Uromodulin Promoter Research Articles

DETECTION SINGLE NUCLEOTIDE POLYMORPHISMS IN UROMODULIN PROMOTER REGION ASSOCIATED WITH RENAL DISEASES USING SINGLE-STRAND CONFORMATION POLYMORPHISM-POLYMERASE CHAIN POLYMORPHISMS TECHNIQUE

Objective: The uromodulin, a glycoprotein, expressed and secreted by epithelial kidney cells lining the thick ascending limb of the Henle’s loop. It is encoded by the UMOD gene in humans. Our objective was to analyze single nucleotide polymorphisms (SNPs) in the UMOD promoter region in patients with chronic kidney disease (CKD) and end-stage renal disease (ESRD).Methods: The blood samples were collected from 100 patients with CKD (50) and ESRD (50), who admitted at Merjan Teaching Hospital in Babylon Province, Iraq (February-July 2016). In addition, 50 blood samples of healthy control. The SNPs of UMOD promoter region was investigated using single-strand conformation polymorphism-polymerase chain polymorphisms (SSCP-PCR) and DNA sequencing techniques.Results: UOMD promoter region polymorphisms using PCR-SSCP and sequencing DNA appeared three different conformational haplotypes, including A\G 49 haplotype (5 bands), A\G 49 and C\A 247 haplotype (5 bands), and C\G 45 and A\G 49 haplotype (6 bands) distributed among CKD and ESRD cases, due to the presence of three SNPs. There was no association between band numbers of PCR-SSCP with ESRD and CKD compared with a control group.Conclusion: SSCP-PCR is a good screening method to detect genetic variations in an uromodulin promoter region.

DETECTION SINGLE NUCLEOTIDE POLYMORPHISMS IN UROMODULIN PROMOTER REGION ASSOCIATED WITH RENAL DISEASES USING SINGLE-STRAND CONFORMATION POLYMORPHISM-POLYMERASE CHAIN POLYMORPHISMS TECHNIQUE

Objective: The uromodulin, a glycoprotein, expressed and secreted by epithelial kidney cells lining the thick ascending limb of the Henle’s loop. It is encoded by the UMOD gene in humans. Our objective was to analyze single nucleotide polymorphisms (SNPs) in the UMOD promoter region in patients with chronic kidney disease (CKD) and end-stage renal disease (ESRD).Methods: The blood samples were collected from 100 patients with CKD (50) and ESRD (50), who admitted at Merjan Teaching Hospital in Babylon Province, Iraq (February-July 2016). In addition, 50 blood samples of healthy control. The SNPs of UMOD promoter region was investigated using single-strand conformation polymorphism-polymerase chain polymorphisms (SSCP-PCR) and DNA sequencing techniques.Results: UOMD promoter region polymorphisms using PCR-SSCP and sequencing DNA appeared three different conformational haplotypes, including A\G 49 haplotype (5 bands), A\G 49 and C\A 247 haplotype (5 bands), and C\G 45 and A\G 49 haplotype (6 bands) distributed among CKD and ESRD cases, due to the presence of three SNPs. There was no association between band numbers of PCR-SSCP with ESRD and CKD compared with a control group.Conclusion: SSCP-PCR is a good screening method to detect genetic variations in an uromodulin promoter region.

307. Hitting the Kidney from All Angles: Using Direct Intrarenal Pelvis Injection Combined with Serotype and Promoter Targeting to Achieve Renal AAV Gene Delivery

Using adeno-associated virus (AAV)-based gene therapy to treat renal diseases is an underdeveloped area. Renal anatomy and physiology make gene delivery to this organ especially difficult. Multiple AAV serotypes and routes of entry in rodents have been tried, with the majority of combinations targeting the renal tubules. To improve upon the current methods of AAV renal transduction for future use in the clinical setting, we employed direct intrarenal pelvis injections of AAV2, 6, 8 and 9 with the luciferase (Luc) or GFP transgene under the control of the cytomegalovirus (CMV) or putative kidney-specific promoters from myo-inositol oxygenase(MIOX), acyl-CoA synthetase medium-chain family member 2 (Acsm2), uromodulin (UMOD), kidney androgen-regulated protein (Kap) and solute carrier family 34 (sodium phosphate), member 1 (Slc34a1) mouse genes in C57Bl/6J mice. Renal luciferase transgene expression was strongest with AAV6, 8 and 9-CMV-Luc. AAV6 and 8-CMV-Luc resulted in stronger expression in the injected kidney, whereas AAV9-CMV-Luc showed equal, bilateral renal expression. AAV9 with the UMOD promoter gave the strongest and most highly renal-specific luciferase expression among all serotype and kidney promoter combinations. Using the GFP transgene we further established AAV transduction of multiple cell types in the kidney. AAV8-CMV-GFP transduced both glomerular and tubular renal cells, while AAV9-CMV-GFP most strikingly transduced periglomerular tubule cells. Both AAV8 and AAV9-UMOD-GFP solely transduced renal tubule cells. We are currently testing AAV variants isolated from clinical urine samples to determine their ability to transduce renal cells in vivo. Our results demonstrate efficient renal transduction by direct intrarenal pelvis injection of AAV vectors. Coupling specific renal cell tropisms of AAV serotypes and renal cell-type-specific promoters, along with the intrarenal pelvis injection strategy would pave the way for a renal-targeted AV gene therapy.

Single-nucleotide polymorphism of the UMOD promoter is associated with the outcome of chronic kidney disease patients.

Uromodulin (UMOD) is the most abundant protein secreted in urine and the mutated form of the UMOD gene is associated with UMOD-associated kidney disease (UAKD). Although UMOD accumulates in the kidney of UAKD patients, it is unclear whether this also occurred in the chronic kidney disease (CKD) patients. Therefore, the association of single-nucleotide polymorphisms (SNPs) in the promoter region of UMOD gene with the kidney survival time of CKD was investigated. The promoter region of the UMOD gene was sequenced for 111 CKD patients. The Kaplan-Meier method was used to identify the disease outcome associated with SNPs in the promoter region of the UMOD gene in CKD patients. The Cox proportional hazard model was used to identify risk factors for the kidney survival time of CKD. SNPs in reference to GenBank accession NG-000016 were detected at 23 sites of the 481-bp in the UMOD promoter region from the CKD patients and the healthy controls. The 6 SNPs with minor allele frequency >5% in the CKD patients or controls were used for disease risk and outcome analysis. The frequent allele rs13333226AA was associated with a shorter period of kidney survival time in CKD patients (P=0.005). The length of kidney survival time in CKD patients with the rs13333226AA genotype was significantly shorter than that of patients with the frequent allele rs13333226AG+GG (relative risk, 0.361; 95% confidence interval, 0.171-0.761; P=0.007). In conclusion, analysis of genetic polymorphisms in UMOD may help to identify the CKD patient subgroups at a high risk for a disease outcome, thereby helping to refine therapeutic decisions in CKD patients.

Common genetic variants of the human uromodulin gene regulate transcription and predict plasma uric acid levels

Uromodulin (UMOD) genetic variants cause familial juvenile hyperuricemic nephropathy, characterized by hyperuricemia with decreased renal excretion of UMOD and uric acid, suggesting a role for UMOD in the regulation of plasma uric acid. To determine this, we screened common variants across the UMOD locus in one community-based Chinese population of 1000 individuals and the other population from 642 American twins and siblings of European and Hispanic ancestry. Transcriptional activity of promoter variants was estimated in luciferase reporter plasmids transfected into HEK-293 cells and mIMCD3 cells. In the primary Chinese population, we found that carriers of the GCC haplotype had higher plasma uric acid, and three promoter variants were associated with plasma uric acid. UMOD promoter variants displayed reciprocal effects on urine uric acid excretion and plasma uric acid concentration, suggesting a primary effect on renal tubular handling of urate. These UMOD genetic marker-on-trait associations for uric acid were replicated in the independent American cohort. Site-directed mutagenesis at trait-associated UMOD promoter variants altered promoter activity in transfected luciferase reporter plasmids. Thus, UMOD promoter variants seem to initiate a cascade of transcriptional and biochemical changes influencing UMOD secretion, leading to altered plasma uric acid levels.

Common variants of the UMOD promoter associated with blood pressure in a community-based Chinese cohort

Hypertension is an important risk factor for chronic kidney disease. The kidney, in turn, has an important role in the regulation of blood pressure (BP). On the basis of a genome-wide association study, single-nucleotide polymorphisms in the uromodulin (UMOD) promoter region had been considered to influence both renal sodium reabsorption and BP. In this study, we asked whether common variants across the UMOD gene influence BP in a community-based Chinese cohort. We screened seven common variants across the UMOD locus in a community-based population from Beijing, including 1000 individuals with 48% males and an average age of 63.7±9.0 years. The urinary UMOD concentration was measured by enzyme-linked immunosorbent assay. We then analyzed the association of common variants of UMOD with BP. The UMOD promoter common variant rs13333226 G/A is associated with diastolic BP (DBP), and G allele carriers have higher DBP compared with A/A homozygotes (P=0.035). The variant rs6497476 C/T predicted the DBP level, with C homozygotes having a higher DBP compared with CT heterozygotes and T homozygotes (P=0.025). Urinary UMOD excretion was correlated with urinary sodium excretion (R=0.239, P=0.656*10(-13)). We determined that common variants of UMOD are associated with DBP level in a community-based Chinese cohort.

Expression of Heme Oxygenase-1 in Thick Ascending Loop of Henle Attenuates Angiotensin II-Dependent Hypertension

Kidney-specific induction of heme oxygenase-1 (HO-1) attenuates the development of angiotensin II (Ang II) -dependent hypertension, but the relative contribution of vascular versus tubular induction of HO-1 is unknown. To determine the specific contribution of thick ascending loop of Henle (TALH) -derived HO-1, we generated a transgenic mouse in which the uromodulin promoter controlled expression of human HO-1. Quantitative RT-PCR and confocal microscopy confirmed successful localization of the HO-1 transgene to TALH tubule segments. Medullary HO activity, but not cortical HO activity, was significantly higher in transgenic mice than control mice. Enhanced TALH HO-1 attenuated the hypertension induced by Ang II delivered by an osmotic minipump for 10 days (139 ± 3 versus 153 ±2 mmHg in the transgenic and control mice, respectively; P<0.05). The lower blood pressure in transgenic mice associated with a 60% decrease in medullary NKCC2 transporter expression determined by Western blot. Transgenic mice also exhibited a 36% decrease in ouabain-sensitive sodium reabsorption and a significantly attenuated response to furosemide in isolated TALH segments. In summary, these results show that increased levels of HO-1 in the TALH can lower blood pressure by a mechanism that may include alterations in NKCC2-dependent sodium reabsorption.

Tissue-specific and transcriptional activity of uromodulin promoter

Objective To investigate tissue-specific and transcriptional activity of Uromodulin (URO) gene promoter in cell lines. Methods Enhanced green fluorescent protein (EGFP) was used as reporter to construct the expression vector pEGFP-URO and transfected into cell lines of HK-2, ACHN, T24 and HBZY-1.Positive cells were observed as a green fluorescence under excitation light. EGFP activity of the cells was detected by fluorescence microscopy and flow cytometry ( FCM ) , and compared to the vector pEGFP-N3.Results The activity of EGFP in HK-2 and ACHN cell lines was stronger than that in the other cell lines (5-10/HP versus 0-1/HP), with positive rate being 13.2% and 11.4% in HK-2 and ACHN cells respectively, but no positive cells were found in the other cell lines. The vector pEGFP-N3 highly expressed EGFP in all cell lines, with positive rate being 23.08% (HK-2) and 19.24% (ACHN) respectively. Conclusion Mouse URO gene promoter can be expressed in a tissue-specific way in human HK-2 and ACHN cells. The URO promoter transcriptional capacity is half of CMV promoter. Key words: Uromodulin; Promoter; Tissue-specific

Role of Angiotensin II Receptor (AT1aR) in Thick Ascending Limb of Henle's Loop and Distal Tubules

The intrarenal renin‐angiotensin system (RAS) plays an important role in the regulation of renal hemodynamics and tubular transport, mainly mediated by AT1aR. However the role of AT1aR in the thick ascending limb of Henle's loop (TAL) and distal convoluted tubules (DCT) remains unknown. To examine this, we generated a transgenic mouse expressing a constitutively active form of HA‐tagged AT1aR (N111G) under the control of the human uromodulin promoter. Tissue‐ and cell‐specific expression was confirmed using quantitative real‐time PCR and immunohistochemistry. There was no difference in baseline blood pressure of transgenic (Tg) and wild type (WT) littermates assessed by radiotelemetry and tail cuff. However, baseline urine osmolality (Os) and sodium concentration [Na] in Tg were significant decreased (Os: 800±90 vs 1100±90 mOsM, P&lt;0.05; [Na]: 48±6 vs 65±9 mM, P&lt;0.05). Preliminary studies revealed increased localization of sodium potassium chloride co‐transporter (NKCC2) to the apical membrane. Expression of the urea transporter (UT‐A) was also increased in Tg. Water loading and deprivation revealed no serious impairment in the urine concentrating and diluting capacity of the Tg mice. We are currently examining the responses to high salt diet and Ang‐II infusion. Our data suggest that AT1aR in the TAL and DCT, like other nephron segments, may play a role in regulating sodium reabsorption.

Goat uromodulin promoter directs kidney-specific expression of GFP gene in transgenic mice.

BackgroundUromodulin is the most abundant protein found in the urine of mammals. In an effort to utilize the uromodulin promoter in order to target recombinant proteins in the urine of transgenic animals we have cloned a goat uromodulin gene promoter fragment (GUM promoter) and used it to drive expression of GFP in the kidney of transgenic mice.ResultsThe GUM-GFP cassette was constructed and transgenic mice were generated in order to study the promoter's tissue specificity, the GFP kidney specific expression and its subcellular distribution. Tissues collected from three GUM-GFP transgenic mouse lines, and analyzed for the presence of GFP by Western blotting and fluorescence confirmed that the GUM promoter drove expression of GFP specifically in the kidney. More specifically, by using immuno-histochemistry analysis of kidney sections, we demonstrated that GFP expression was co-localized, with endogenous uromodulin protein, in the epithelial cells of the thick ascending limbs (TAL) of Henle's loop and the early distal convoluted tubule in the kidney.ConclusionThe goat uromodulin promoter is capable of driving recombinant protein expression in the kidney of transgenic mice. The goat promoter fragment cloned may be a useful tool in targeting proteins or oncogenes in the kidney of mammals.

Kidney-specific activity of the bovine uromodulin promoter.

A 10-kilobase (kb) lambda bacteriophage bovine genomic clone containing 5.4 kb of the 5'-flanking region, exons, and introns of bovine uromodulin gene was isolated. Transgenic mice containing 3.9 kb of the bovine uromodulin promoter and a lacZ reporter gene were generated by pronuclear microinjection. RT-PCR and northern blot analyses of transgene expression in various tissues of founder and F1 mice showed that the transgene was expressed exclusively in the kidney. In situ hybridization and histochemistry for lacZ demonstrated that transgene expression was restricted to tubule epithelial cells of the loop of Henle in the kidney. Stepwise 5' deletion analysis revealed that transfection of luciferase reporter constructs fused to various proximal 5'-flanking regions of the bovine uromodulin gene markedly increased luciferase activity in mouse renal epithelial cells but not in mesenchymal cells and that the most critical cis elements of the uromodulin gene are located within the 600 bp upstream region.

Uromodulin promoter directs high-level expression of biologically active human alpha1-antitrypsin into mouse urine.

We have recently shown that the regulatory sequence of the uromodulin gene, containing the 3.7 kb promoter, exon 1 and a part of exon 2, provided for kidney-specific expression of the reporter lacZ gene in transgenic mice [Zbikowska, Soukhareva, Behnam, Chang, Drews, Lubon, Hammond and Soukharev (2002) Transgenic Res., in the press]. In the present study, we generated transgenic mice harbouring the regulatory sequence of the uromodulin gene to direct the expression of human alpha1-antitrypsin (alpha1AT) into urine. Of the 13 founder mice that tested positive by PCR, seven showed the presence of the human protein in their urine. The concentration of the recombinant human (rh) alpha1AT in the urine, estimated by using ELISA, ranged from 0.5 to 14 microg/ml in the F(0)-generation mice, and reached up to 65 microg/ml in the F1 generation. The transgenically produced rh alpha1AT was found to be N-glycosylated and biologically active. The N-terminal sequence analysis confirmed the identity of the human protein and revealed that the recombinant alpha1AT was correctly processed with the signal peptide cleaved off. Our results demonstrate for the first time that the uromodulin regulatory sequence provides a very attractive option for the potential large-scale production of functional therapeutic proteins in livestock.

The use of the uromodulin promoter to target production of recombinant proteins into urine of transgenic animals.

A uromodulin promoter has been isolated, sequenced, and used to generate two sets of transgenic mice for expression of the lacZ marker gene and for production of the human recombinant erythropoietin (rhEPO) in urine. We demonstrated that the 5.6-kb fragment of the uromodulin gene containing the 3.7-kb promoter area and, both the first exon and part of the second exon, were sufficient to provide kidney-specific expression of the lacZ gene. Histological analysis of the lacZ expression pattern revealed beta-galactosidase activity specifically in the thick limb of Henle's loop. However, due to random integration of the transgene, ectopic expression was detected in some transgenic lines. Analysis of the EPO-transgenic mice showed that rhEPO was secreted into the urine of founder mice (up to 6 ng/ml). We were able to breed and analyze only two sublines with a very low expression level of rhEPO (up to 260 pg/ml). All of our transgenic mice expressing rhEPO in urine developed disease symptoms similar to polycythemia in humans. These included a considerable increase in red blood cell counts, hemoglobin concentration, and hematocrit concomitant with severe thrombocytopenia, all of which were detected in the rhEPO-expressing mice. Although our model did not prove to be beneficial for commercial production of rhEPO, we concluded that the uromodulin promoter could be useful for expression of other important therapeutic proteins into the urine of transgenic animals.