Combur test

Urine samples were collected by cystocentesis, guided by ultrasonography, or urethral catheterization. Samples were kept refrigerated and stored within 12 h after collection for AA measurements. Urinary density was established by refractometry, and physicochemical characteristics of urine were measured with reagent tapes (Combur-Test^®, Roche, Basel, Switzerland). For blood collection, animals underwent a 12 h fasting period, and venous blood samples were collected and placed in a coagulation activator tube (BD Vacutainer^® SST ™ Advance, BD, Franklin Lakes, NJ, USA). The samples were centrifuged, and serum was obtained afterward. Amounts of 0.5 mL of serum and 1.0 mL of urine were stored at −80 °C until analysis. Samples were analyzed by high-performance liquid chromatography (HPLC) (Agilent 1200 Series, Santa Clara, CA, USA) on LUNA C18 100Å 5 u 250 × 4.6 mm 00G-4252-EQ column for determination of the total AA. Free AAs were analyzed on Luna 3u C18 (2) 100A 250 × 4.6 mm HPLC column 00G-4251-E0, based on the technique of White et al., 1986 [41 (link)]. The reading was performed at a wavelength of 254 nm. Diet AA profile was also determined by HPLC, and previous acid hydrolysis by the use of hydrochloric acid 6N has been performed for 24 h. All analyzes were performed in duplicate.

Blood samples were withdrawn by means of tail artery prick method to measure BG level (mmol/L) using glucometer (Accu-Check Performa, Roche). Body weight (g) of rats was measured gravimetrically using analytical balance. Urine sugar levels were detected by Combur test from Roche Diagnostics. HbA1c (%) levels were determined by using Cobas Integra 800 analyser based on Turbidimetric Inhibition Immunoassay (TINIA) method. Lipid, renal function and kidney function profiles were determined by using Architect (Abbott brand, Ci8000) analyser based on enzymatic photometric method.

As a measure for schistosome-induced pathology, haematuria was determined in urine samples collected at baseline and 2 years following treatment with 40 mg/kg praziquantel. A Combur-Test (Roche Diagnostics GmbH, Mannheim, Germany) reagent strip was used to detect the presence of blood in the urine.

The first urine sample in the morning and the first urine sample after SC were taken from all the subjects. They were collected in polyethylene tubes previously washed with diluted nitric acid and frozen at −80 °C until analyzed, and once the container was handed over, it was measured and codified. Prior to analyses, samples were thawed and homogenized by shaking.
A 10 mL quantity was used to obtain the different evaluated parameters. Specific gravity was analyzed in situ with a precalibrated refractometer (URC-Ne, Atago, Japan), as previously described [32 (link)]. Biochemical variables (pH, microalbuminuria (MA), erythrocytes) were measured by placing a reagent strip (Combur Test, Roche, Spain) in a small portion of urine samples. Next, the strip was placed inside an automatic reflection photometer (Urisys 1100, Roche, Spain) to measure the parameters after a 1 min incubation time.

We used the strain WT31 [12 (link)]x LU898 [13 (link)] which was kindly provided by W. Marwan. Microplasmodia were grown in liquid shaking culture as described in [14 ]. After 6 days, the slime molds have depleted the medium of glucose, which was confirmed by a glucose monitoring strip test (Combur-Test, Roche). The microplasmodia were then taken out of the liquid culture and plated onto an agar plate containing glucose-deficient SDM-agar (prepared after [15 ]). Once on the agar surface, microplasmodia form aggregates and fuse. At ∼ 3 hours after plating, small plasmodia begin to leave the boundaries of the patch and move outwards in a star-shaped fashion. We call those autonomous, steadily migrating units mesoplasmodia. After approximately 10 hours, mesoplasmodia cease their straight, outward migration and transition into stationary networks in a topological transition creating numerous holes and handles. Directional persistence is evident for approximately 7 hours. All mesoplasmodia were observed and recorded within this seven-hour interval.

Mice were maintained on 12 h light : dark cycles at a temperature of 22 °C with a humidity of 55%, and fed a standard diet. Meg^lox/lox and Nph2^lox/lox mice were crossed with tamoxifen‐inducible UBC‐cre/ERT2 transgenic mice (The Jackson Laboratory, Bar Harbor, ME, USA) [27 (link)]. Tail DNA was analyzed by PCR for genotyping. All mice were of a mixed C57BL/6‐129/Svj background. At age 8 to 12 weeks, female inducible megalin knock out (KO) (Meg^lox/lox; Cre⁺), podocin KO (Nph2^lox/lox; Cre⁺) and littermate control mice (Meg^lox/lox, Nph2^lox/lox; Cre⁻), were induced by i.p. injection of tamoxifen (Cat.No.: T5648, Sigma‐Aldrich) at a dose of 50 mg·kg⁻¹ body weight for 5 consecutive days. The knockdown of the target genes in the kidney was assessed by quantitative real‐time PCR at termination, 4 weeks after the first day of tamoxifen induction. Mice with less than 70% megalin or podocin gene knock out were excluded. Mouse breeding and experiments were carried out in a certified animal facility according to provisions from the Danish Animal Experiments Inspectorate (2020‐15‐0201‐00400). Urine was collected at week 3 by housing the mice in metabolic cages for 24 h, and the urinary protein excretion was estimated by urine dipstick test (Roche Combur‐Test).