Differently, was expressed equally in all groups. patients had a serum creatinine less than 1.1 mg/dl, and daily urinary protein excretion less than 0.150 g/24 h. The measure of urine creatinine was 1.29 0.82 and 1.42 0.55 g/L in women and men, respectively. Hematuria, proteinuria, and bacteriuria were assessed by dipstick urinalysis and, additionally, the presence of bacteria and yeasts was further excluded by microscopy inspection. 2.2. Patients The cohort of patients considered in our study was composed of RTx (= 20) and N (= 18), (Table 1). Table 1 Clinical parameters and characteristics of analyzed patients. RBx: renal biopsy; M: Males; F: Females; Prot-U: urinary protein excretion; UC: urinary creatinine; sCr: serum creatinine; mGFR: measured glomerular filtration rate. for 15 min at 4 degrees, as previously described [23], to obtain a pellet containing cells originating from kidney and bladder but lacking in subcellular particles. 2.4. Biochemical Analysis Biochemical analyses for the evaluation of kidney function were performed in the central laboratory of our institution and were measured the day of biopsy. mGFR was measured by means of 24 h urinary collection and by the measurement of creatinine clearance. The study was approved by the Institutional Review Board of Fondazione IRCCS Ca Granda Ospedale Maggiore Policlinico of Milan, protocol code 4759-1837/19 19 November 2019 and conducted according to the guidelines of the Declaration of Helsinki. Informed consent was obtained from all subjects involved in the study. 2.5. Human Immortalized Podocytes Culture Human immortalized podocytes (hPODO) (University of Bristol, Bristol, UK) were used to check primers positivity and to compare the melting curve, as described in the supplementary section. 2.6. Immunofluorescence Staining For immunofluorescence staining, the cells were fixed in cold 4% PFA and/or acetone as appropriate and permeabilized by 0.3% Triton X-100. Dehydroepiandrosterone The detailed process is reported in the supplementary section. To prevent unspecific binding, we used 5% bovine serum albumin for 30 min at room temperature. The following primary antibodies were applied: rabbit anti-podocin (and and and whose expressions were lower than that of because it was too high compared to that of and and activation or inactivation have been proposed to induce opposite mechanisms, leading to cell survival or death, as a form of regulation of homeostasis or apoptosis. We selected genes characteristic of differentiated podocytes: [20,23,26]. In addition, as markers particularly involved in podocyte functionality, we considered and detected normalization as the ratio between two podocyte markers belonging to different cell compartments and further normalized to UC. We chose in representation of the slit diaphragm and as molecules bound to actin cytoskeleton, and in addition, as a nuclear marker. 2.10. Statistical Analysis We expressed RTqPCR data as mean fold SE relative to CTRL, repeating the determination of each sample at least three times. Variables with skewed distribution were transformed in their base log10 + 1 to obtain normal distribution. To compare data, we used the unequal variance two-tailed Students 0.05, ** 0.01, or *** 0.001 was used. 3. Results Dehydroepiandrosterone 3.1. Cell Morphology and Growth To ascertain if live cells were present in the urine, the urine pellet Dehydroepiandrosterone was maintained in culture; the presence of cells able to adhere was demonstrated by optical microscopy inspection day-by-day for the time of observation. After about two or three days, we noted cell adhesion and we could identify some podocytes [28,29] Rabbit Polyclonal to ADCK5 showing a large cytoplasm with first and secondary processes or long processes adhering to each other and with double nucleus in the same cytoplasm (Supplementary Figure S1: urinary cells culture). We obtained podocyte cultures from both N and RTx urine, but not from CTRL, probably because of the low number along with the absence of proliferative capacity that characterized these terminally differentiated cells that were also described as senescent in healthy individuals. The cells identified as podocytes did not proliferate over.
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