Uromodulin Expression Research Articles

Uromodulin and progression of IgA nephropathy.

This study investigates the link between genetic variants associated with kidney function and immunoglobulin A (IgA) nephropathy (IgAN) progression. We recruited 961 biopsy-proven IgAN patients and 651 non-IgAN end-stage renal disease (ESRD) patients from Ruijin Hospital. Clinical and renal pathological data were collected. The primary outcome was the time to ESRD. A healthy population was defined as estimated glomerular filtration rate >60mL/min/1.73 m2 without albuminuria or hematuria. Fifteen single-nucleotide polymorphisms (SNPs) were selected from a genome-wide association study of kidney function and genotyped by the SNaPshot. Immunohistochemistry in renal tissue and ELISA in urine samples were performed to explore the potential functions of genetic variations. The rs77924615-G was independently associated with an increased risk for ESRD in IgAN patients after adjustments for clinical and pathologic indices, and treatment (adjusted hazard ratio 2.10; 95% confidence interval 1.14-3.88). No significant differences in ESRD-free survival time were found among different genotypes in non-IgAN ESRD patients (log-rank, P=.480). Moreover, rs77924615 exhibited allele-specific enhancer activity by dual-luciferase reporter assay. Accordingly, the urinary uromodulin-creatinine ratio (uUCR) was significantly higher in healthy individuals with rs77924615 AG or GG than in individuals with AA. Furthermore, uromodulin expression in tubular epithelial cells was higher in patients with rs77924615 AG or GG. Finally, we confirmed that an increased uUCR (P=.009) was associated with faster IgAN progression. The SNP rs77924615, which modulates the enhancer activity of the UMOD gene, is associated with renal function deterioration in IgAN patients by increasing uromodulin levels in both the renal tubular epithelium and urine.

#2062 Protein interactome and trafficking of uromodulin in HEK and MDCK cell model

Abstract Background and Aims Uromodulin (UMOD) is a kidney-specific glycoprotein and the most abundant urinary protein in healthy individuals. Genome-wide association studies have shown an association between UMOD variants, kidney diseases, and hypertension. UMOD exhibits bidirectional secretion as it releases into the renal interstitium and circulation. However, the molecular trafficking of UMOD and its interaction remains poorly defined. We aimed to establish a stable Human Embryonic Kidney (HEK293) cell line expressing human UMOD to identify the potential regulators of UMOD expression or secretion. This study will provide significant implications for conditions such as hypertension, chronic kidney diseases, acute kidney injury, or UMOD-autosomal dominant tubulointerstitial kidney disease (UMOD-ADTKD). Method A HEK-UMOD-expressing cell line was generated by introducing human UMOD transcript 1 expression and using a tetracycline-inducible expression vector (pcDNA™FRT⁄TO vector) with yellow fluorescent protein tag at the C-terminal and previously established MDCK-UMOD cell line was also used. An untargeted proteomic approach was used to identify proteins, using GFP trap co-immunoprecipitation assay followed by mass spectrometry analysis. Results We identified 1367 and 1140 UMOD interacting proteins in HEK and MDCK cell models respectively. There were 620 proteins common to both these models. Through gene ontology, we unravel the molecular mechanisms involved in the biosynthesis, folding, and transport of UMOD. We observed several proteins associated with clathrin-mediated transporting. To study the role of clathrin in UMOD transport, we treated the polarised MDCK-UMOD cells with pitstop 2. There was significantly more secretion of UMOD with pitstop 2 treatment in the apical compartment compared to untreated cells however there was no significant difference in UMOD secretion in the basolateral compartment. Conclusion These results suggested that clathrin-coated vesicles are involved in transporting and sorting UMOD. Further studies need to investigate the functional importance of these proteins in UMOD trafficking and their implications in different physiological processes.

Does Uromodulin Play a Role in Tacrolimus-Mediated Upregulation of the Sodium Chloride Cotransporter?

Background: Tacrolimus, a calcineurin inhibitor (CNI), is a medication commonly prescribed to prevent organ transplant rejection. However, chronic CNI use has several adverse effects, including hypertension. Recent studies have revealed that the renal sodium chloride cotransporter (NCC) mediates tacrolimus-induced hypertension. However, the mechanisms underlying NCC upregulation are not fully understood. Previous studies indicate that uromodulin plays a key role in renal ion channel regulation. As such, we hypothesize that uromodulin mediates tacrolimus induced NCC upregulation. Experimental Design: To test this hypothesis, male C57BL/6 mice received tacrolimus (10 mg/kg/day) or vehicle (ethanol/DMSO/saline) for 21 consecutive days via intraperitoneal injection. The impact of tacrolimus on systolic blood pressure (BP) was monitored by tail-cuff plethysmography. To examine uromodulin and NCC expression, kidneys were harvested and processed for immunohistochemistry. Results: Compared to the BP of vehicle-treated mice (120 mm Hg ± 4.252), mice administered tacrolimus had significant increases in BP (170 mm Hg ± 3.528). Furthermore, tacrolimus treatment increased phosphorylated NCC (pNCC) and uromodulin expression. Conclusions: These results indicate that tacrolimus-induced hypertension is accompanied by pNCC and uromodulin upregulation in distal convoluted tubules. Significance: Overall, these findings highlight uromodulin as a possible regulator of NCC. Funding: R21 DK119879, R01 DK-133698, R25DK07838. This is the full abstract presented at the American Physiology Summit 2024 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.

SiRNA as potential therapeutic strategy for hypertension

Hypertension, a well-known cardiovascular disorder noticed by rise in blood pressure, poses a significant global health challenge. The development RNA interfering (RNAi)-based therapies offers a ground-breaking molecular tool, holds promise for addressing hypertension's intricate molecular mechanisms. Harnessing the power of small interfering RNA (siRNA), researchers aim to selectively target and modulate genes associated with hypertension. Furthermore, they aim to downregulate the levels of mRNA by activating cellular nucleases in response to sequence homology between the siRNA and the corresponding mRNA molecule. As a result, genes involved in the cause of disorders linked to a known genetic background can be silenced using siRNA strategy. In the realm of hypertension, siRNA therapy emerges as a potential therapy for prognostics, diagnostics and treatments. It plays an important role in execution of targeting suppression of genes involved in vascular tone regulation, sodium handling, and pathways contributing to high blood pressure. A clinical trial involving intervention like angiotensinogen siRNA (AGT siRNA) is currently being carried out to treat hypertension. Genetic correlations between uromodulin (UMOD) and hypertension are investigated as emerging Non AGT siRNA target. Furthermore, expression of UMOD is responsible for regulation of sodium by modulating the tumor necrosis factor-α and regulating the Na + -K + -2Cl-cotransporter (NKCC2) in the thick ascending limb, which makes it an important target for blood pressure regulation.

Uromodulin — Biological Significance and Prospects for Clinical Use

Uromodulin is a unique protein produced in the kidneys by epithelial cells of the ascending thick portion of the loop of Henle. It implements physiological mechanisms not only at the tubular level, but also participates in the coordination of general body processes. The main functions of uromodulin are an obstacle to prevent stone formation due to a violation of the aggregation of calcium salts and water reabsorption, coordination of electrolyte balance, and an obstacle to inflammatory processes locally and systemically. Moreover, the expression of uromodulin and its qualitative characteristics are under genetic control. In this regard, the pathology of the tubular apparatus or mutations in the genes encoding uromodulin lead to the development of primary or secondary tubulopathies with dysfunction of other organs and systems. At the same time, it is known that uromodulin is an incompletely studied protein both in terms of structure and features of the functions it performs. A thorough analysis of research data, including experimental work on the study of uromodulin in domestic and international literature sources, was carried out, with a presentation of the material in this manuscript.

Role of Uromodulin in Salt-Sensitive Hypertension.

The exclusive expression of uromodulin in the kidneys has made it an intriguing protein in kidney and cardiovascular research. Genome-wide association studies discovered variants of uromodulin that are associated with chronic kidney diseases and hypertension. Urinary and circulating uromodulin levels reflect kidney and cardiovascular health as well as overall mortality. More recently, Mendelian randomization studies have shown that genetically driven levels of uromodulin have a causal and adverse effect on kidney function. On a mechanistic level, salt sensitivity is an important factor in the pathophysiology of hypertension, and uromodulin is involved in salt reabsorption via the NKCC2 (Na+-K+-2Cl− cotransporter) on epithelial cells of the ascending limb of loop of Henle. In this review, we provide an overview of the multifaceted physiology and pathophysiology of uromodulin including recent advances in its genetics; cellular trafficking; and mechanistic and clinical studies undertaken to understand the complex relationship between uromodulin, blood pressure, and kidney function. We focus on tubular sodium reabsorption as one of the best understood and pathophysiologically and clinically most important roles of uromodulin, which can lead to therapeutic interventions.

Phenome-Wide Association Study of UMOD Gene Variants and Differential Associations With Clinical Outcomes Across Populations in the Million Veteran Program a Multiethnic Biobank

IntroductionCommon variants in the UMOD gene are considered an evolutionary adaptation against urinary tract infections (UTIs) and have been implicated in kidney stone formation, chronic kidney disease (CKD), and hypertension. However, differences in UMOD variant-phenotype associations across population groups are unclear.MethodsWe tested associations between UMOD/PDILT variants and up to 1528 clinical diagnosis codes mapped to phenotype groups in the Million Veteran Program (MVP), using published phenome-wide association study (PheWAS) methodology. Associations were tested using logistic regression adjusted for age, sex, and 10 principal components of ancestry. Bonferroni correction for multiple comparisons was applied.ResultsAmong 648,593 veterans, mean (SD) age was 62 (14) years; 9% were female, 19% Black, and 8% Hispanic. In White patients, the rs4293393 UMOD risk variant associated with increased uromodulin was associated with increased odds of CKD (odds ratio [OR]: 1.22, 95% CI: 1.20–1.24, P = 5.90 × 10−111), end-stage kidney disease (OR: 1.17, 95% CI: 1.11–1.24, P = 2.40 × 10−09), and hypertension (OR: 1.03, 95% CI: 1.05–1.05, P = 2.11 × 10−06) and significantly lower odds of UTIs (OR: 0.94, 95% CI: 0.92–0.96, P = 1.21 × 10−10) and kidney calculus (OR: 0.85, 95% CI: 0.83–0.86, P = 4.27 × 10−69). Similar findings were observed across UMOD/PDILT variants. The rs77924615 PDILT variant had stronger associations with acute cystitis in White female (OR: 0.73, 95% CI: 0.59–0.91, P = 4.98 × 10−03) versus male (OR: 0.99, 95% CI: 0.89–1.11, P = 8.80 × 10−01) (P interaction = 0.01) patients. In Black patients, the rs77924615 PDILT variant was significantly associated with pyelonephritis (OR: 0.65, 95% CI: 0.54–0.79, P = 1.05 × 10−05), whereas associations with UMOD promoter variants were attenuated.ConclusionRobust associations were observed between UMOD/PDILT variants linked with increased uromodulin expression and lower odds of UTIs and calculus and increased odds of CKD and hypertension. However, these associations varied significantly across ancestry groups and sex.

Role of uromodulin and complement activation in the progression of kidney disease.

Uromodulin (UMOD) is a glycoprotein that is selectively expressed on the epithelial cells of the thick ascending limb of Henle's loop and the early distal renal tubule. The present study aimed to investigate whether UMOD was associated with complement activation in patients with renal diseases. In addition, its biological function was examined in vitro. The expression levels of UMOD and complement components, including C1q, C3, C4 and C3a, and membrane attack complex (MAC) in the plasma of patients with IgA nephropathy (IgAN; n=58) and lupus nephritis (LN; n=36) were detected using ELISA, which was used to determine the association between UMOD expression and complement components. In addition, a simulated hypoxia-reoxygenation (H/R) model was used to stimulate UMOD expression in mouse inner medullary collecting duct cells. Additionally, the association between UMOD expression and complement components C1q and C3d at the cellular level was identified using western blotting and immunofluorescence, respectively. It was revealed that the plasma UMOD concentration was significantly decreased in patients with IgAN and LN compared with in healthy controls, and the levels of C3a and MAC were significantly increased in the plasma of patients with IgAN and LN. Furthermore, the plasma levels of C1q, C3 and C4 in patients with LN, but not in patients with IgAN, were significantly decreased compared with in healthy controls. The plasma levels of UMOD were negatively correlated with the plasma C3a and MAC concentrations. However, the plasma levels of UMOD were significantly and positively correlated with the plasma C1q concentration, but not with that of C3 and C4. It was identified that UMOD expression started to increase after 1 h of simulated H/R, and continued to increase at 6 and 12 h. In addition, cells with lower UMOD expression had higher C3d expression in vitro. Collectively, the present results suggested that UMOD was associated with severe complement activation and may be involved in complement-mediated immune protection by inhibiting complement activation in renal disease.

Abstract 08: Functional Characterisation Of Uromodulin Promoter Risk Variants Associated With Blood Pressure

Using a genome wide association study (GWAS), we identified a locus on the promoter of the human uromodulin (UMOD) gene associating with primary hypertension. In this study we aimed to identify genotype-differential protein binding partners which influence UMOD expression and to examine pathways linking UMOD expression to blood pressure variability in humans.We examined a variant on this locus, by incubating 50mer oligonucleotides featuring alternate alleles (C/G) with HEK-293 nuclear protein extract. Subsequently, we performed an electrophoretic mobility shift assay (EMSA) and purified protein complexes to analyse by Tandem-mass-spectroscopy (MS2). Filtering for peptide matches >20, we interpreted these data by Ingenuity Pathway Analysis (IPA). From a bank of 98 human renal biopsy samples, we assessed UMOD expression by qRT-PCR. RNA-seq was performed using the NextSeq-500 system on a subset of high and low UMOD expressors (n=3/ group). Differentially expressed (false-discovery-rate (FDR) <0.05) genes were identified, with strong correlations in these data validated by qRT-PCR. Implicated pathways were interpreted using both IPA and gene set enrichment analysis (GSEA).The C allele at our risk variant displayed greater binding affinity versus the G allele; 0.77 (0.13, 1.40, 95%CI). In contrast no difference in binding affinity was observed between the T and C alleles of our control variant, 0.08 ( -1.22, 1.39, 95%CI). Mass spectroscopy of EMSA complexes identified PARP1 binding at this target. By IPA we identified a complex between PARP1 and HNF1β as driving expression of UMOD. RNA-sequencing identified positive correlation in expression of UMOD and the Na-K-Cl cotransporter (NKCC2) (FDR=2.58e-9, log2-fold-change [L2FC]= 3.3), the renal outer medullary potassium channel (ROMK) (FDR=3.69e-2, L2FC= 2.79) and the epithelial sodium channel (ENaC) (FDR=1.83e-2, L2FC= 1.25). We validated this coexpression between UMOD and NKCC2 by qRT-PCR and show a significant correlation (p=2.2e-16, R2= 0.844, n=84).Together these data suggest our risk variant contributes to differences in UMOD expression via a PARP1:HNF1β transcriptional complex and these differences associate with blood pressure through sodium homeostasis in the human kidney.

Abstract P232: Pregnancy Induced Changes In Urinary Uromodulin Excretion In Normotensive And Chronic Hypertensive Rodent Models

Hypertensive disorders complicate 1-4% of pregnancies and are the leading cause of maternal death. Pregnancy involves major adaptions in renal hemodynamics, tubular and endocrine functions. Uromodulin (UMOD) is a renal protein that plays a role in renal sodium and divalent cation transport and is associated with hypertension and kidney diseases. We aimed to understand the role of blood pressure (BP) in urinary UMOD excretion during pregnancy. Pregnant Wistar Kyoto (WKY) and Stroke Prone Spontaneously Hypertensive (SHRSP) rats (n=8) were studied for 18.5 gestational days (GD). A group of pregnant SHRSP were treated with the beta-blocker propranolol (100mg/kgBW/day; n=8) and the calcium channel blocker nifedipine (25mg/kgBW/day; n=7) from GD 0.5. Systolic BP (SBP; tail cuff plethysmography), urine and plasma characteristic were monitored at baseline, GD3.5, GD12.5 and GD 18.5. UMOD concentration were measured in 24hr urine with ELISA. Pregnant SHRSP were hypertensive throughout the gestation compared to WKY (delta SBP 22.6 ± 3.6 mmHg, p<0.0001). At baseline (pre-pregnancy), urinary UMOD levels were 2-fold higher in WKY compared to SHRSP (p<0.05). During pregnancy, the urinary UMOD gradually decreased in WKY (2-fold decrease, p<0.05), whereas a 1.5-fold increase (p<0.05) was observed in hypertensive SHRSP compared to pre-pregnancy. The changes observed in urinary levels corresponded to total kidney UMOD levels at GD18.5, wherein SHRSP kidney shows 1.3-fold (p<0.01) greater expression of UMOD compared to WKY. Nifedipine reduced BP in pregnant SHRSP (delta SBP between GD18.5 and baseline: 44.3 ± 7.4 mmHg, p<0.0001) while propranolol did not affect BP. However, neither nifedipine nor propranolol changed urinary UMOD excretion in pregnant SHRSP. BP was not significantly correlated with urinary uromodulin levels in pregnant WKY (Pearson, r=-0.01, p 0.95) and SHRSP without (r=-0.16, p 0.46) or with (nifedipine r=0.26, p 0.31; propranolol r=0.17, p 0.45) antihypertensive treatment. We demonstrate differences in UMOD excretion between hypertensive and normotensive pregnancy and changes during pregnancy that are at least in part BP independent. Our data provide novel insights into molecular changes associated with hypertensive pregnancy.

Commentary: Use of BACTRAC Proteomic Database-Uromodulin Protein Expression During Ischemic Stroke

Uromodulin (UMOD) is a glycoprotein expressed by the epithelial cells of the thick ascending limb of Henle's loop in the kidney. Research has shown that increased uromodulin expression may be associated with lower risk of cardiovascular disease in adults. Utilizing the Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) (clinicaltrials.gov NCT03153683), a continuously enrolling tissue bank, we aimed to examine the associations between serum uromodulin, age, and high BMI (BMI>25) and its relationship to stroke in patients. Arterial blood distal and proximal to the thrombus was collected during a thrombectomy procedure using the BACTRAC protocol and sent to Olink (Boston, MA) to determine proteomic expression via proximity extension assay. Uromodulin expression was recorded and analyzed using two tailed T-tests and linear regressions. The relationship between systemic and intracranial uromodulin, age, high BMI and hypertension were assessed. Systemic and intracranial uromodulin decreased with age (p<0.0001 and r2=0.343, p=0.0416 and r2=0.102) respectively. Systemic uromodulin expression increased with BMI>25 (p=0.014). Presence of hypertension decreased uromodulin's expression systemically (p=0.018) and intracranially (p=0.007). Uromodulin was increased significantly in overweight patients, decreased significantly in older patients, and decreased in patients with hypertension. The increase in uromodulin in people with high BMI could be a protective reaction of the kidney to worsening conditions that make ischemic stroke more likely, with a goal of delaying dangerous outcomes. The decreased expression of uromodulin in older adults could be associated with the decline of general kidney function that accompanies aging. Hypertension can contribute to an AKI by decreasing perfusion to the kidney, therefore decreasing kidney function and uromodulin production. Further analyses are needed to understand the role of uromodulin following ischemic stroke.

Upregulation of renal Na–K–2Cl cotransporter 2 in obese diabetes mellitus via a vasopressin receptor 2-dependent pathway

Na–K–2Cl cotransporter 2 (NKCC2) in thick ascending limb (TAL) in the kidney plays a central role in tubuloglomerular feedback (TGF) system by sensing NaCl delivery to the distal tubules. Although accumulating data indicate that dysregulated TGF contributes to the progression of diabetic complications, the regulation of NKCC2 in diabetes mellitus (DM) remains unclear. We here show that NKCC2 is overactivated via a vasopressin receptor 2 (V2R)-dependent mechanism in db/db mice, a mouse model of obese DM. Compared with db/+ mice, we found that both aquaporin 2 and NKCC2 levels were significantly increased in the kidney in db/db mice. Immunohistochemical analysis of V2R and NKCC2 in the kidney demonstrated that V2R is present in the TAL, as well as in the collecting duct. Moreover, the administration of tolvaptan, a selective V2R antagonist, sharply decreased aquaporin 2 and NKCC2 in db/db mice, confirming the causal role of V2R signaling in NKCC2 induction in this model. Although tolvaptan reduced aquaporin 2 abundance also in db/+ mice, its effect on NKCC2 was modest compared with db/db mice. In total kidney lysates, uromodulin expression was not altered between db/+ and db/db mice, suggesting that V2R signaling alters NKCC2 without altering uromodulin levels. These data implicate the dysregulation of NKCC2 in the pathophysiology of type 2 DM, and underscore the complex nature of fluid volume disorders in diabetic kidney disease.

The apical membrane localization and trafficking of uromodulin (UMOD) is critically dependent on nonmuscle myosin II isoforms Myh9 and Myh10 in mouse thick ascending limb epithelial cells

Mutations that alter the trafficking and function of uromodulin, a membrane associated GPI anchored protein expressed in the TAL segment, result in autosomal dominant tubulointerstitial kidney disease (ADTKD)1. Proper localization and function of GPI‐anchored proteins requires intact ER to Golgi to apical membrane transport. Recently Nonmuscle myosin II (NM2) have been linked to vesicular transport2, and we have identified a novel function for NM2 isoforms Myh9 (NM2A) and Myh10 (NM2B) in regulating membrane remodeling in mammalian cells3. Due to the differential expression and unique localization of NM2 isoforms in the murine renal tubules4, we hypothesized that NM2 isoforms are involved in the specialized transport pathways of membrane associated proteins in the TAL epithelial cells. Conditional genetic knockout of Myh9 &amp; Myh10 within the renal tubules of mice results in initial mis‐localization and gradual loss of the TAL‐specific protein: Uromodulin. These mice develop rapidly progressing tubulointerstitial kidney disease and are moribund by 5–6 months of age. In order to understand the mechanisms underlying the mis‐localization of the Uromodulin, we generated an immortalized TAL cell line from the adult kidneys of Pax8‐&gt;rtTA; Tet‐o‐cre; Myh9F/F; Myh10F/F mice crossed to mice harboring Immorto +/Tg transgene. These cells can be maintained at 33C indefinitely due to the expression of the temperature sensitive SV40 T‐antigen transgene. To promote polarization and endogenous expression of uromodulin and other membrane‐associated proteins in these cells, we have modified a recently established culture protocol with cells grown on polyester membranes at 37C on an orbital shaker5. We observe robust expression of endogenous uromodulin detected by immunohistochemistry and western blot analysis. Furthermore, induction of Myh9 and Myh10 inactivation by doxycycline treatment of these cells results in altered levels of uromodulin in the cells possibly indicating changes in N‐glycosylation of uromodulin along the ER‐Golgi transport pathway. Loss of NM2 also decreases the intracellular sodium levels in the knockout cells, observed using sodium green™ indicator. Our studies indicate a novel relationship between NM2 isoforms and uromodulin transport along the TAL segment and are suggestive of a role for MYH9 and MYH10 in tubulointerstitial kidney disease associated with abnormal renal epithelial transport.Support or Funding InformationFunding support from NIH Grant P20GM103620 and Sanford Research Program Funds.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reduced asymmetric dimethylarginine accumulation through inhibition of the type I protein arginine methyltransferases promotes renal fibrosis in obstructed kidneys.

The role of asymmetric dimethylarginine (ADMA) in chronic kidney disease (CKD) is unclear. Through inhibition of type I protein arginine methyltransferases (PRMTs), a novel strategy, we aimed to determine the effect of ADMA on renal fibrosis and explore its underlying working mechanisms. After sham or unilateral ureter ligation (UUO) operation, 20-25 g male c57 mice were treated with vehicle or PT1001B, an inhibitor of type I PRMTs, for 13 d. Moreover, human kidney 2 (HK2) and normal rat kidney 49F (NRK-49F) cells were treated with various concentrations of PT1001B or ADMA in the presence of 2.5 ng/ml TGF-β. We found that treatment with PT1001B increased the deposition of extracellular matrix proteins, the expression of α smooth muscle actin, and connective tissue growth factor in UUO-induced fibrotic kidneys, which is correlated with reduced expression of PRMT1, reduced the production of ADMA, and increased expression of uromodulin. In TGF-β-stimulated HK2 and NRK-49F cells, PT1001B dose-dependently inhibited ADMA production, increased NO concentrations, and enhanced the expression of profibrotic proteins. Exogenous addition of ADMA inhibited the expression of profibrotic proteins dose-dependently and attenuated the profibrotic effect of PT1001B. Moreover, ADMA reduced the NO concentration in PT1001B-treated HK2 cells. Finally, we conclude that ADMA has an antifibrotic effect in obstructed kidneys, and future application of type I PRMT inhibitor should be done cautiously for patients with CKD.-Wu, M., Lin, P., Li, L., Chen, D., Yang, X., Xu, L., Zhou, B., Wang, C., Zhang, Y., Luo, C., Ye, C. Reduced asymmetric dimethylarginine accumulation through inhibition of the type I protein arginine methyltransferases promotes renal fibrosis in obstructed kidneys.

Salt stress in the renal tubules is linked to TAL-specific expression of uromodulin and an upregulation of heat shock genes.

Previously, our comprehensive cardiovascular characterization study validated Uromodulin as a blood pressure gene. Uromodulin is a glycoprotein exclusively synthesized at the thick ascending limb of the loop of Henle and is encoded by the Umod gene. Umod−/− mice have significantly lower blood pressure than Umod+/+ mice, are resistant to salt-induced changes in blood pressure, and show a leftward shift in pressure-natriuresis curves reflecting changes of sodium reabsorption. Salt stress triggers transcription factors and genes that alter renal sodium reabsorption. To date there are no studies on renal transcriptome responses to salt stress. Here we aimed use RNA-Seq to delineate salt stress pathways in tubules isolated from Umod+/+ mice (a model of sodium retention) and Umod−/− mice (a model of sodium depletion) ± 300 mosmol sodium chloride (n = 3 per group). In response to salt stress, the tubules of Umod+/+ mice displayed an upregulation of heat shock transcripts. The greatest changes occurred in the expression of: Hspa1a (Log2 fold change 4.35, P = 2.48 e−12) and Hspa1b (Log2 fold change 4.05, P = 2.48 e−12). This response was absent in tubules of Umod−/− mice. Interestingly, seven of the genes discordantly expressed in the Umod−/− tubules were electrolyte transporters. Our results are the first to show that salt stress in renal tubules alters the transcriptome, increasing the expression of heat shock genes. This direction of effect in Umod+/+ tubules suggest the difference is due to the presence of Umod facilitating greater sodium entry into the tubule cell reflecting a specific response to salt stress.

Uromodulin is expressed in the distal convoluted tubule, where it is critical for regulation of the sodium chloride cotransporter NCC

Uromodulin, the most abundant protein in normal urine, is essentially produced by the cells lining the thick ascending limb. There it regulates the activity of the cotransporter NKCC2 and is involved in sodium chloride handling and blood pressure regulation. Conflicting reports suggested that uromodulin may also be expressed in the distal convoluted tubule (DCT) where its role remains unknown. Using microdissection studies combined with fluorescent insitu hybridization and co-immunostaining analyses, we found a significant expression of uromodulin in mouse and human DCT at approximately 10% of thick ascending limb expression levels, but restricted to the early part of the DCT (DCT1). Genetic deletion of Umod in mouse was reflected by a major shift in NCC activity from the DCT1 to the downstream DCT2 segment, paralleled by a compensatory expansion of DCT2. By increasing the distal sodium chloride and calcium ion load with chronic furosemide administration, an intrinsic compensatory defect in the DCT from Umod-/- compared to wild type mice was found manifested as sodium wasting and hypercalciuria. In line, co-expression studies in HEK cells suggested a facilitating role for uromodulin in NCC phosphorylation, possibly via SPAK-OSR1 modulation. These experiments demonstrate a significant expression of uromodulin in the early part of mouse and human DCT. Thus, biosynthesis of uromodulin in the DCT1 is critical for its function, structure and plasticity, suggesting novel links between uromodulin, blood pressure control and risk of kidney stones.

Striking Differences in Urinary Uromodulin, Salt‐sensitive Hypertension and Proteinuria in Dahl SS vs. SS.BN1 Consomic Rats

Uromodulin (UMOD) is a protein made exclusively by epithelial cells of the thick ascending limb. Numerous clinical studies have demonstrated that rare missense mutations in UMOD result in autosomal dominant tubulointerstitial kidney diseases manifest by tubulointerstitial fibrosis, tubular cysts and a rapid progression to renal failure. In addition, several genome wide association studies reported that common single nucleotide polymorphisms in the UMOD gene are associated with a 20% and 15% increased risk of chronic kidney disease (CKD) and hypertension, respectively, in the general population. Interestingly, Dahl salt‐sensitive (SS) rats exhibit many of the same pathologies observed in these clinical populations; however, whether UMOD is altered in Dahl SS rats has not been examined.The goal of this study was to assess the qualitative and quantitative aspects of urinary UMOD via western blotting and the extent of SS hypertension and proteinuria in Dahl SS vs. a consomic rat strain in which chromosome 1 of the salt‐resistant Brown‐Norway (BN) rat, harboring the UMOD gene, has been introgressed into the Dahl SS background (SS.BN1). We hypothesized that differences in urinary UMOD would be apparent in SS vs. SS.BN1 rats maintained on a low salt‐diet and that the extent of SS hypertension and proteinuria would be attenuated in SS.BN1 vs SS rats. Western blot of urinary UMOD (mouse monoclonal, sc‐271022) was performed in 16 week old SS (n=5), SS.BN1 (n=7) and BN (n=6) rats maintained on a 0.4% NaCl diet since birth. BP (radiotelemetry) and proteinuria were assessed during 0.4% NaCl feeding and during three weeks of 4.0% NaCl feeding in a different group of 8–10 week old SS (n=5) and SS.BN1 (n=8) rats.For western blotting, loading of urine was normalized to urinary creatinine content and the density of the most abundant occurring 85 kDa band in SS and SS.BN1 samples was normalized to the average density observed in BN rats. The 85 kDa UMOD band was 4.5 fold higher (p&lt;0.01) in SS.BN1 vs. SS rats with similar levels observed between SS.BN1 and BN rats. Interestingly, a unique ~45 kDa UMOD band was only observed in SS rats, suggestive of structural alterations in UMOD. During 0.4% NaCl feeding, substantially higher levels of systolic BP (161±1 vs. 141±2 mmHg, P&lt;0.001) and proteinuria (153±15 vs. 30±5 mg/day) were observed in SS vs. SS.BN1 rats, respectively. Administration of a 4% NaCl diet for three weeks exacerbated these differences in BP (183±3 vs. 149±1 mmHg, P&lt;0.001) and proteinuria (218±24 vs. 68±15 mg/day, P&lt;0.001) in SS vs. SS.BN1 rats, respectively.In summary, these data demonstrate striking qualitative and quantitative differences in urinary UMOD between SS and SS.BN1 rats. The pattern of urinary UMOD expression in SS rats is consistent with that observed in some patient populations of UMOD associated kidney disease. Finally, the evidence that SS.BN1 rats, harboring the UMOD gene from BN rats, exhibit significant protection against SS hypertension and proteinuria is consistent with the notion that an alteration in UMOD structure or function may, in part, be responsible for such pathologies in SS rats.Support or Funding InformationEast Tennessee State University, American Society of Nephrology, American Heart Association, American Physiological SocietyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mutant uromodulin expression leads to altered homeostasis of the endoplasmic reticulum and activates the unfolded protein response.

Uromodulin is the most abundant urinary protein in physiological conditions. It is exclusively produced by renal epithelial cells lining the thick ascending limb of Henle’s loop (TAL) and it plays key roles in kidney function and disease. Mutations in UMOD, the gene encoding uromodulin, cause autosomal dominant tubulointerstitial kidney disease uromodulin-related (ADTKD-UMOD), characterised by hyperuricemia, gout and progressive loss of renal function. While the primary effect of UMOD mutations, retention in the endoplasmic reticulum (ER), is well established, its downstream effects are still largely unknown. To gain insight into ADTKD-UMOD pathogenesis, we performed transcriptional profiling and biochemical characterisation of cellular models (immortalised mouse TAL cells) of robust expression of wild type or mutant GFP-tagged uromodulin. In this model mutant uromodulin accumulation in the ER does not impact on cell viability and proliferation. Transcriptional profiling identified 109 genes that are differentially expressed in mutant cells relative to wild type ones. Up-regulated genes include several ER resident chaperones and protein disulphide isomerases. Consistently, pathway enrichment analysis indicates that mutant uromodulin expression affects ER function and protein homeostasis. Interestingly, mutant uromodulin expression induces the Unfolded Protein Response (UPR), and specifically the IRE1 branch, as shown by an increased splicing of XBP1. Consistent with UPR induction, we show increased interaction of mutant uromodulin with ER chaperones Bip, calnexin and PDI. Using metabolic labelling, we also demonstrate that while autophagy plays no role, mutant protein is partially degraded by the proteasome through ER-associated degradation. Our work demonstrates that ER stress could play a central role in ADTKD-UMOD pathogenesis. This sets the bases for future work to develop novel therapeutic strategies through modulation of ER homeostasis and associated protein degradation pathways.

Urinary proteomic pattern in female stress urinary incontinence: a pilot study.

Introduction and hypothesisPrevious studies aiming to identify specific pre-defined urine protein biomarkers for stress urinary incontinence (SUI) have not identified clinically important differences. The hypothesis of our study was that the global distribution of urinary proteins, the proteome, differs between women with and those without SUI.MethodsIn this age-matched case–control study, we compared the urinary proteome of 20 women with SUI and 20 controls. Proteins were identified by applying high-performance liquid chromatography separation and tandem mass spectrometry detection. Data analysis was performed using Mascot 2.4.1 embedded in ProteinScape 3.1.ResultsWe identified 828 different proteins. The concentration of six of those showed a significant difference between urine samples of SUI patients and those of controls (q value < 0.25). Four proteins showed a higher abundance in SUI samples compared with controls: plasma serine protease inhibitor (logFC 1.11), leucine-rich alpha-2-glycoprotein (logFC 3.91), lysosomal alpha-glucosidase (logFC 1.24), and peptidyl-prolyl cis- trans isomerase A (logFC 1.96). We identified two proteins in lower abundance in SUI samples compared with controls: uromodulin (logFC −4.87) and TALPID3 (logFC −1.99).ConclusionsOverexpression of plasma serine protease inhibitor, leucine-rich alpha-2-glycoprotein, lysosomal alpha-glucosidase, and peptidyl-prolyl cis- trans isomerase A, and lower expression of uromodulin and TALPID3, in urine may be associated with female SUI.Electronic supplementary materialThe online version of this article (doi:10.1007/s00192-016-3033-5) contains supplementary material, which is available to authorized users

Reduced Renal Methylarginine Metabolism Protects against Progressive Kidney Damage.

Nitric oxide (NO) production is diminished in many patients with cardiovascular and renal disease. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthesis, and elevated plasma levels of ADMA are associated with poor outcomes. Dimethylarginine dimethylaminohydrolase-1 (DDAH1) is a methylarginine-metabolizing enzyme that reduces ADMA levels. We reported previously that a DDAH1 gene variant associated with increased renal DDAH1 mRNA transcription and lower plasma ADMA levels, but counterintuitively, a steeper rate of renal function decline. Here, we test the hypothesis that reduced renal-specific ADMA metabolism protects against progressive renal damage. Renal DDAH1 is expressed predominately within the proximal tubule. A novel proximal tubule-specific Ddah1 knockout (Ddah1(PT-/-)) mouse demonstrated tubular cell accumulation of ADMA and lower NO concentrations, but unaltered plasma ADMA concentrations. Ddah1(PT-/-) mice were protected from reduced kidney tissue mass, collagen deposition, and profibrotic cytokine expression in two independent renal injury models: folate nephropathy and unilateral ureteric obstruction. Furthermore, a study of two independent kidney transplant cohorts revealed higher levels of human renal allograft methylarginine-metabolizing enzyme gene expression associated with steeper function decline. We also report an association among DDAH1 expression, NO activity, and uromodulin expression supported by data from both animal and human studies, raising the possibility that kidney DDAH1 expression exacerbates renal injury through uromodulin-related mechanisms. Together, these data demonstrate that reduced renal tubular ADMA metabolism protects against progressive kidney function decline. Thus, circulating ADMA may be an imprecise marker of renal methylarginine metabolism, and therapeutic ADMA reduction may even be deleterious to kidney function.