Urine Pellet Research Articles

The Rab-Rabphilin system in injured human podocytes stressed by glucose overload and angiotensin II.

Kidney injury in hypertension and diabetes entails, among in other structures, damage in a key cell of the glomerular filtration barrier, the podocyte. Podocytes are polarized and highly differentiated cells in which vesicular transport, partly driven by Rab GTPases, is a relevant process. The aim of the present study was to analyze Rab GTPases of the Rab-Rabphilin system in human immortalized podocytes and the impact of high glucose and angiotensin II. Furthermore, alterations of the system in urine cell pellets from patients with hypertension and diabetes were studied. Apoptosis was analyzed in podocytes, and mRNA level quantification, Western blot analysis, and immunofluorescence were developed to quantify podocyte-specific molecules and Rab-Rabphilin components (Rab3A, Rab27A, and Rabphilin3A). Quantitative RT-PCR was performed on urinary cell pellet from patients. The results showed that differentiated cells had reduced protein levels of the Rab-rabphillin system compared with undifferentiated cells. After glucose overload and angiotensin II treatment, apoptosis was increased and podocyte-specific proteins were reduced. Rab3A and Rab27A protein levels were increased under glucose overload, and Rabphilin3A decreased. Furthermore, this system exhibited higher levels under stress conditions in a manner of angiotensin II dose and time treatment. Immunofluorescence imaging indicated different expression patterns of podocyte markers and Rab27A under treatments. Finally, Rab3A and Rab27A were increased in patient urine pellets and showed a direct relationship with albuminuria. Collectively, these results suggest that the Rab-Rabphilin system could be involved in the alterations observed in injured podocytes and that a mechanism may be activated to reduce damage through the vesicular transport enhancement directed by this system.

Podocyte stress and detachment measured in urine are related to mean arterial pressure in healthy humans

Hypertension-associated progressive glomerulosclerosis is a significant driver of both de novo and all-cause chronic kidney disease leading to end-stage kidney failure. The progression of glomerular disease proceeds via continuing depletion of podocytes from the glomeruli into the ultrafiltrate. To non-invasively assess injury patterns associated with mean arterial pressure (MAP), we conducted an observational study of 87 healthy normotensive individuals who were cleared for living kidney donation. Urine pellet podocin and aquaporin2 mRNAs normalized to the urine creatinine concentration (UPod:Creat ratio and UAqp2:Creat ratio) were used as markers of podocyte detachment and tubular injury, respectively. The ratio of two podocyte mRNA markers, podocin to nephrin (UPod:Neph) as well as the ratio of podocin to the tubular marker aquaporin2 (UPod:Aqp2) estimated the relative rates of podocyte stress and glomerular vs. tubular injury. The MAP was positively correlated with the UPod:Neph and UPod:Aqp2, thereby confirming the relationship of MAP with podocyte stress and the preferential targeting of the glomerulus by higher MAP. In multivariable linear regression analysis, both UPod:Neph and UPod:Creat, but not UAqp2:Creat or proteinuria, were both significantly related to a range of normal MAP (70 to 110 mmHg). Systolic, as opposed to diastolic or pulse pressure was associated with UPod:Creat. Thus, higher podocyte stress and detachment into the urine are associated with MAP even in a relatively "normal" range of MAP. Hence, urine pellet mRNA monitoring can potentially identify progression risk before the onset of overt hypertension, proteinuria or chronic kidney disease.

Comparative Analysis of Urine Fractions for Optimal Bladder Cancer Detection Using DNA Methylation Markers.

DNA methylation analysis of full void urine and urine pellet seems promising for bladder cancer (BC) detection and surveillance. Urinary cell-free DNA from urine supernatant is now gaining interest for other molecular tests in BC. This study aims to evaluate which urine fraction is preferred for BC diagnosis using methylation markers: full void urine, urine pellet or supernatant. Methylation levels of nine markers were determined in the three urine fractions and correlated with their respective tumor tissues in BC patients and compared to controls. For all markers and marker panel GHSR/MAL, diagnostic performance was determined by calculating the area under the curve (AUC) of the respective receiver operating characteristic curves. For most of the markers, there was a significant correlation between the methylation levels in each of the urine fractions and the matched tumor tissues. Urine pellet was the most representative fraction. Generally, AUCs for BC diagnosis were comparable among the fractions. The highest AUC was obtained for GHSR/MAL in urine pellet: AUC 0.87 (95% confidence interval: 0.73–1.00), corresponding to a sensitivity of 78.6% and a specificity of 91.7%. Our results demonstrate that cellular and cell-free DNA in urine can be used for BC diagnosis by urinary methylation analysis. Based on our comparative analysis and for practical reasons, we recommend the use of urine pellet.

PD04-01 ANTIBIOTICS AFFECT THE GUT MICROBIOME AND ALTER KIDNEY STONE FORMATION IN GENETIC HYPERCALCIURIC STONE-FORMING RATS

You have accessJournal of UrologyStone Disease: Basic Research & Pathophysiology I (PD04)1 Apr 2020PD04-01 ANTIBIOTICS AFFECT THE GUT MICROBIOME AND ALTER KIDNEY STONE FORMATION IN GENETIC HYPERCALCIURIC STONE-FORMING RATS Joshua Stern*, John Asplin, Nancy Krieger, Sylvia Suadicani, Luojing Chen, Jennifer Becker, Michaela Chan, Justin Lee, Yi Wang, and David Bushinsky Joshua Stern*Joshua Stern* More articles by this author , John AsplinJohn Asplin More articles by this author , Nancy KriegerNancy Krieger More articles by this author , Sylvia SuadicaniSylvia Suadicani More articles by this author , Luojing ChenLuojing Chen More articles by this author , Jennifer BeckerJennifer Becker More articles by this author , Michaela ChanMichaela Chan More articles by this author , Justin LeeJustin Lee More articles by this author , Yi WangYi Wang More articles by this author , and David BushinskyDavid Bushinsky More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000824.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Recent evidence suggests both antibiotic exposure and the gut microbiome (GMB) are associated with stone disease. Antibiotics can cause rapid alterations in the GMB. We utilized genetic hypercalciuric stone-forming (GHS) rats that form calcium phosphate (CaP) stones, to determine the effect of antibiotics on the gut microbiome, urine ion excretion and stone formation. METHODS: 116th generation GHS rats were fed a fixed amount of a normal Ca (1.2%) and P (0.65%) diet, housed in metabolic cages and divided into 3 groups (n=10): control (CTL) diet, or supplemented with ciprofloxacin (Cipro, 5 mg/d) or Bactrim (250 mg/d). Urine and fecal pellets were collected at 6, 12 and 18 wks for analyses. DNA from fecal pellets were amplified on the 16S rRNA V4 region using primers on an Illumina platform. QIIME was used for analysis. At 18 wks kidney stone formation was determined by Faxitron analysis and assessed by 3 blinded reviewers. RESULTS: After 18 wks, urine Ca decreased with Bactrim (CTL=13.7± 0.4, Bactrim=12.1±0.4 mg/d, p<0.05) as did urine oxalate (CTL=1.2±0.04, Bactrim=0.8±0.02 mg/d, p<0.05). CaP supersaturation increased with Bactrim (CTL=6.8±0.4, Bactrim=8.4±0.5, p<0.05) while CaOx supersaturation fell (CTL=16.2± 0.6, Bactrim=12.0±0.4, p<0.05). Calcification was increased with Bactrim (CTL=1.0±0.2, Bactrim=2.98±0.3, p<0.05). Cipro was not different from CTL for any parameter. Principal component analysis of the GMB showed the Bactrim group and controls clustered separately (p=0.001). Microbial diversity negatively correlated with urinary oxalate in all animals (R=-0.46, p=0.006) and positively correlated with urinary pH in the Bactrim group (R=0.76, p=0.01). CONCLUSIONS: Bactrim altered the GMB of GHS rats, decreased urine Ca, increased CaP supersaturation and increased calcification, while Cipro had no effects. Whether the alteration in the GMB is mechanistically related to the changes in urine ion excretion and calcification remains to be determined. Source of Funding: NIDDK © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e78-e78 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Joshua Stern* More articles by this author John Asplin More articles by this author Nancy Krieger More articles by this author Sylvia Suadicani More articles by this author Luojing Chen More articles by this author Jennifer Becker More articles by this author Michaela Chan More articles by this author Justin Lee More articles by this author Yi Wang More articles by this author David Bushinsky More articles by this author Expand All Advertisement PDF downloadLoading ...

Podocyte-associated messenger ribonucleic acids profile in urine of patients with idiopathic nephrotic syndrome and lupus nephritis.

Background: Podocytes associated proteins are essential in the maintenance of a healthy glomerular filtration barrier, a spectrum of different glomerular diseases occurs due to podocyte abnormalities. Urinary podocytes mRNAs are a more accurate tool for monitoring the progression of different glomerular diseases than proteinuria.Methods: Quantification of podocyte mRNAs in urinary sediment by real time polymerase chain reaction (PCR) of idiopathic nephrotic syndrome, lupus nephritis patients (LN) and the healthy controls, each group contains 15 individuals to be correlated with proteinuria level and eGFR (by MDRD equation).Results: the urinary podocyte mRNAs in both idiopathic nephrotic syndrome and LN groups differed significantly in comparison to that of controls, there was a significant correlation when comparing renal function tests and estimated glomerular filtration rate (by MDRD equation) between nephrotic, LN groups and control group, the value of podocin showed significant correlation in active LN subgroup with its corresponding value in the non-active LN subgroup.Conclusions: This study has revealed that urine pellet podocyte mRNAs can be used as a tool for monitoring the progression of idiopathic nephrotic syndrome and lupus nephritis patients and that urinary podocin can be used as a marker for lupus nephritis activity.

Migration of an Intra Uterine Device (IUD) on a Bilharzian Bladder: An Exceptional Fact About a Case

Intra uterine device (IUD) migration is a rare complication of female contraception. The mechanism for this migration is still poorly understood. Its association with a bilharzian bladder is exceptional. Urinary schistosomiasis is an endemic disease in our regions. A real public health problem, it affects poor populations of farmers and fishermen. And also women when they do their household chores in infested water. Through an observation, we analyze the clinical and therapeutic aspects of the intra vesical migration of an IUD on a bilharzian bladder. The diagnosis was made on a 31 years old patient who had an IUD 1 year 3 months ago. She had presented some urinary disorders such as dysuria, micturition burns and intermittent hematuria, evolving for 3 months. A urine pellet had highlighted Schistosoma haematobium eggs. After medical treatment and sterilization of the urine, the symptoms remained. A cystoscopy was performed which found the IUD in the bladder. There followed a surgical exploration by an under umbilical laparotomy for the removal of the IUD. Migration of the IUD into the bladder and bilharzian cystitis are two distincts nosologies afections but similar clinical manifestations. In both cases, a good clinical and paraclinical analysis makes it possible to make their diagnosis and ensure adequate management.

Podocyte hypertrophic stress and detachment precedes hyperglycemia or albuminuria in a rat model of obesity and type2 diabetes-associated nephropathy

Type2 diabetes-associated nephropathy is the commonest cause of renal failure. Mechanisms responsible are controversial. Leptin-deficient hyperphagic Zucker (fa/fa) rats were modeled to test the hypothesis that glomerular enlargement drives podocyte hypertrophic stress leading to accelerated podocyte detachment, podocyte depletion, albuminuria and progression. By 6weeks, prior to development of either hyperglycemia or albuminuria, fa/fa rats were hyperinsulinemic with high urinary IGF1/2 excretion, gaining weight rapidly, and had 1.6-fold greater glomerular volume than controls (P < 0.01). At this time the podocyte number per glomerulus was not yet reduced although podocytes were already hypertrophically stressed as shown by high podocyte phosphor-ribosomal S6 (a marker of mTORC1 activation), high urinary pellet podocin:nephrin mRNA ratio and accelerated podocyte detachment (high urinary pellet podocin:aquaporin2 mRNA ratio). Subsequently, fa/fa rats became both hyperglycemic and albuminuric. 24 hr urine albumin excretion correlated highly with decreasing podocyte density (R2 = 0.86), as a consequence of both increasing glomerular volume (R2 = 0.70) and decreasing podocyte number (R2 = 0.63). Glomerular podocyte loss rate was quantitatively related to podocyte detachment rate measured by urine pellet mRNAs. Glomerulosclerosis occurred when podocyte density reached <50/106um3. Reducing food intake by 40% to slow growth reduced podocyte hypertrophic stress and “froze” all elements of the progression process in place, but had small effect on hyperglycemia. Glomerular enlargement caused by high growth factor milieu starting in pre-diabetic kidneys appears to be a primary driver of albuminuria in fa/fa rats and thereby an under-recognized target for progression prevention. Progression risk could be identified prior to onset of hyperglycemia or albuminuria, and monitored non-invasively by urinary pellet podocyte mRNA markers.

Detection and molecular characterization of urinary tract HIV-1 populations

BackgroundIdentification of all possible HIV reservoirs is an important aspect in HIV eradication efforts. The urinary tract has however not been well studied as a potential HIV reservoir. In this pilot study we molecularly characterized HIV-1 viruses in urine and plasma samples to investigate HIV-1 replication, compartmentalization and persistence in the urinary tract.MethodsProspectively collected urine and blood samples collected over 12–36 months from 20 HIV-1 infected individuals were analysed including sampling points from prior to and after ART initiation. HIV-1 pol gene RNA and DNA from urine supernatant and urine pellets respectively were analysed and compared to plasma RNA viruses from the same individual.ResultsHIV-1 nucleic acid was detected in urine samples from at least one time point in 8/20 (40%) treatment-naïve subjects compared to 1/13 (7.7%) individuals on antiretroviral treatment (ART) during periods of plasma viral suppression and 1/7 (14.3%) individuals with virological failure. HIV-1 RNA was undetectable in urine samples after ART initiation but HIV-1 DNA was detectable in one patient more than 6 months after treatment initiation. There was co-clustering of urine-derived pol sequences but some urine-derived sequences were interspersed among the plasma-derived sequences.ConclusionsSuppressive ART reduces HIV-1 replication in the urinary tract but HIV-1 DNA may persist in these cells despite treatment. A larger number of sequences would be required to confirm HIV compartmentalization in the urinary tract.

A Prospective Study of the Urinary and Gastrointestinal Microbiome in Prepubertal Males

OBJECTIVETo determine if urinary microbial communities similar to those described in adults exist in children and to profile the urinary and gastrointestinal microbiome in children presenting to urology for both routine and complex urologic procedures. METHODSPrepubertal boys (n = 20, ages 3 months-8 years; median age 15 months) who required elective urologic procedures were eligible. Urine samples were collected via sterile catheterization and fecal samples were obtained by rectal swabs. DNA was extracted from urine pellet and fecal samples and subjected to bacterial profiling via 16S rDNA Illumina sequencing and 16S rDNA quantitative polymerase chain reaction. We assessed within and between sample diversity and differential species abundance between samples. RESULTSUrine samples had low bacterial biomass that reflected the presence of bacterial populations. The most abundant genera detected in urine samples are not common to skin microbiota and several of the genera have been previously identified in the urinary microbiome of adults. We report presumably atypical compositional differences in both the urinary and gastrointestinal microbiome in children with prior antibiotic exposure and highlight an important case of a child who had undergone lifelong antibiotic treatment as prophylaxis for congenital abnormalities. CONCLUSIONThis study provides one of the first characterizations of the urinary microbiome in prepubertal males. Defining the baseline healthy microbiome in children may lay the foundation for understanding the long-term impact of factors such as antibiotic use in the development of a healthy microbiome as well as the development of future urologic and gastrointestinal diseases.

Urinary exosomal microRNA panel unravels novel biomarkers for diagnosis of type 2 diabetic kidney disease

Abstract Background A potential approach adopted in the current study is to design a panel based on in silico retrieval of novel miRNAs related to diabetic kidney disease and to evaluate its usefulness in disease diagnosis. Patient and Methods In the current study, we measured the differential expression of a 6 miRNA panel in urine pellet and exosome in an initial screening group using syber green-based PCR array. Also, we performed pathway enrichment analysis of the key target genes of these miRNAs. Finally, we selected the most significantly up-regulated miRNAs in DKD, exosomal miR-15b, miR-34a and miR-636, that were measured by real-time PCR in a larger independent set of 180 participants to evaluate their usefulness as novel urine biomarkers for diagnosis diabetic kidney disease. Results PCR array analysis showed that miR-15b, miR-34a, and miR-636 were upregulated in both urine pellet and exosome of type 2DKD patients. qRT-PCR validation in the larger independent set of participants confirmed the significant up-regulation of these urinary exosomal miRs ( P Conclusion We identified urinary exosomal miR-15b, miR-34a, and miR-636 as a novel diagnostic panel and a major contributor in the pathogenesis of diabetic kidney disease.

Accelerated podocyte detachment early after kidney transplantation is related to long-term allograft loss of function.

Kidney allograft half-life has not improved despite excellent short-term survival. Recent long-term surveillance biopsy studies identify accumulating glomerulosclerosis (GS) to be associated with late allograft loss. While podocyte depletion is well known to drive proteinuria and GS in animal models and human glomerular diseases, its role in renal allograft loss of function is generally not recognized. To address these questions, we collected urine from 125 kidney allograft recipients in the first posttransplant year for urine pellet messenger RNA (mRNA) and protein analysis, with a median follow up of 4.5years. Using multivariable linear models adjusted for proteinuria, transplant, recipient and donor factors, we observed that the average urine pellet podocin mRNA normalized to urine creatinine (UPodCR) in the first posttransplant year was significantly associated with an estimated glomerular filtration rate (eGFR) decline (P = 0.001). The relationship between UPodCR and eGFR decline persisted even among recipients who were nonproteinuric and who had no recurrent or de novo glomerular disease identified on 1-year protocol biopsy. Finally, we identified recipient, donor and recipient:donor body surface area mismatch ratio to be independently associated with UPodCR early after transplantation. A larger donor was protective, while a larger recipient and increased recipient:donor size mismatch ratio were associated with increased UPodCR. These findings support the concept that in kidney allografts, accelerated podocyte loss precedes proteinuria and is associated with inferior long-term allograft outcomes as measured by eGFR decline and may be initiated by recipient:donor size mismatch. Modulating factors driving early podocyte detachment after kidney transplantation may help improve long-term outcomes.

Profiling the Urinary Microbiota in Male Patients With Bladder Cancer in China.

Mounting evidence indicates that microbiome plays an important role in the development and progression of cancer. The dogma that urine in healthy individuals must be sterile has been overturned. Dysbiosis of the urinary microbiome has been revealed responsible for various urological disorders, including prostate cancer. The link between chronic inflammation, microbiome and solid tumors has been established for various neoplastic diseases. However, a detailed and comprehensive analysis of urinary microenvironment of bladder cancer has not been yet reported. We performed this study to characterize the potential urinary microbial community possibly associated with bladder cancer. Mid-stream urine was collected from 31 male patients with bladder cancer and 18 non-neoplastic controls. DNA was extracted from urine pellet samples and processed for high throughput 16S rRNA amplicon sequencing of the V4 region using Illumina MiSeq. Sequencing reads were filtered using QIIME and clustered using UPARSE. We observed increased bacterial richness (Observed Species, Chao 1 and Ace indexes; cancer vs. control; 120.0 vs. 56.0; 134.5 vs. 68.3; and 139.6 vs. 72.9, respectively), enrichment of some bacterial genera (e.g., Acinetobacter, Anaerococcus, and Sphingobacterium) and decrease of some bacterial genera (e.g., Serratia, Proteus, and Roseomonas) in cancer group when compared to non-cancer group. Significant difference in beta diversity was found between cancer and non-cancer group, among different risk level, but not among different tumor grade. Enrichment of Herbaspirillum, Porphyrobacter, and Bacteroides was observed in cancer patients with high risk of recurrence and progression, which means these genera maybe potential biomarkers for risk stratification. The PICRUSt showed that various functional pathways were enriched in cancer group, including Staphylococcus aureus infection, glycerolipid metabolism and retinol metabolism. To our knowledge, we performed the most comprehensive study to date to characterize the urinary microbiome associated with bladder cancer. A better understanding of the role of microbiome in the development and progression of bladder cancer could pave a new way for exploring new therapeutic options and biomarkers.

Toward Personalised Liquid Biopsies for Urothelial Carcinoma: Characterisation of ddPCR and Urinary cfDNA for the Detection of the TERT 228 G>A/T Mutation.

Background:TERT promotor mutations are present in >75% of bladder tumours; these mutations are also detectable in urine. Previous studies have used urinary pellet DNA, and semi-quantitative methods unsuitable for detecting very low mutant allele frequencies.Objective:In this proof-of-principle study we use ddPCR to count the DNA molecules with wt and mutant TERT sequences in urinary cfDNA from patients whose bladder cancers harbour TERT mutations.Methods:Urinary cfDNA prepared from the urine from 104 bladder cancer patients was analysed. We determined the mutant allele frequency across stages and grades of disease, analysed concordance between cfDNA and tumour DNA, compared cfDNA with pellet DNA, and analysed the quantity and size distribution of cfDNA.Results:In 71 of 77 patients with a 228 G>A/T mutant tumour, the mutation was also detected in urinary cfDNA by ddPCR; all 6 “false negatives” were low grade pTa tumours. Overall concordance between tissue and cfDNA mutation status was 92%, and 100% was achieved for high grade disease. Median mutant allele frequencies in urinary cfDNA were 3.4, 13.4 and 32.1% in grade 1, 2 and 3 disease. The 228 G>A/T mutation was not detected in urinary cfDNA in 26 out of 27 mutation-negative patients (96% specificity).Conclusions:Concordance between tumour DNA and urinary cfDNA is high, and TERT 228 G>A/T ddPCR may prove useful for monitoring patients that harbour this mutation. Mutant allele frequencies in cfDNA are often high, but assays capable of detecting very low mutant allele frequencies will be required to achieve high sensitivity in low grade disease.

Urinary miR-155-5p and CXCL10 as prognostic and predictive biomarkers of rejection, graft outcome and treatment response in kidney transplantation.

MicroRNAs (miRNAs) may be useful biomarkers of rejection and allograft outcome in kidney transplantation. Elevated urinary CXCL10 levels have been associated with acute rejection (AR) and may predict allograft failure. We examined the correlation of miRNA, CXCL10 levels and immunosuppressive drug exposure with AR and graft function in kidney transplant recipients. Eighty de novo kidney transplant recipients were recruited from four European centres. All patients received tacrolimus, mycophenolate mofetil, and methylprednisolone. Urinary pellet expression of miR-142-3p, miR-210-3p and miR-155-5p was assessed by quantitative real-time polymerase chain reaction and urinary CXCL10 levels by enzyme-linked immunosorbent assay at the 1st week and the 1st , 2nd , 3rd and 6th months post-transplantation. Eight patients experienced AR. Before and during AR, patients showed a significant increase of urinary miR-142-3p, miR-155-5p and CXCL10 levels and a decrease of miR-210-3p levels. Receiver operating characteristic curve analysis showed that miR-155-5p (area under the curve=0.875; P=0.046) and CXCL10 (area under the curve=0.865; P=0.029) had excellent capacity to discriminate between rejectors and nonrejectors. The optimal cut-off values for the prognosis of AR were 0.51, with 85% sensitivity and 86% specificity for miR-155-5p and 84.73pgml-1 , with 84% sensitivity and 80% specificity for CXCL10. miR-155-5p and CXCL10 levels correlated with glomerular filtration rate. Levels of both biomarkers normalized after recovery of graft function. The regular early post-transplantation monitoring of urinary miR-155-5p and CXCL10 can help in the prognosis of AR and graft dysfunction. Large prospective randomized multicentre trials are warranted to refine our cut-off values and validate the clinical usefulness of these biomarkers.

Profiling the Urinary Microbiome in Men with Positive versus Negative Biopsies for Prostate Cancer.

Studies demonstrating bacterial DNA and cultivable bacteria in urine samples have challenged the clinical dogma that urine is sterile. Furthermore, studies now indicate that dysbiosis of the urinary microbiome is associated with pathological conditions. We propose that the urinary microbiome may influence chronic inflammation observed in the prostate, leading to prostate cancer development and progression. Therefore, we profiled the urinary microbiome in men with positive vs negative biopsies for prostate cancer. Urine was collected from men prior to biopsy for prostate cancer. DNA was extracted from urine pellet samples and subjected to bacterial 16S rDNA Illumina® sequencing and 16S rDNA quantitative polymerase chain reaction. We determined the association between bacterial species and the presence or absence of cancer, cancer grade, and type and degree of prostate inflammation. Urine samples revealed diverse bacterial populations. There were no significant differences in α or β diversity and no clear hierarchical clustering of benign or cancer samples. We identified a cluster of pro-inflammatory bacteria previously implicated in urogenital infections in a subset of samples. Many species, including known uropathogens, were significantly and differentially abundant among cancer and benign samples, in low vs higher grade cancers and in relation to prostate inflammation type and degree. To our knowledge we report the most comprehensive study to date of the male urinary microbiome and its relationship to prostate cancer. Our results suggest a prevalence of pro-inflammatory bacteria and uropathogens in the urinary tract of men with prostate cancer.

Accelerated podocyte detachment and progressive podocyte loss from glomeruli with age in Alport Syndrome

Podocyte depletion is a common mechanism driving progression in glomerular diseases. Alport Syndrome glomerulopathy, caused by defective α3α4α5 (IV) collagen heterotrimer production by podocytes, is associated with an increased rate of podocyte detachment detectable in urine and reduced glomerular podocyte number suggesting that defective podocyte adherence to the glomerular basement membrane might play a role in driving progression. Here a genetically phenotyped Alport Syndrome cohort of 95 individuals [urine study] and 41 archived biopsies [biopsy study] were used to test this hypothesis. Podocyte detachment rate (measured by podocin mRNA in urine pellets expressed either per creatinine or 24-hour excretion) was significantly increased 11-fold above control, and prior to a detectably increased proteinuria or microalbuminuria. In parallel, Alport Syndrome glomeruli lose an average 26 podocytes per year versus control glomeruli that lose 2.3 podocytes per year, an 11-fold difference corresponding to the increased urine podocyte detachment rate. Podocyte number per glomerulus in Alport Syndrome biopsies is projected to be normal at birth (558/glomerulus) but accelerated podocyte loss was projected to cause end-stage kidney disease by about 22 years. Biopsy data from two independent cohorts showed a similar estimated glomerular podocyte loss rate comparable to the measured 11-fold increase in podocyte detachment rate. Reduction in podocyte number and density in biopsies correlated with proteinuria, glomerulosclerosis, and reduced renal function. Thus, the podocyte detachment rate appears to be increased from birth in Alport Syndrome, drives the progression process, and could potentially help predict time to end-stage kidney disease and response to treatment.

Rapid Filter-Based Detection and Culture of Burkholderia pseudomallei from Small Volumes of Urine.

Clinical outcomes of melioidosis patients improve when the infecting agent, Burkholderia pseudomallei, is rapidly detected and identified by laboratory testing. Detection of B. pseudomallei DNA or recovery of the pathogen by culture from urine can support a diagnosis of melioidosis and guide patient care. Two new methods, designated filter-capture DNA isolation (FCDI) and filter cellular recovery (FCR), were developed to increase the sensitivity of detection and recovery of viable B. pseudomallei cells from small volumes (0.45 ml) of urine. DNA from eight strains of B. pseudomallei that were spiked into synthetic urine at low concentrations (1 × 102 CFU/ml) was detected in FCDI cell lysates using real-time PCR with greater consistency than with preparations from a QIAamp DNA Blood minikit. The FCR method showed greater B. pseudomallei detection sensitivity than conventional urine culture methods and resulted in typical colony growth at 24 h from as few as 1 × 102 CFU/ml. In addition, the FCR method does not rely on precipitation of a urine pellet by centrifugation and requires a smaller volume of urine. The FCDI and FCR methods described here could improve time-to-results and decrease the number of negative B. pseudomallei reports that are currently observed from urine culture as a consequence of samples containing low or variable bacterial cell concentrations.

Detection of prostate cancer-specific transcripts in extracellular vesicles isolated from post-DRE urine.

The measurement of gene expression in post-digital rectal examination (DRE) urine specimens provides a non-invasive method to determine a patient's risk of prostate cancer. Many currently available assays use whole urine or cell pellets for the analysis of prostate cancer-associated genes, although the use of extracellular vesicles (EVs) has also recently been of interest. We investigated the expression of prostate-, kidney-, and bladder-specific transcripts and known prostate cancer biomarkers in urine EVs. Cell pellets and EVs were recovered from post-DRE urine specimens, with the total RNA yield and quality determined by Bioanalyzer. The levels of prostate, kidney, and bladder-associated transcripts in EVs were assessed by TaqMan qPCR and targeted sequencing. RNA was more consistently recovered from the urine EV specimens, with over 80% of the patients demonstrating higher RNA yields in the EV fraction as compared to urine cell pellets. The median EV RNA yield of 36.4 ng was significantly higher than the median urine cell pellet RNA yield of 4.8 ng. Analysis of the post-DRE urine EVs indicated that prostate-specific transcripts were more abundant than kidney- or bladder-specific transcripts. Additionally, patients with prostate cancer had significantly higher levels of the prostate cancer-associated genes PCA3 and ERG. Post-DRE urine EVs are a viable source of prostate-derived RNAs for biomarker discovery and prostate cancer status can be distinguished from analysis of these specimens. Continued analysis of urine EVs offers the potential discovery of novel biomarkers for pre-biopsy prostate cancer detection.

Hypoxia Inducible Factor 1: A Urinary Biomarker of Kidney Disease.

Identifying noninvasive biomarkers of kidney disease is valuable for diagnostic and therapeutic purposes. Hypoxia inducible factor 1 (HIF‐1) expression is known to be elevated in the kidneys in several renal disease pathologies. We hypothesized that the urinary HIF‐1a mRNA level may be a suitable biomarker for expression of this protein in chronic kidney disease (CKD). We compared HIF‐1a mRNA levels from urine pellets of CKD and healthy subjects. To ensure that urinary HIF‐1a mRNA is of kidney origin, we examined colocalization of HIF‐1a mRNA with two kidney specific markers in urine cells. We found that HIF‐1a mRNA is readily quantifiable in urine pellets and its expression was significantly higher in CKD patients compared with healthy adults. We also showed that the urinary HIF‐1a mRNA comes primarily from cells of renal origin. Our data suggest that urinary HIF‐1a mRNA is a potential biomarker in CKD and can be noninvasively assessed in patients.

Comprehensive Metaproteomic Analyses of Urine in the Presence and Absence of Neutrophil-Associated Inflammation in the Urinary Tract.

Inflammation in the urinary tract results in a urinary proteome characterized by a high dynamic range of protein concentrations and high variability in protein content. This proteome encompasses plasma proteins not resorbed by renal tubular uptake, renal secretion products, proteins of immune cells and erythrocytes derived from trans-urothelial migration and vascular leakage, respectively, and exfoliating urothelial and squamous epithelial cells. We examined how such proteins partition into soluble urine (SU) and urinary pellet (UP) fractions by analyzing 33 urine specimens 12 of which were associated with a urinary tract infection (UTI). Using mass spectrometry-based metaproteomic approaches, we identified 5,327 non-redundant human proteins, 2,638 and 4,379 of which were associated with SU and UP fractions, respectively, and 1,206 non-redundant protein orthology groups derived from pathogenic and commensal organisms of the urogenital tract. Differences between the SU and UP proteomes were influenced by local inflammation, supported by respective comparisons with 12 healthy control urine proteomes. Clustering analyses showed that SU and UP fractions had proteomic signatures discerning UTIs, vascular injury, and epithelial cell exfoliation from the control group to varying degrees. Cases of UTI revealed clusters of proteins produced by activated neutrophils. Network analysis supported the central role of neutrophil effector proteins in the defense against invading pathogens associated with subsequent coagulation and wound repair processes. Our study expands the existing knowledge of the urinary proteome under perturbed conditions, and should be useful as reference dataset in the search of biomarkers.