Slippery Viscosity-Sensing Platform with Time Readout for the Detection of Hyaluronidase and Its Inhibitor.

Hyaluronidase (HAase) is a biomarker for cancer, and its detection is of great significance for early diagnosis. However, the requirement of sophisticated instruments, tedious operation procedures, and labeled molecules of conventional HAase biosensing methods hampers their widespread applications. Herein, we report a portable slippery viscosity-sensing platform with time readout for the first time and demonstrate HAase and tannic acid (TA, HAase inhibitor) detection as a model system. HAase specifically cleaves hyaluronic acid (HA) and decreases HA solution viscosity, thereby shortening the aqueous droplet's sliding time on a slippery surface. Thus, the HA solution viscosity alteration due to enzymatic hydrolysis is used to quantify the HAase concentration through the difference in the sliding time of the aqueous droplets on a slippery surface. The developed HAase sensing platform exhibits high sensitivity with a minimum detection limit of 0.23 U/mL and excellent specificity without the use of specialized instruments and labeled molecules. HAase detection in actual urine samples by a standard addition method is performed as well. Moreover, the quantitative detection of TA with an IC50 value of 37.68 ± 1.38 µg/mL is achieved. As an equipment-free, label-free, and high-portability sensing platform, this method holds promise in developing a user-friendly and inexpensive point-of-care testing (POCT) device for HAase detection, and its use can be extended to analyze other analytes with different stimuli-responsive polymers for great universality and expansibility in biosensing applications.

Paper-Based Flow Sensor for the Detection of Hyaluronidase via an Enzyme Hydrolysis-Induced Viscosity Change in a Polymer Solution.

Hyaluronidase (HAase) is implicated in inflammation, cancer development, and allergic reaction. The detection of HAase is significantly important in clinical diagnosis and medical treatment. Herein, we propose a new principle for the development of equipment-free and label-free paper-based flow sensors based on the enzymatic hydrolysis-induced viscosity change in a stimuli-responsive polymer solution, which increases the water flow distance on the pH indicator paper. The detection of HAase is demonstrated as an example. This facile and versatile method can overcome the potential drawbacks of traditional hydrogel-based sensors, including complex preparation steps, slow response time, or low sensitivity. Moreover, it can also avoid the use of specialized instruments, labeled molecules, or functionalized nanoparticles in the sensors developed using the polymer solutions. Using this strategy, the detection of HAase is achieved with a limit of detection as low as 0.2 U/mL. Also, it works well in human urine. Additionally, the detection of tannic acid, which is an inhibitor of HAase, is also fulfilled. Overall, a simple, efficient, high-throughput, and low-cost detection method is developed for the rapid and quantitative detection of HAase and its inhibitor without the use of labeled molecules, synthetic particles, and specialized instruments. As only minimal reagents of HAase, HA, and paper are used, it is very promising in the development of commercial kits for point-of-care testing.

One-pot synthesis of hyaluronic acid-coated gold nanoparticles as SERS substrate for the determination of hyaluronidase activity.

A novel ultrasensitive surface-enhanced Raman spectroscopy (SERS)-based method was developed for the determination of hyaluronidase (HAase), which was based on hyaluronic acid-coated gold nanoparticles (HA-AuNPs) as a substrate, via a facile one-pot method. The detection mechanism is based on HAase which can hydrolyze HA on HA-AuNPs into hyaluronic acid oligomers, causing the originally uniformly dispersed HA-AuNPs to be disintegrated into many smaller HA-AuNPs. These oligomers in turn increase the surface shielding of AuNPs, resulting in high aggregation tendencies. As a result, the original SERS substrate was disassembled, leading to a weakening of the SERS signal at 1173 cm-1. Malachite green was also used as a Raman probe to detect the change of SERS peak intensity and to quantify HAase. Compared with other methods for the determination of HAase, this method is more convenient and efficient; its determination limit was 0.4 mU mL-1. The recoveries of HAase spiked into human urine samples ranged from 97.2 to 103.9%.

A novel surface-enhanced Raman scattering-based ratiometric approach for detection of hyaluronidase in urine.

A ratiometric surface-enhanced Raman scattering (SERS) based method is described for the determination of the activity of hyaluronidase (HAase). Gold nanorods (AuNRs) were functionalized with 4-thiobenzonitrile (TBN) to act as the Raman reporter (TBN-AuNRs), and 4-thiophenylacetylene-functionalized gold-silver alloy nanoparticles (TPA-AuAgNPs) were used as the reference. Hyaluronic acid (HA) acts as the HAase recognition element. The TBN-modified AuNRs aggregate in the presence of HA due to the strong electrostatic interaction between the positively charged TBN-AuNRs and negatively charged HA. This strongly enhances the Raman signal of TBN at 2220 cm-1. However, HA has no significant effect on the dispersion of the modified AuAg NPs which are electroneutral. Hence, no change can be seen in the Raman intensity of TPA at 1974 cm-1. In the presence of HAase, HA is digested into smaller fragments. This results in good dispersion of the TBN-AuNRs and a weaker TBN Raman signal. Hence, the ratio of the Raman peaks at 1974 and 2220 cm-1 increases. Under the optimized conditions, the ratio changes in the 5-70 U mL-1 HAase activity range, and the detection limit is 1.7 U mL-1 (based on the 3σ rule). Moreover, this method has been successfully applied in the determination of the activity of HAase in artificial urine and it is expected to be a new method for the diagnosis of cancer, especially bladder cancer.

A dual-readout nanosensor based on biomass-based C-dots and chitosan@AuNPs with hyaluronic acid for determination of hyaluronidase.

A dual-signal strategy is proposed based on fluorescent biomass-based carbon dots (BC-dots) and chitosan stabilized AuNPs (CS@AuNPs) to determine hyaluronidase (HAase). BC-dots can induce aggregation of CS@AuNPs nanoparticles with a colour change from red to blue. Positively charged CS@AuNPs interacted with the negatively charged hyaluronic acid (HA) through electrostatic adsorption, and CS@AuNPs maintained stability due to the semirigid coil conformation of HA. However, in the presence of HAase, due to enzymatic hydrolysis of HA by HAase, the CS@AuNPs agglomerated. Based on the change of fluorescence and colour, quantitative analysis of HAase was achieved. Linear ranges for the fluorometric and colorimetric determinations were 2.0-70 U mL-1 and 8-60 U mL-1 , respectively, with a detection limit of 0.27 U mL-1 . This dual-signal sensing system possesses high potential for determination of HAase in biological matrices.

A high sensitivity background eliminated fluorescence sensing platform for hyaluronidase activity detection based on Si QDs/HA-δ-FeOOH nanoassembly.

Using fluorescent sensors for highly sensitive and selective detection of biomolecules is a very important strategy in clinical diagnoses as well as biomedical applications. But fluorescent sensors usually suffer from high background signal, which greatly hinders their detection sensitivity. In this work, a novel background-eliminated fluorescence assay for sensitive and selective detection of biomolecule has been developed by coupling feroxyhyte nanosheets (δ-FeOOH) with amino-functionalized silicon quantum dot (Si QDs). We select hyaluronidase (HAase) as the modal target to verify the concept. Si QDs/HA-δ-FeOOH nanoassembly was fabricated by self-assembly of positive Si QDs together with negative HA-δ-FeOOH through electrostatic adsorption. By the introduction of hyaluronidase, the nanoassembly exhibits obviously fluorescence signal recovered. Research suggests that under optimized conditions, this strategy exhibits a good linear response to the concentration of HAase in the range of 0.1 to 12 ng/mL. The detection limit for HAase was 0.02 ng/mL (based on 3σ/S), which was three-order lower than most of the reported fluorescence biosensors for the detection of HAase. Furthermore, this new biosensor has already been applied in the study of urine samples, and the detection results were consistent with those obtained by the clinical tests.

Sensitive Hyaluronidase Biosensor Based on Target-Responsive Hydrogel Using Electronic Balance as Readout.

The development of simple but sensitive methods for hyaluronidase (HAase) detection has been paid a great deal of attention because HAase is a potential cancer marker. In this work, a novel system coupled with a controlled release system has been designed for HAase determination without complex analytical instruments and skilled technicians. Pt@SiO2 nanoparticles (NPs), which can catalyze the breakdown of H2O2 into O2 and H2O, was embedded in the hydrogel constructed by polyethylenimine (PEI) and hyaluronic acid (HA). In the presence of HAase, the hydrogel was broken down as HAase can catalyze the degradation of HA and hence the Pt@SiO2 NPs in the hydrogel was released. The released Pt@SiO2 NPs mixed with H2O2 solution in a drainage device, and then O2 was generated due to the decomposition of H2O2, resulting in an enhancement of pressure in the drainage device because of the low solubility of O2. A certain amount of H2O overflowed from the drainage device because the difference of the pressure between the inner and outer of the drainage device. The overflowed H2O was collected by a tube, and its amount was easily measured by an electronic balance. The weight of the H2O has a linear relationship with the HAase concentration in the range of 1-60 U/mL (120 min enzymatic hydrolysis time) and 0.2-10 U/mL (240 min enzymatic hydrolysis time). The developed system has been applied to detect the activity of HAase in urine samples with satisfied results.

Analyzing Hyaluronidases in Biological Fluids.

Hyaluronidases are a group of enzymes responsible for the degradation of hyaluronan. They seem to be associated with a plethora of pathological conditions, as it has been showcased by numerous studies over the past years. The emerging role of hyaluronidases in various pathological states, especially cancer, is of a great interest. Thus, they are considered as important research targets.In this chapter the popular assay for hyaluronidase analysis in biological fluids is presented and discussed in detail. The assay is divided into two steps; the first is zymography that aims mainly to detect different hyaluronidase enzymes in a biological sample, and the second is the direct quantitative measurement of enzymatic activity by a microtiter plate assay. Both steps are characterized by high sensitivity, simplicity, and limited time consumption.

Hyaluronic acid and hyaluronidase as possible novel urine biomarkers for the diagnosis of prostate cancer.

The goal of the study is to examine the possible use of HA (hyaluronic acid) and HAase (hyaluronidase) as novel urine biomarkers for the early diagnosis for prostate cancer (Pca). After a prostatic massage, the urine of 118 high-risk patients for Pca was collected, and the patients were submitted to ultrasound-guided transrectal biopsy. HA and HAase were detected and analyzed with Enzyme-Linked Immunosorbent Assay, and a statistical analysis of the urine levels of the two biomarkers according to the histology results was performed. HAase and HA were independently associated with Pca, and both HAase and HA showed significant predictive ability for prostate cancer. With an optimal cut-off point of 183.71 HAase had 70% sensitivity maintaining at the same time a 55.2% specificity, while the optimal cut-off point for HA was 50.13 with 65% sensitivity and 53.9% specificity. Patients with HAase more than 183.71 ng/ml had 3.67 times greater likelihood for prostate cancer and Patients with HA more than 50.13 ng/ml had 2.31 times greater likelihood for prostate cancer. The need of novel biomarkers that will improve the efficacy of PSA is urgent. HAase and HA showed significant predictive ability for prostate cancer and were independently associated with Pca, and greater levels were associated with greater odds for prostate cancer. To Our Knowledge, this is the first study referring to the detection of HAase and HA as potential urine biomarkers for the early diagnosis of Pca.

A ratiometric fluorescent probe for hyaluronidase detection via hyaluronan-induced formation of red-light emitting excimers.

Hyaluronidase (HAase), which is involved in various physiological and pathological processes, can selectively degrade hyaluronan (HA) into small fragments, and it has been reported as a diagnostic and prognostic biomarker for bladder cancer. Herein, a facile ratiometric fluorescent sensing system for HAase has been developed, which is based on hyaluronan-induced formation of red-light emitting excimers and can realize sensitive detection of HAase with a detection limit of 0.007 U/mL. A positively-charged pyrene analog (N-Py) has been synthesized and then mixed with the negatively-charged HA, due to electrostatic interaction between the two components, aggregation along with the N-Py excimers readily form which emits red light. While in the presence of HAase, the enzyme catalyzes the hydrolysis of HA into small fragments, which in turn triggers disassembly of excimers; consequently the N-Py excimer emission turns into monomer emission. The emission ratio resulted from the excimer-monomer transition can be used as the sensing signal for detecting HAase. The probe features visible-light excitation and red light emission (excimer), which is conducive to reducing possible interference from autofluorescence of biological samples. Furthermore, the assay system can be successfully used to determine HAase in human urine samples with satisfactory accuracy. This strategy may provide a suitable sensitive and accurate assay for HAase as well as an effective approach for developing fluorescent ratiometric assays for other enzymes.

Cationic Conjugated Polymer/Hyaluronan-Doxorubicin Complex for Sensitive Fluorescence Detection of Hyaluronidase and Tumor-Targeting Drug Delivery and Imaging.

Hyaluronidase (HAase) is becoming a new type of tumor marker since it has been demonstrated to be overexpressed in various kinds of cancer cells. In this study, we described a novel fluorescence method for sensitive, rapid, and convenient HAase detection and tumor-targeting drug delivery and imaging, using a probe prepared by electrostatic assembly of a cationic conjugated polymer (CCP) and anionic hyaluronan (HA) conjugated with the anticancer drug doxorubicin (Dox). The CCP we used was poly{[9,9-bis(6'-(N,N,N-diethylmethylammonium)hexyl)-2,7-fluorenylene ethynylene]-alt-co-[2,5-bis(3'-(N,N,N-diethylmethylammonium)-1'-oxapropyl)-1,4-phenylene]} tetraiodide (PFEP). HA is a natural mucopolysaccharide that can be hydrolyzed by HAase into fragments with low molecular weights. In the PFEP/HA-Dox complex, the fluorescence of PFEP was efficiently quenched due to electron transfer from PFEP to Dox. After the PFEP/HA-Dox complex was exposed to HAase or was taken up by cancer cells through the specific binding between HA and CD44 receptor, HA was degraded by HAase to release the Dox, leading to the recovery of PFEP fluorescence to the "turn-on" state. Moreover, the degree of fluorescence recovery was quantitatively correlated with the concentrations of HAase. Compared with many previously reported methods, our work did not require laborious multiple modifications of HA that may affect the activity of HAase. This point, combined with the excellent optoelectronic property of conjugated polymer, endowed this method with high sensitivity (detection limit: 0.075 U/mL), high specificity, and rapid response, making it applicable for reliable and routine detection of HAase. This fluorescent probe was successfully utilized to detect HAase levels in human urine samples; furthermore, it can also be employed as a multifunctional system by realizing tumor-targeting drug delivery and cell imaging simultaneously. The development of this fluorescence method showed promising potential for early tumor diagnosis and therapy based on HAase detection.

Integrative functional genetic-epigenetic approach for selecting genes as urine biomarkers for bladder cancer diagnosis.

Early screening for bladder cancer (BC) holds the key to combat and control the increasing global burden of BC mortality. We presented a simple approach to characterize, analyze, and validate a panel of biomarkers in BC and their relationship to bilharziasis. We investigated voided urine and blood samples from patients with bladder cancer (n = 94), benign bladder lesions (n = 60), and age-matched normal controls (n = 56). This study was divided into the following phases. (1) We analyzed the expression of urinary Hyaluronoglucosaminidase 1 (HYAL1) protein in BC and control samples by zymography. (2) We performed bioinformatics analysis to retrieve a set of epigenetic regulators of HYAL1. (3) This set of three selected genes [long non-coding RNA-urothelial cancer associated 1(lncRNA-UCA1), microRNA-210, and microRNA-96] was then analyzed in the same urine samples used in phase I by quantitative real-time PCR. (4) A high reproducibility of gene selection results was also determined from statistical validation. The urinary expression of HYAL1 protein and its epigenetic regulators were higher in BC patients (P < .001). The receiver-operating characteristic curve analyses demonstrated that each one had good sensitivity and specificity for distinguishing BC patients from non-BC ones (HYAL1, 89.4 and 91.2 %; miR-210, 76.6 and 93 %; miR-96, 76.6 and 89.4 %; and lncRNA-UCA1, 91.5 and 96.5 %). There was a significant positive correlation between HYAL1 and the selected epigenetic biomarkers. The performance of this urine biomarker panel reached 100 % sensitivity and 89.5 % specificity for bladder cancer diagnosis.

Ratiometric fluorescent biosensor for hyaluronidase with hyaluronan as both nanoparticle scaffold and substrate for enzymatic reaction.

Hyaluronidases (HAase) are involved in various physiological and pathological processes and have been reported as urinary marker for bladder cancer. In this study, a novel ratiometric fluorescent sensing system based on both aggregation-induced emission (AIE) and aggregation-induced quenching (ACQ) was developed to quantitatively assess hyaluronidase level. First, a tetraphenylethylene derivative with positive charges (TPE-2N(+), typical AIE molecule) at both ends and an anthracene derivative with positive charge at one end (AN-N(+), typical ACQ molecule) was synthesized. These two positively charged compounds were then mixed with a negatively charged hyaluronan (HA), which induced the aggregation of the compounds as well as the nanoparticles formation as a result of electrostatic complexation, with TPE-2N(+) acting as cross-linking agent. The aggregation also caused the efficient quenching of the emission of AN-N(+) due to ACQ effect, as well as the fluorescence enhancement of TPE-2N(+) due to AIE effect. In the presence of HAase, the enzymatic reaction led to the degradation of HA and triggered disassembly of the nanoparticles; as a result, the emission of AN-N(+) was restored and that of TPE-2N(+) was suppressed. This fluorescence variation affords the system a robust ratiometric biosensor for HAase, and the ratio of fluorescence intensity for AN-N(+) (I414) to that for TPE-2N(+) (I474) can be used as the sensing signal for detecting HAase activity. In this system, hyaluronan serves not only as the scaffold for nanoparticle formation but also as the substrate for enzymatic reaction. This assay system is operable in aqueous media with very low detection limit of 0.0017 U/mL and is capable of detecting HAase in biological fluids such as serum and urine. This strategy may provide a new and effective approach for developing other enzyme assays.

Sensitivity and specificity of urinary hyaluronic acid and hyaluronidase in detection of bladder transitional cell carcinoma.

PURPOSE: To the assess sensitivity and specificity of urinary levels of hyaluronic acid (HA) and hyaluronidase (HAase) as an individual or a combined test to diagnose bladder transitional cell carcinoma (TCC). MATERIALS AND METHODS: One hundred and ninety-four urine specimens were collected from individuals between July 2007 and March 2008. The urinary level of hyaluronic acid (HA) was measured by Enzyme-linked immunosorbent assay. Thereafter, the urinary levels of HA and HAase were normalized to urinary creatinine level and expressed as ng/mg and µ/mg. RESULTS: Eighty percent of patients with bladder cancer had urinary HA level < 500 ng/mg, and 90% of controls showed HA level < 500 ng/mg (P < .001). The mean urinary levels of HA in controls did not vary significantly (P < .05), whereas they significantly increased (2.5 to 6.5 folds) in all grades of TCC. More than 80% of patients with grades 2 and 3 TCC had urinary HAase level < 10 µ/mg and over 80% of controls showed HAase level < 10 µ/mg (P < .05). Hyaluronidase levels increased in patients with grades 2 and 3 bladder TCC. CONCLUSION: Measurement of urinary levels of HA and HAase (with 89% sensitivity and 83% specificity) appears to be a highly accurate and non-invasive method for detecting bladder TCC and evaluating its grade.

Direct detection of hyaluronidase in urine using cationic gold nanoparticles: a potential diagnostic test for bladder cancer.

Hyaluronidase (HAase) was reported as a urinary marker of bladder cancer. In this study, a simple colorimetric gold nanoparticle (AuNP) assay was developed for rapid and sensitive detection of urinary HAase activity. Charge interaction between polyanionic hyaluronic acid (HA) and cationic AuNPs stabilized with cetyl trimethyl ammonium bromide (CTAB) led to formation of gold aggregates and a red to blue color shift. HAase digests HA into small fragments preventing the aggregation of cationic AuNPs. The nonspecific aggregation of AuNPs in urine samples was overcome by pre-treatment of samples with the polycationic chitosan that was able to agglomerate all negatively charged interfering moieties before performing the assay. The developed AuNP assay was compared with zymography for qualitative detection of urinary HAase activity in 40 bladder carcinoma patients, 11 benign bladder lesions patients and 15 normal individuals, the assay sensitivity was 82.5% vs. 65% for zymography, while the specificity for both assays was 96.1%. The absorption ratio, A530/A620 of the reacted AuNP solution was used to quantify the HAase activity. The best cut off value was 93.5 µU/ng protein, at which the sensitivity was 90% and the specificity was 80.8%.The developed colorimetric AuNP HAase assay is simple, inexpensive, and can aid noninvasive diagnosis of bladder cancer.

[Characteristic of virulence potential of clinical isolates of enterococci].

AIM: Determination of virulence of enterococci strains isolated from clinical material from humans on pheno- and genotype levels. MATERIALS AND METHODS: 30 strains of enterococci isolated from wound exudate, urine, newborn skin lavage were used in the study. Strain identification was carried out by multiplex PCR. Hemolytic activity was determined by dish method, gelatinase - by dissolution of gelatin column, proteolytic--by biuret method; genes coding virulence factor synthesis (gelE, sprE, cylM, cylB, cylA, cylLs, cylL1, ESP, HYL, ASA)--by using PCR. RESULTS: Clinical isolates of enterococci were assigned to E. faecalis and E. faecium species. Virulence factors on phenotype and genotype levels were detected in both species. CONCLUSION: Genetic determinants of virulence are more widespread among clinical isolates of E.faecalis species. Set of genes coding virulence factors in E. faecalis depends on biotope. Gene coding hyaluronidase synthesis is characteristic for E. faecium. A correlation between phenotypic manifestation of features and enterococci genotype was detected.

FRET based ratio-metric sensing of hyaluronidase in synthetic urine as a biomarker for bladder and prostate cancer.

Elevated hyaluronidase levels are found in the urine of bladder and prostate cancer patients. Therefore, HA-ase is regarded as an important biomarker for the detection of these cancers. In this report, we use a FRET based ratiometric sensing approach to detect the level of HA-ase in synthetic urine. For this, we have used a HA-FRET probe (hyaluronan) labeled with fluorescein as a donor and rhodamine as an acceptor. We monitor the digestion of our HA-FRET probe with different concentrations of HA-ase in synthetic urine via fluorescence emission. The extent to which FRET is released depends on the concentration of HA-ase. Our fluorescence intensity results are also supported with time resolved fluorescence decay data. This assay can be used to develop a non-invasive technique for the detection of bladder and/or prostate cancer progression.

Detection of hyaluronidase RNA and activity in urine of schistosomal and non-schistosomal bladder cancer.

Diagnosis of bladder cancer is done by cystoscopy and cytology. In the last decade, many urine-based tests for bladder cancer have been developed and tested in different populations. Hence, it was relevant to assess the diagnostic significance of urinary hyaluronidase RNA and its enzyme activity in bladder cancer. Seventy patients with bladder cancer, 56 patients with benign bladder lesions, and 49 healthy controls were enrolled in this study. Voided urine samples from all subjects were used for estimation of urinary HAase RNA by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and determination of its enzymatic activity by zymography. There was a significant difference in the mean ranks and positivity rates of HAase RNA expression (P < 0.01) and its enzymatic activity among the three investigated groups: malignant, benign, and normal (P < 0.01). In detecting bladder cancer, the sensitivity of urine cytology (42.83 %) was improved to 100 % when combined with urinary Hyal RNA or Hyal enzyme activity. Detection of urinary Hyal RNA and its enzyme activity is promising noninvasive tests with high sensitivities and specificities for detection of bladder cancer.

Urinary retinoic acid receptor-ß2 gene promoter methylation and hyaluronidase activity as noninvasive tests for diagnosis of bladder cancer.

OBJECTIVES: We evaluated the significance of urinary retinoic acid receptor-ß2 (RAR-ß2) gene promoter methylation and hyaluronidase activity in comparison with voided urine cytology (VUC) in diagnosis of bladder cancer. DESIGN AND METHODS: This study included 100 patients diagnosed with bladder cancer, 65 patients with benign urological disorders and 51 healthy volunteers. Urine supernatant was used for determining hyaluronidase activity by zymography while urine sediment was used for cytology and detection of methylated RAR-ß2 gene promoter by methylation specific nested PCR. RESULTS: The sensitivity and specificity were 53% and 90.5% for VUC, 65% and 89.7% for percent methylation fraction of RAR-ß2 gene promoter, and 89% and 90.5% for hyaluronidase activity; combination of the three parameters increased sensitivity to 95%. A significant association was observed between investigated markers and advanced grade tumor. CONCLUSIONS: Combined use of RAR-ß2 gene promoter methylation, hyaluronidase activity and VUC is promising non-invasive tool for bladder cancer detection.

Angiotensin converting enzyme inhibition blocks interstitial hyaluronan dissipation in the neonatal rat kidney via hyaluronan synthase 2 and hyaluronidase 1.

A functional renin-angiotensin system (RAS) is required for normal kidney development. Neonatal inhibition of the RAS in rats results in long-term pathological renal phenotype and causes hyaluronan (HA), which is involved in morphogenesis and inflammation, to accumulate. To elucidate the mechanisms, intrarenal HA content was followed during neonatal completion of nephrogenesis with or without angiotensin converting enzyme inhibition (ACEI) together with mRNA expression of hyaluronan synthases (HAS), hyaluronidases (Hyal), urinary hyaluronidase activity and cortical lymphatic vessels, which facilitate the drainage of HA from the tissue. In 6-8days old control rats cortical HA content was high and reduced by 93% on days 10-21, reaching adult low levels. Medullary HA content was high on days 6-8 and then reduced by 85% to 12-fold above cortical levels at day 21. In neonatally ACEI-treated rats the reduction in HA was abolished. Temporal expression of HAS2 corresponded with the reduction in HA content in the normal kidney. In ACEI-treated animals cortical HAS2 remained twice the expression of controls. Medullary Hyal1 increased in controls but decreased in ACEI-treated animals. Urine hyaluronidase activity decreased with time in control animals while in ACEI-treated animals it was initially 50% lower and did not change over time. Cells expressing the lymphatic endothelial mucoprotein podoplanin in ACEI-treated animals were increased 18-fold compared to controls suggesting compensation. In conclusion, the high renal HA content is rapidly reduced due to reduced HAS2 and increased Hyal1 mRNA expressions. Normal angiotensin II function is crucial for inducing these changes. Due to the extreme water-attracting and pro-inflammatory properties of HA, accumulation in the neonatally ACEI-treated kidneys may partly explain the pathological renal phenotype of the adult kidney, which include reduced urinary concentration ability and tubulointerstitial inflammation.