD articles were published in 193 academic journals by 2346 authors from 50 different countries/regions and 396 institutions. The United States published the most papers. Professor Devuyst O from University of Zurich not only published the greatest number of UMOD-related papers but also is among the top 10 co-cited authors. KIDNEY INTERNATIONAL published the most necroptosis studies, and it was also the most cited journal. High-frequency keywords mainly included ‘chronic kidney disease’, ‘Tamm Horsfall protein’ and ‘mutation’.ConclusionsThe number of UMOD-related articles has steadily increased over the past decades Current UMOD studies focused on Biological relevance of the UMOD to kidney function and potential applications in the risk of CKD mechanisms, these might provide ideas for further research in the UMOD field.

#4952 CLINICOPATHOLOGIC CORRELATIONS OF URINARY PROTEOMIC AND METABOLOMIC ANALYSIS IN PATIENTS WITH RENAL AA AMYLOIDOSIS AND MEMBRANOUS NEPHROPATHY

Abstract Background and Aims AA Amyloidosis is a multisystemic amyloidosis subtype that develops on the background of various chronic inflammatory etiologies. Urinary omics studies have become a promising tool for elucidating pathophysiology and prognosis of glomerular diseases. However, no urinary omics analysis has been performed focusing on renal AA amyloidosis in literature to the best of our knowledge. Our main aim in this study is to perform a comparative urine proteomic and metabolomic analysis of recently diagnosed renal AA amyloidosis and to investigate the correlation of bioinformatic results with clinical and pathological data. Method Urine samples of 8 recently diagnosed AA amyloidosis (AA), 8 membranous nephropathy (MN) and 6 healthy control group patients were collected before kidney biopsy procedure. Proteomic analyzes were performed with nLC/Q-TOF MS/MS and metabolomic analyzes were performed by GC/MS in all patients. Biopsy specimens were scored according to glomerulosclerosis (G), tubular atrophy (TA) and interstitial fibrosis (IF) grades by two pathologists. Raw spectroscopic data was analyzed using MaxQuant and MS-DIAL programs for proteomic and metabolomic studies, respectively. Statistical analysis of the differences in molecules between study groups were performed with ANOVA and HSD-Tukey tests. Principal component (PCA) and heatmap analyzes were made in R language, while gene ontology (GO), network and functional enrichment analysis of bioinformatic results were performed with PANTHER, STRING and MetaboAnalyst databases. Results In comparison between AA and MN groups, median eGFR values tend to be lower in the AA group (67.6 vs 112 ml/min/1.73 m2 respectively, p = 0.08). Median 24-hour urine protein levels did not show statistically significant difference (9499 vs 9512 mg/day respectively, p = 0.9). Percentage of patients with moderate/severe IF/TA was higher and G score was tend to be in AA group compared to MN group (p values 0.02 and 0.07 for IF/TA and G scores, respectively). As a result of proteomic analysis, a total of 859 proteins were determined. Statistical analysis showed 51 proteins that were significantly differ in AA group compared to the control group. GO and functional enrichment analyzes showed that statistically most significant sub-domains were mainly related with cell-cell adhesion (Figure 1 & 2). In comparative analysis between AA and MN patients, uromodulin (UMOD) was lower in the AA group than in the MN group (log2FC -3.37), whereas ribonuclease 1 (RNASE1) and α-1-microglobulin/bikunin precursor protein (AMBP) were higher in the AA group (log2FC 3.41 and 3.07, respectively). In Spearman correlation analyzes, significant negative correlations were demonstrated between UMOD-proteinuria (r = -0.48, p = 0.03) and between AMBP-eGFR (r = -0.69, p = 0.003) variables. Metabolomic analysis showed 9 metabolites that were significantly different between AA and other study groups. Myo-inositol and urate were higher in AA group compared to MN group, while D-mannitol and N-acetylglutamate were higher in AA group compared to the control group. Significant positive correlation independent of GFR was detected between RNASE1 and urate (r = 0.63, p = 0.01). Conclusion Our study is the first urinary comparative omics analysis performed on renal AA amyloidosis patients to the best of our knowledge. We demonstrated specific protein and metabolites that distinguish AA group from the control and MN groups. Enrichment and GO analyzes between AA and the control group showed a negative enrichment in cell-cell adhesion related sub-domains, suggesting a possible increased urinary shear stress resulting in downregulation of cadherins in AA amyloidosis. In comparative analysis between AA and MN groups, UMOD and AMBP proteins and myo-inositol were thought to be associated with high tubulointerstitial damage, whereas RNASE1 and urate were believed to be related with systemic inflammation and endothelial damage [1].

#2850 EFFICIENCY OF THE GENETIC STUDY IN THE DIAGNOSIS OF AUTOSOMAL DOMINANT TUBULOINTERSTITIAL NEPHROPATHY

Abstract Background and Aims Autosomal dominant tubulointerstitial nephropathy (ADTKD) is a rare underdiagnosed cause of chronic kidney disease (CKD). ADTKD is a renal disease with a non-specific phenotypic expression that cause a slow progression over time. Given the great variability in its inter-familial presentation and among the same family members carrying the same mutation, its diagnosis is a challenge. Five genes have been identified to date such as Uromodulin (UMOD), Renin (REN), Hepatocyte Nuclear Factor 1b (HNF1β), Mucin-1 (MUC1) and, more recently, new genes, SEC61A1(encoding protein transport protein Sec61 subunit α and atypical (DNAJB11). Our main objective is to analyze the diagnostic yield of the genetic study in cases of clinical suspicion of ADTKD in patients with CKD of unknown etiology. We present a case series of patients from our center with CKD of unknown etiology and clinical suspicion of ADTKD. Genetic analysis of the gene SEC61A1 and DNAJB11 was not available at that time. Method This is a descriptive observational retrospective study from 2016 to 2021 at the Virgen del Rocio University Hospital in Seville, Spain. A total of 26 cases had phenotypic presentation of ADTKD. We collected clinical, laboratory, radiologic, and pathologic data. We performed in all the cases genetic testing for UMOD, MUC1, REN and HNF1β mutations. We collected data in a database, including age of presentation of kidney disease, sex, family history of kidney disease, the presence of hypertension at early age (defined as under 40 years of age), hyperuricemia (defined as uric acid >7.5 mg/dl) at early age (<40 years), hemoglobin at the time of diagnosis, age at last follow up with renal function and estimated glomerular filtration rate (eGFR) using the CKD-EPI (CKD Epidemiology Collaboration), urine elemental, renal ultrasound and renal biopsy if it had been performed. Descriptive results of quantitative variables are expressed as median. For categorical data, frequency and percentage are reported. Results Of the 26 cases with suspected ADTKD, 4 cases presented a genetic study that confirmed the diagnosis, that is, 15% of the cases. The median age at the time of the diagnosis was 41 years (IQR 32.7–48). 84% of the cases were male; 34% of cases had a family history of CKD of unknown etiology. 65% percent of the cases had a history of hyperuricemia at early age (<40 years), with a personal history of hypertension <40 years in 30% of the cases; 44% of the cases had renal lithiasis. The median glomerular filtration rate (CKD-EPI) at the time of diagnosis was 70 ml/min/1.73 m2 (46.4–70.7). In most of the patients, the urine elemental was normal. Renal cysts were found on ultrasound in 46% of patients. In 5 of the twenty-six patients a renal biopsy was performed, in which 60% presented data compatible with ADTKD with interstitial fibrosis and tubular atrophy. 4 of the patients with clinical suspicion of ADTKD obtained a genetic study that confirmed the diagnosis. The most frequently identified gene was HNF1β, being present in two of the four patients. The other genes identified were UMOD and MUC. The results are summarized in Table 1. Conclusion Adequate selection of patients based on clinical suspicion is essential to give greater profitability to the genetic study of this entity. The negativity of the study cannot exclude the diagnosis, since at the moment not all the causative genes of this pathology have been identified and it is necessary to use massive sequencing techniques or even the analysis of the complete exome to see the possibility of other variants of the disease.

#3271 EXPRESSION OF THE COLLAGEN IV α345 MOLECULE ALONG THE RENAL TUBULE IN HEALTH AND DISEASE

Abstract Background and Aims Patients suffering from Alport Syndrome (AS) have a high life time risk of kidney failure and hearing loss. The underlying molecular cause is a germline mutation in one of the COL4A3/4/5 genes, whose gene products collectively form an important component of the glomerular basement membrane and the inner ear. Recent genetic studies have shown that the AS is much more prevalent than clinically recognized, suggesting that atypical clinical cases are frequent. Thus, patients with AS may phenocopy other kidney diseases. To date, pathomechanistic studies of the AS have focused exclusively on the glomerular membrane, yet equally strong expression of collagen IV can be found along the distal renal tubule. Structural disintegration of the distal tubular membrane may induce inflammation and fibrosis, thus progression of kidney disease. We hypothesize that genetic loss of the tubular collagen IV molecule contributes to kidney failure and may drive atypical phenotypes. Method Immunohistochemistry (IHC) and in situ hybridization (ISH, RNAscope) for COL IV α5, where IHC was performed on healthy human tissues as well as biopsies from patients with AS. Tubular marker staining for localization of COL IV α5 expression as well as spatial changes on consecutive sections. Human urinary primary tubular cells (huPTC) from healthy probands and patients with AS were generated and cultivated in cell culture, with analysis of COL IV mRNA by qPCR and detection of specifically expressed proteins by immunoblotting. Results COL IV α5 was detected within the tubular basement membrane (TBM) of the distal segments of renal tubules by IHC, including the thick ascending limb (TAL) of Henle (Tamm-Horsfall Protein), the distal convoluted tubule (11ß-Hydroxysteroid-Dehydrogenase) and the collecting duct (aquaporin 2). Spacial vicinity of these areas showed a predominance of interstitial fibrosis and inflammation in patient samples with AS, but not control samples with primary proteinuric diseases. Collagen IV protein is lost along the TBM of patients with AS, even in the hemizygous male and heterozygous female carriers of the hypomorphic but frequent COL4A5 mutation G624D. By ISH, COL4A5 expression was mainly observed in the glomerulus (most likely podocytes) and broadly in tubular epithelial cells, with only few tubulointerstitial cells showing mRNA expression. Likewise, the huPTC showed COL IV expression in qPCR, as well as COL IV α5 protein in immunoblotting. Conclusion Our studies show that the COL IV molecule is situated within the TBM of the complete distal tubule, starting from the TAL throughout the distant collecting duct. Although the epithelial cells have no detectable protein, in situ hybridization and primary cell cultures clearly suggest that tubular cells are a major source of the COL IV α345 molecule within the TBM. Renal biopsies of AS patients with diagnostic genetic COL IV variants indicate that tubulointerstitial fibrosis and inflammation starts in the vicinity of the distal tubule. Therefore, we postulate that the pathogenesis of AS may in part stem from the (distal) tubular apparatus, which is already known from other diseases such as ADTKD and nephronophthisis.

#3639 WHEN WHOLE EXOME SEQUENCING IS NOT GOOD ENOUGH: BACK TO THE ROOTS OR FORWARD TO THE FUTURE

Abstract Background and Aims Over the last few years, massively parallel sequencing (MPS) has been established in genetic diagnostics, where large virtual panels of candidate genes can be screened on a whole exome basis. This has enabled large analyses for most medical indications, including renal genetic diseases. The 25 year old, index female patient was referred for disease clarification. Both non-related parents suffered from end-stage renal disease, with kidney failure in mid adulthood and familiar cystic disease in both the maternal and paternal lines. An external genetic analysis applying MPS on a restricted panel of genes retrieved no conclusive result, apart from two variants in the UMOD gene, classified as class 3 and 4 following ACMG criteria. Method Whole exome sequencing (WES), linkage analysis, haplotype reconstruction, whole genome sequencing (WGS), long-range PCR (LR-PCR). Results The detailed pedigree of the family displayed an autosomal dominant disease with several family members reaching end-stage kidney disease on both the maternal and paternal sides. The maternal family is characterized by polycystic disease of the kidney and most profoundly the liver, with two female members already liver transplanted and one on the waiting list. The paternal family shows more unspecific chronic kidney disease (CKD) with multiple cysts in the kidney, but not in the liver. Two male members reported gout attacks, before the knowledge of CKD. We consented 12 family members for research studies and collected blood for DNA. Contemporary MPS of a virtual panel for cystic diseases (> 60 genes, i.e. PKD1 and PKD2, all known genes for cystic liver disease) in the index patient did not yield a conclusive result, apart from the previously reported UMOD variants [c.464G>A, p.(Cys155Tyr) and c.907G>C, p.(Asp303His)]. These variants both segregated in the paternal family, defining them as lying in cis on one allele. WES was performed on a trio of three affected family members of the maternal family, yielding no further result. Linkage analysis showed 9 peaks with a LOD score above 2.0. One peak is situated on chromosome 16, containing the candidate genes PKD1 and IFT140. WGS revealed the heterozygous variant c.2180T>C, p.(Leu727Pro) in exon 11 of PKD1, which has been reported as causative numerous times in the past. Conventional LR-PCR, which is designed to acknowledge the highly homologous regions of the 6 pseudogenes of PKD1, also retrieved the PKD1 variant, which was not called by MPS. Comparing the sequence reads of the WES with the WGS, the PKD1 variant was detected in 12% in the former, whereas complete detection was achieved in the latter. Since the cutoff for calling variants in WES was set to 20%, the PKD1 variant was not visible to the genetic analyst. The index patient indeed inherited both diseases from her parents, giving her the simultaneous diagnoses of ADPKD and ADTKD(-UMOD). Conclusion ADPKD is one of the most frequent genetic diseases of the human, with PKD1 being involved in > 80% of cases. With the apparent ease of modern technology, increasingly ADPKD families are presented for genetic consultation, also to non-specialized centers. It is tempting to use MPS considering the number of candidate genes and the size of PKD1 (46 exons). However, a negative result should be supported either by LR-PCR or WGS, taking the profound difficulties with the 6 pseudogenes of PKD1 into consideration. It is not known whether the predominance of liver disease in this family with the PKD1 c.2180T>C variant implicates a genotype-phenotype correlation, or whether other (genetic) modifiers may press for a liver phenotype. The renal prognosis of the index case is feared to be worse baring both diseases, even with the pathogeneses being different with a ciliopathy (ADPKD) and a toxic proteinopathy (ADTKD-UMOD), respectively. Since both affected genes are situated on the respective chromosomes 16, the patient bares a 100% risk of receiving children with an adult onset kidney disease, where pre-implantation technology will not solve this specific problem.

#6786 ADTKD DUE TO MUC-1 GENE MUTATION: A MAJOR DIAGNOSTIC CHALLENGE, NOT IDENTIFIED BY EXOME SEQUENCING

Abstract Background and Aims Autosomal dominant tubulointerstitial renal disease (ADTKD) is a rare cause of end-stage renal disease (ESKD) in adulthood. In addition to its rarity, ADTKD is probably underdiagnosed due to the very non-specific and heterogeneous clinical-laboratory-histopathological-instrumental picture and because of the diagnostic need to use specific methods of genetic investigation in order to highlight the mutations within the VNTR sequences of the MUC1 gene, not recognized by the most common exome sequencing. Mutations in the MUC1 gene, together with those in the UMOD gene, are the main mutations implicated in the pathogenesis of ADTKD. Method We analyzed the clinical, laboratory and histopathological characteristics of four families followed by our center with suspected diagnosis of ADTKD and detection of mutation of the MUC1 gene. Results Our data (Table 1) revealed a marked phenotypic variability, even within members of the same family (Fig. 1), and in particular as regards the age of disease onset and the different rate of progression towards ESKD (range 16 – 88 years). From a laboratory point of view, in 10 out of 13 patients the sediment was negative, and only in 3 patients proteinuria >1 g/24h was highlighted. In 6/13 the onset occurred at the age of less than 35 years and among these, 4 of them presented ESKD early. In the few cases in which renal biopsy was performed (4/17), representing 0.4% of all biopsies in our centre, the most frequent finding was the presence of tubular atrophy and interstitial fibrosis (3/4), with cystic ectasia of the tubular lumen at the cortical-medullary level in half of the cases. Among the 10 patients subjected to genetic analysis, only in one case the mutation was highlighted by exome sequencing. Conclusion The large phenotypic variability, in particular of age at achievement of ESKD, suggests that other factors (modifier or environmental genes) could also affect disease progression. Common gene sequencing methods (e.g. exome sequencing, Sanger) have proved unable, in most cases, to identify mutations within the VNTR sequences of the MUC1 gene.

#5302 REVISITING THE PATHOGENIC ROLE OF THE SLC8A1 GENE IN A FAMILY WITH A NOVEL TERMINAL GROSS DELETION IN THE UMOD GENE

Abstract Background and Aims Autosomal dominant tubulointerstitial kidney disease (ADTKD) - the third most common genetic disorder in adults with CKD - comprises a heterogeneous group of rare, hereditary kidney diseases characterized by family history of progressive chronic kidney disease (CKD) with bland urine sediment, absence of significant proteinuria and normal or small-sized kidneys. Biopsy findings are nonspecific and the gold standard for definitive diagnosis is identification of a pathogenic variant in the genes UMOD, MUC1, HNF1β, REN or SEC61A1. Method Case report. Results The proband was a 27-year-old white male referred for CKD, KDIGO stage 3a. His medical history was relevant for nocturnal enuresis until age 10; arterial hypertension diagnosed at age 19; and asymptomatic hyperuricemia, disproportional to the level of renal function impairment. Urinalysis was negative for protein, red blood cells or leucocytes and magnetic resonance imaging showed normal sized kidneys with bilateral cysts in the medulla and the corticomedullary junction. The family pedigree (Figure 1) illustrates that hyperuricemic nephropathy and CKD segregated in this family with an autosomal dominant pattern of inheritance. A custom NGS-based gene panel allowed to identify in the proband (IV-2) and his affected brother (IV-1): (i) a previously unreported large deletion in the UMOD gene, encompassing the entire exon 11, classified as “likely pathogenic”; ii) an ultra-rare nonsense SLC8A1 allele — c.16C>T p.(Arg6*), classified as “variant of uncertain clinical significance (VUS)”; (iii) an ultra-rare missense variant in the gene encoding polycystin-1 (PKD1) — c.9170T>C p.(Val3057Ala), classified as VUS. Since the latter was inherited from their healthy mother (III-1) it was considered a benign variation, similar to the more common p.Val3057Met variant affecting the same codon. Other affected family member (III-5) was heterozygous for the UMOD exon 11 deletion, which was excluded in two healthy relatives (III-1, III-4), establishing the primary diagnosis of ADTKD-UMOD. It is noteworthy that the only two gross UMOD deletions associated with ADTKD affected evolutionarily conserved residues in exon 4 [1]. The presence of the SLC8A1 p.(Arg6*) allele in patients IV-1 and IV-2, who had early clinical presentations, together with its absence in patient III-5, who had a significantly later clinical onset than her older brother (III-2), suggests that it exerted a pathogenic or disease modifier role in this family, in accordance with the predicted protein truncating effect. Indeed, genome-wide linkage analysis in 5 multiplex families with ADTKD identified involvement of chromosome 2p22.1-p21; SLC8A1 was the most likely gene but no pathogenic variants could be identified; copy number variants might have been missed [2]. Unfortunately, in our family study, we could not identify any subject segregating only the SLC8A1 p.(Arg6*) allele, which would have been indispensable to demonstrate its pathogenicity. Conclusion Herein, we report a family with hyperuricemic nephropathy where genetic testing identified a novel, likely pathogenic UMOD variant. The mechanisms underlying the pathogenicity of exon 11 deletion require further study. Additionally, the co-segregation of an ultra-rare nonsense SLC8A1 variant in some individuals was associated with earlier onset and faster progression of CKD, suggesting once again a role for SLC8A1 in the pathogenesis of ADTKD.

#5158 AUTOSOMAL DOMINANT TUBULAR KIDNEY DISEASE: FROM PHENOTYPE TO GENOTYPE

Abstract Background and Aims Autosomal dominant tubulointerstitial kidney disease (ADTKD) represent a recently described group of kidney diseases, characterized by chronic interstitial nephritis (CIN), in most cases hereditary, with an autosomal dominant inheritance pattern, subclassified according to its genetic cause, when identified. Mutations in UMOD, MUC1, REN and HNF1B genes have been implicated in their pathogenesis. Recent molecular diagnostic technologies, such as Next-Generation Sequencing (NGS), and particularly the use of restriction and Snapshot method for the detection of a cytosine insertion on the coding variable-number tandem repeat (VNTR) sequence in the MUC1 gene encoding mucin 1, have allowed the nephrologists to diagnose renal diseases of previously unknown cause, with a real prevalence probably underestimated. We present the cohort of patients identified with ADTKD in the Nephrogenetics’ clinic from the Nephrology department of our hospital from 2015 to 2022. Method Patients with chronic kidney disease (CKD) of unknown etiology and renal phenotype suggesting CIN (bland urine analysis, hyperuricemia and/or anemia unrelated to the degree of CKD, hypo/hypercalcemia, acidemia, urinary concentration defects, absent hypertension, renal cysts), especially with a family history of similar CKD, were referred for the Nephrogenetics’ clinic and ADTKD was investigated. In these cases, we performed a genetic study of ADTKD by the following steps: 1) UMOD, REN, HNF1B mutations studied through NGS; 2) if negative, search for the insertion of a single cytosine of the repeat unit comprising the extremely long (∼1.5-5 kb), GC-rich (>80%) coding variable-number tandem repeat (VNTR) sequence in the MUC1 gene encoding mucin 1 (see Kirby et al Nat Genet. 2013). It is used a restriction and Snapshot method. 3) if negative, search for HNF1B Large deletion, detected by MLPA (kit P241-D2, MRC-Holland). Pre-genetic counselling was performed by the nephrologist, and post-genetic counselling was performed by the nephrologist and geneticist, namely to proceed with family screening of potentially affected family members when a pathogenic mutation was identified or when segregation studies were necessary. Results 31 families were studied and a confirmed genetic diagnosis was obtained in 11 (35%), allowing the identification of 30 patients with ADTKD; the mutations identified were in MUC1 in 4 families (13 patients), UMOD in 4 families (11 patients), HNF1B (2 families, 5 patients) and REN: 1 patient with a phenotype compatible with ADTKD but with a genotype compatible with renal tubular dysgenesis (two mutations in REN gene from each of the parents). One patient with HNF1B deletion also presented with a heterozygous variant of unknown significance in PKD1 gene, and a pathogenic variant of SLC12A3, heterozygous. Three families are still under study: 2 families are being studied with segregation studies to evaluate the pathogenicity of mutations in UMOD (1 family) and HNF1B (1 family), and 1 family with a phenotype of ADTKD (3 young patients with positive family history) with negative results for mutations in ADTKD genes is in study, with a negative result in WES. The study will be pursued in a specialized laboratory to better study MUC1, but at this moment we classify this family as ADTKD-NOS. If positive results are obtained for these 3 families, the percentage of families identified using this protocol will increase to 43,3% of the families studied through this model. Conclusion In our cohort, a genetic diagnosis was established in 35% of the families studied. MUC1 and UMOD are the most prevalent genes implicated in ADTKD at our center, similarly to what has been reported in other cohorts of ADTKD. Given the rarity of these diseases and the fact that they require specific molecular techniques for accurate diagnosis, we believe that Nephrogenetics’ clinics with dedicated nephrologists and geneticists in close cooperation are critical for an accurate diagnosis.

Urinary Kidney Injury Biomarkers Are Associated with Ischemia-Reperfusion Injury Severity in Kidney Allograft Recipients.

We explored the potential of emerging and conventional urinary kidney injury biomarkers in recipients of living donor (LD) or donation after circulatory death (DCD) kidney transplantation, patients with chronic kidney disease (CKD), and individuals from the general population. Urine samples from kidney allograft recipients with mild (LD; n = 199) or severe (DCD; n = 71) ischemia-reperfusion injury (IRI) were analyzed for neutrophil gelatinase-associated lipocalin (NGAL), insulin-like growth factor-binding protein 7 (IGFBP7), tissue inhibitor of metalloproteinases 2 (TIMP2), kidney injury molecule-1 (KIM-1), chemokine C-X-C motif (CXCL9), solute carrier family 22 member 2 (SLC22A2), nephrin, and uromodulin (UMOD) by quantitative multiplex LC-MS/MS analysis. The fold-change in biomarker levels was determined in mild and severe IRI and in patients with CKD stage 1-2 (n = 127) or stage ≥3 (n = 132) in comparison to the general population (n = 1438). Relationships between the biomarkers and total protein, β2-microglobulin (B2M), creatinine, and osmolality were assessed. NGAL, IGFBP7, TIMP2, KIM-1, CXCL9, and UMOD were quantifiable, whereas nephrin and SLC22A2 were below the limit of detection. Kidney injury biomarkers were increased up to 6.2-fold in allograft recipients with mild IRI and 8.3-fold in recipients with severe IRI, compared to the reference population, with the strongest response observed for NGAL and B2M. In CKD stage 1-2, B2M, NGAL, IGFBP7, TIMP2, KIM-1, UMOD, and CXCL9 were not altered, but in individuals with CKD stage ≥3, B2M, NGAL, and KIM-1 were increased up to 1.3-fold. IGFBP7, TIMP2, NGAL, and CXCL9 were strongly correlated (all r ≥ 0.8); correlations with B2M and TP were smaller (all r ≤ 0.6). IRI, but not stable CKD, was associated with increased urinary levels of kidney injury biomarkers determined by LC-MS/MS. Absolute and multiplexed protein quantitation by LC-MS/MS is an effective strategy for biomarker panel evaluation for translation toward the clinical laboratory.

Integrating automated liquid handling in the separation workflow of extracellular vesicles enhances specificity and reproducibility

BackgroundExtracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for numerous diseases. Major impediments of EV-based biomarker discovery include the specificity and reproducibility of EV sample preparation as well as intensive manual labor. We present an automated liquid handling workstation for the density-based separation of EV from human body fluids and compare its performance to manual handling by (in)experienced researchers.ResultsAutomated versus manual density-based separation of trackable recombinant extracellular vesicles (rEV) spiked in PBS significantly reduces variability in rEV recovery as quantified by fluorescent nanoparticle tracking analysis and ELISA. To validate automated density-based EV separation from complex body fluids, including blood plasma and urine, we assess reproducibility, recovery, and specificity by mass spectrometry-based proteomics and transmission electron microscopy. Method reproducibility is the highest in the automated procedure independent of the matrix used. While retaining (in urine) or enhancing (in plasma) EV recovery compared to manual liquid handling, automation significantly reduces the presence of body fluid specific abundant proteins in EV preparations, including apolipoproteins in plasma and Tamm-Horsfall protein in urine.ConclusionsIn conclusion, automated liquid handling ensures cost-effective EV separation from human body fluids with high reproducibility, specificity, and reduced hands-on time with the potential to enable larger-scale biomarker studies.Graphical

Nonmuscle myosin II proteins, Myh9 and Myh10, interacts with unconventional myosin Myo18A in immortalized mouse thick ascending limb epithelial cells

Conventional nonmuscle myosin II (NM2) and unconventional myosin 18A (Myo18A) belong to an evolutionarily conserved family of molecular motor proteins that slide or walk on the actin cytoskeleton to facilitate a myriad of cellular functions including cargo transport pathways. NM2 and Myo18A are known to copolymerize into filaments in vitro in cell-free systems and in cells. Our previous work demonstrated the crucial role for Myh9&10 proteins in regulating transport of uromodulin (UMOD) and the sodium, potassium, chloride cotransporter (NKCC2) in the thick ascending limb (TAL) of mouse kidney using a Myh9&10 conditional knockout mouse model (Otterpohl, et al; 2020). Myo18A is expressed in the proximal tubule and TAL cells in the murine kidney and localizes to the apical membrane. Our results show reduced expression of Myo18A in the TAL cells of the Myh9&10 cKO mouse kidney sections. Furthermore, we identified Myo18A as a major interaction partner to the Myh10 heavy chain in a BioID screen performed in renal epithelial cells. Given that Myo18A alpha is the only myosin with a PDZ domain, we hypothesize that Myo18A coordinates with Myh9&10 in the TAL cells to regulate membrane remodeling during vesicular transport, including the targeting and stabilization of TAL proteins like UMOD and NKCC2. To test this hypothesis, we stably expressed GFP-tagged Myo18A alpha construct in immortalized, mouse thick ascending limb (imTAL) cells generated in the lab. We performed coimmunoprecipitation (Co-IP) experiments that showed interaction between Myo18A alpha, Myh9 and Myh10 proteins. Immunostaining experiments show colocalization of GFP-Myo18A alpha with Myh9 and Myh10 in imTAL cells. These results strongly support the coordinated functions of Myo18A and Myh9&10 proteins in TAL epithelia and warrants further experiments to determine their role in regulating UMOD and NKCC2 transport in the TAL cells. 1R01DK131020-01A1 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Associations of Urine Biomarkers of Kidney Tubule Health With Incident Hypertension and Longitudinal Blood Pressure Change in Middle-Aged Adults: The CARDIA Study.

Urine biomarkers of kidney tubule injury associate with incident hypertension in older adults with comorbidities, but less is known about these associations in younger adults. In 1170 participants of the CARDIA study (Coronary Artery Risk Development in Young Adults; mean age, 45 years; 40% Black people; 56% women) without hypertension, cardiovascular disease, or kidney disease at baseline, we examined associations of urine MCP-1 (monocyte chemoattractant protein-1), α1m (alpha-1-microglobulin), KIM-1 (kidney injury molecule-1), EGF (epidermal growth factor), IL (interleukin)-18, YKL-40 (chitinase-3-like protein 1), and UMOD (uromodulin) with incident hypertension (onset of systolic blood pressure [BP] ≥130 mm Hg or diastolic BP ≥80 mm Hg or initiation of hypertension medications) and longitudinal BP change in models adjusted for hypertension risk factors, estimated glomerular filtration rate, and albuminuria. After a median 9.9 (interquartile range, 5.9-10.2) years, 376 participants developed incident hypertension. In demographic-adjusted analyses, higher tertiles of EGF associated with lower risk of incident hypertension in both Black and White participants. After multivariable adjustment, the risk of incident hypertension remained lower in tertile 2 (hazard ratio, 0.70 [95% CI, 0.50-0.97]) and tertile 3 (hazard ratio, 0.58 [0.39-0.85]) of EGF versus tertile 1. In fully adjusted models, participants in EGF tertile 3 had smaller 10-year increases in systolic (-3.4 [95% CI, -6.1 to -0.7] mm Hg) and diastolic BP (-2.6 [95% CI, -4.6 to -0.6] mm Hg) than tertile 1. Other biomarkers showed inconsistent associations with incident hypertension and BP change. In middle-aged adults without hypertension, cardiovascular disease, or kidney disease, higher urine EGF associated with lower risk of incident hypertension and lower 10-year BP elevations.

Kidney stone matrix proteins: Role in stone formation.

Stone formation is induced by an increased level of urine crystallization promoters and reduced levels of its inhibitors. Crystallization inhibitors include citrate, magnesium, zinc, and organic compounds such as glycosaminoglycans. In the urine, there are various proteins, such as uromodulin (Tamm-Horsfall protein), calgranulin, osteopontin, bikunin, and nephrocalcin, that are present in the stone matrix. The presence of several carboxyl groups in these macromolecules reduces calcium oxalate monohydrate crystal adhesion to the urinary epithelium and could potentially protect against lithiasis. Proteins are the most abundant component of kidney stone matrix, and their presence may reflect the process of stone formation. Many recent studies have explored the proteomics of urinary stones. Among the stone matrix proteins, the most frequently identified were uromodulin, S100 proteins (calgranulins A and B), osteopontin, and several other proteins typically engaged in inflammation and immune response. The normal level and structure of these macromolecules may constitute protection against calcium salt formation. Paradoxically, most of them may act as both promoters and inhibitors depending on circumstances. Many of these proteins have other functions in modulating oxidative stress, immune function, and inflammation that could also influence stone formation. Yet, the role of these kidney stone matrix proteins needs to be established through more studies comparing urinary stone proteomics between stone formers and non-stone formers.

Selected Serum Markers Associated with Pathogenesis and Clinical Course of Type 1 Diabetes in Pediatric Patients-The Effect of Disease Duration.

Biochemical abnormalities in the course of type 1 diabetes (T1D) may cause the production/activation of various proteins and peptides influencing treatment and causing a risk of complications. The aim of this study was to assess concentrations of selected serum substances involved in the pathogenesis and course of T1D and to correlate their concentrations with the duration of T1D. The study included patients with T1D (n = 156) at the age of 3-17, who were divided according to the duration of the disease into those newly diagnosed (n = 30), diagnosed after 3-5 (n = 77), 6-7 (n = 25), and over 7 (n = 24) years from the onset of T1D, and age-matched healthy controls (n = 30). Concentrations of amylin (IAPP), proamylin (proIAPP), catestatin (CST), chromogranin A (ChgA), nerve growth factor (NFG), platelet-activating factor (PAF), uromodulin (UMOD), and intestinal fatty acid binding protein (I-FABP) were measured in sera using immunoenzymatic tests. There were significant differences in concentrations of all the substances except UMOD and NGF between T1D patients and healthy children. The duration of the disease affected concentrations of CST, ChgA, PAF, and NGF, i.e., proteins/peptides which could have an impact on the course of T1D and the development of complications. In long-term patients, a decrease in concentrations of CST and ChgA, and an increase in PAF concentrations were found. In the case of NGF, a decrease was observed after the initial high values, followed by an increase over 7 years after T1D diagnosis. Concluding, the results show that concentrations of selected serum indicators may change in the course of T1D. Further studies are needed to establish whether these indicators could be used in the context of predicting long-term complications.

Anti-urolithiatic effect of Cucumis melo L. var inodorous in male rats with kidney stones.

Melon seed extracts have high antioxidant activities and are effective against a variety of diseases, including kidney stones. In kidney stone model rats, the anti-urolithiatic effects of the hydro-ethanolic extract of melon seed and potassium citrate were studied and compared. After urolithiasis induction by ethylene glycol, the extract and potassium citrate were treated orally for 38days concurrent with ethylene glycol. Then, urine and kidney sampling were done, and the urinary parameter levels were measured. The melon and potassium citrate treatments reduced the kidney index, the levels of urinary calcium and oxalate, calcium oxalate deposit numbers, the score of crystal deposits, histo-pathological damages, and the score of inflammation in the kidney sections, while elevating the urinary pH, magnesium, and citrate levels, and also the expression of the UMOD, spp1, and reg1 genes in the kidney of treated animals. The effect of potassium citrate is the same as the effect of melon in treated animals. So, their effects could be by normalizing urinary parameters, reducing crystal deposits, excreting small deposits from the kidney, reducing the chance of them being retained in the urinary tract, and elevating the expression of the UMOD, spp1, and reg1 genes, which are involved in kidney stone formation.

Photobiomodulation Effect of 850 nm Near-infrared Diode Laser on the Release of M1-related Cytokines Inmonocytic THP-1 Cells

Photobiomodulation (PBM) is a form of the use of visible red and Near-infrared (NIR) light at low power, where a laser light photon is absorbed at the electronic level, without heat production. PBM can be applied in wide range of treatment to help the wound, inflammation, edema, and pain reduction. However, there is a lack of scientific documentation regarding its actual effects. Objectives: This study assesses the impact of PBM on the release of M1-related cytokine in monocyte cells with particular emphasis on interleukin-1β (IL-1β) and Tumour Necrosis Factor α (TNF-α). Methods: Tamm-Horsfall Protein 1 (THP-1) macrophages M1 cells have been exposed to the light from the diode laser of 850nmat different doses (0, 0.6, 1.2 and 3.6 J/cm2). The release of cytokines was determined by enzyme-linked immunosorbent assay, after different periods of incubation (0, 12, 24, and 48 hours) post-irradiation. The proliferation of fibroblast cells suspended in irradiated M1-supernatent was evaluated for the same periods of incubation. Results: The results showed that PBM significantly enhanced M1-related cytokine release (p < 0.05). Obviously, IL-1β increased post-irradiation at 1.2 J/cm2 more than other doses for all incubation periods. TNF-α was decreased significantly after two days of irradiation (p < 0.005) for all doses. A significant increase in fibroblast proliferation (p < 0.005) was observed concomitant with the boost of cytokine release. Conclusion: This in vitro study has demonstrated that the PBM of the 850 nm diode laser therapy can enhance M1-related cytokine release, which in turn increases the proliferation of fibroblast cells. Moreover, PBM at 850 nm plays an anti-inflammatory role, which manifested by decreasing the level of TNF-α. Therefore, this therapy may be able to accelerate the wound healing process.

Comparison of Allele Frequency of Uromodulin Gene rs13333226 and rs13333144 in a Sample of Iraqi Patients on Dialysis

Chronic kidney disease (CKD) is a major public health concern around the world. UMOD gene variants are linked to a higher incidence of hypertension and CKD in the general population. This study aimed to investigate the role of uromodulin rs13333226 and rs13333144 genes association with chronic kidney disease.The study samples were divided into two groups. The first group included 100patient samples and 70 chosen among them were under the dialysis and had kidney failure aged between 18-88 years old. The second group included 30 samples from healthy individuals who were used as a control. One of the ways used to identify the genotype is the tetra-primers amplification refractory mutation system–polymerase chain reaction (ARMS–PCR). Regarding the results of SNP (rs13333226 and rs13333144), the genotypes GG (OR=150.3), and AA (OR=0.01) for rs13333226. The genotypes GG (OR=0.02) and TT(OR=140.4) for rs13333144 when comparing between patients and control (P≤ 0.01), they were observed when the odds ratio is less than 1 means they are preventive factors, while OR more than 1 means the risk increase (P≤ 0.01). It was concluded that there was an association between SNP (rs13333226 and rs13333144) and uromodulin in kidney diseases. Where genotype GG) rs13333226) and TT (rs13333144) represents a risk factor and AA rs13333226 and GG rs13333144 represents a protective factor.

SPP1 and UMOD gene variants are synergistically associated with risk of renal stone disease

ObjectiveCalcium and oxalate are the most abundant metabolites present in the stone matrix. The SPP1 and UMOD gene has specific expression in kidneys and are involved in various stages of stone formation. Therefore, genetic variants in the SPP1 and UMOD genes may enhance the development of renal stone disease. This study has been designed to understand the association of genetic variants of SPP1 and UMOD genes with renal stone disease. Materials and MethodA prospective study has been carried out, including 150 renal stone disease patients and 150 healthy individuals. Biochemical parameters were performed, including serum calcium levels, creatinine, parathyroid hormone, and 24-Hour urine metabolites. The genotyping of SPP1 (rs1126616) and UMOD (rs4293393) gene variants were performed using a customized TaqMan probe. T-test was used for continuous biochemical data analysis. The Chi-square test has been applied to assess the risk of a particular genotype associated with renal stone disease. In addition, correlation analysis for biochemical parameters and genetic variants with the renal stone disease has been performed using Shapley additive explanations (SHAP) values calculated with the help of the pycaret library. ResultRenal stone patients had significantly higher levels of parathyroid hormone (93.37 ± 52.78 pg/ml vs 64.67 ± 31.50 pg/ml, P=<0.0001), serum creatinine (0.94 ± 0.38 mg/dl vs 0.77 ± 0.17 mg/dl, P=<0.0001) and 24hr urine metabolites in comparison to the healthy controls. Heterozygous (CT) variant of SPP1 and homozygous (GG) variant of UMOD genes were significantly associated with an increased risk of developing the renal stone disease (p = 0.0100, OR = 2.06, 95 %CI = 1.13–3.75; p=<0.0001, OR = 5.773, 95 % CI = 2.03–16.38, respectively). Individuals with hyperparathyroidism and CC (SPP1) and GG (UMOD) genotypes have a high risk (P = 0.0055, OR = 2.75, 95 %CI = 1.35–5.67; P = 0.0129, OR = 10.03, 95 %CI = 1.60–110.40, respectively) of developing a renal stone. In addition, individuals with hypercalciuria and TT genotype of SPP1 (P = 0.0112, OR = 2.92, 95 % CI = 1.33–6.35), AG genotype of UMOD (P=<0.0001, OR = 5.45, 95 %CI = 2.24–13.96) and GG genotype of UMOD (P=<0.0001, OR = 10.02, 95 %CI = 3.53–24.63) have high risk of developing renal stones. Moreover, Individuals with hyperoxaluria and AG + GG (UMOD) genotype have a greater risk (P=<0.0001, OR = 7.35, 95 % CI = 3.83–13.68) of developing a renal stone. The renal stone risk was persistent (P=<0.0002, OR = 2.44, 95 % CI = 1.52–3.86) when analyzed for the synergistic effect of risk genotypes of SPP1 (CT) and UMOD (GG) gene. Further, correlation analysis also confirmed the strong association between genetic variants and renal stone development. ConclusionGenetic variants of the SPP1 and UMOD genes were associated with renal stone disease. In the presence of risk genotype and hyperparathyroidism, hypercalciuria, and hyperoxaluria, the susceptibility to develop the renal stone disease risk gets modulated.

Development of a Signature Based on Eight Metastatic-Related Genes for Prognosis of GC Patients.

Gastric cancer (GC) has been a common tumor type with high mortality. Distal metastasis is one of the main causes of death in GC patients, which is also related to poor prognosis. The mRNA profiles and clinical information of GC patients were downloaded from The Cancer Genome Atlas and Gene Expression Omnibus databases. Univariate Cox and LASSO Cox analyses were used to screen the optimal metastasis-related genes (MRGs) to establish a prognostic Risk Score model for GC patients. The nomogram was used to visualize the Risk Score and predict the 1-, 3-, 5-year survival rate. The immune cell infiltration was analyzed by CIBERSORT and the ratio of immune-stromal component was calculated by the ESTIMATE algorithm. A total of 142 differentially expressed genes were identified between metastatic and non-metastatic GC samples. The optimal 8 genes, comprising GAMT (guanidinoacetate N-methyltransferase), ABCB5 (ATP-binding cassette subfamily B member 5), ITIH3 (inter-alpha-trypsin inhibitor heavy chain 3), GDF3 (growth differentiation factor 3), VSTM2L (V-set and transmembrane domain-containing 2 like), CIDEA (cell death inducing DFFA like effector a), NPTX1 (neuronal pentraxin-1), and UMOD (uromodulin), were further screened to establish a prognostic Risk Score, which proved to be an independent prognostic factor. Patients in high-risk group had a poor prognosis. There were significant differences in the proportion of 11 tumor-infiltrating immune cells between high-risk and low-risk subgroups. In addition, the StromalScore, ImmuneScore, and ESTIMATEScore in high-risk group were higher than those in low-risk group, indicating that the tumor microenvironment of the high-risk group was more complex. A Risk Score model based on eight metastasis-related genes could clearly distinguish the prognosis of GC patients. The poor prognosis of patients with high-Risk Score might be associated with the complex tumor microenvironments.

Uromodulin and Vesico-Ureteral Reflux: A Genetic Study

Vesicoureteral reflux (VUR) is associated with urinary tract infections (UTI) and renal scars. The kidney damage is correlated with the grade of reflux and the number of UTI, but other factors may also play a role. Uromodulin (UMOD) is a protein produced by kidney tubular cells, forming a matrix in the lumen. We evaluated whether the common variant rs4293393 in the UMOD gene was associated with febrile UTI (FUTI) and/or scars in a group of children with VUR. A total of 31 patients with primary VUR were enrolled. Renal scars were detected in 16 children; no scar was detected in 15 children. Genotype rs4293393 TC (TC) was present in 8 patients, 7 (88%) had scars; genotype rs4293393 TT (TT) was found in 23 patients, and 9 (39%) had scars. Among children with scars, those with TC compared with those with TT were younger (mean age 77 vs. 101 months), their reflux grade was comparable (3.7 vs. 3.9), and the number of FUTI was lower (2.9 vs. 3.7 per patient). Children with VUR carrying UMOD genotype rs4293393 TC seem more prone to developing renal scars, independent of FUTI.