Acute Kidney Injury and BK Polyomavirus in Urine Sediment Cells.

Polyomaviruses are widespread, with BK viruses being most common in humans who require immunosuppression due to allotransplantation. Infection with BK polyomavirus (BKV) may manifest as BK virus-associated nephropathy and hemorrhagic cystitis. Established diagnostic methods include the detection of polyomavirus in urine and blood by PCR and in tissue biopsies via immunohistochemistry. In this study, 79 patients with pathological renal retention parameters and acute kidney injury (AKI) were screened for BK polyomavirus replication by RNA extraction, reverse transcription, and virus-specific qPCR in urine sediment cells. A short fragment of the VP2 coding region was the target of qPCR amplification; patients with (n = 31) and without (n = 48) a history of renal transplantation were included. Urine sediment cell immunofluorescence staining for VP1 BK polyomavirus protein was performed using confocal microscopy. In 22 patients with acute renal injury, urinary sediment cells from 11 participants with kidney transplantation (KTX) and from 11 non-kidney transplanted patients (nonKTX) were positive for BK virus replication. BK virus copies were found more frequently in patients with AKI stage III (n = 14). Higher copy numbers were detected in KTX patients having experienced BK polyoma-nephropathy (BKPyVAN) in the past or diagnosed recently by histology (5.6 × 109-3.1 × 1010). One patient developed BK viremia following delayed graft function (DGF) with BK virus-positive urine sediment. In nonKTX patients with BK copies, decoy cells were absent; however, positive staining of cells was found with epithelial morphology. Decoy cells were only found in KTX patients with BKPyVAN. In AKI, damage to the tubular epithelium itself may render the epithelial cells more permissive for polyoma replication. This non-invasive diagnostic approach to assess BK polyomavirus replication in urine sediment cells has the potential to identify KTX patients at risk for viremia and BKPyVAN during AKI. This method might serve as a valuable screening tool for close monitoring and tailored immunosuppression decisions.

Cytomegalovirus in urinary sediment in patients with acute kidney injury.

BACKGROUND: Immunosuppression in solid organ transplantation is associated with frequent infections. Renal allograft recipients are susceptible to opportunistic infections and can acquire human cytomegalovirus (HCMV) infections even within the allograft. There, HCMV can be found in both the glomerulus and tubular cells, but is mostly restricted to specific and circumscribed sites. Therefore, not all organ infections are identifiable by immunohistology for HCMV proteins in fine needle core biopsies. Thus, we performed a urinalysis study to search for HCMV-specific RNA transcripts in the urine sediment of patients with acute kidney injury. METHODS: Urinary sediment of 90 patients with acute kidney injury (AKI), including 48 renal transplant recipients (RTX) and 42 non-transplant recipients (nRTX), was collected from morning urine for RNA extraction and reverse transcription. The copy number of HCMV transcripts was evaluated using a UL132 HCMV-specific probe set and by real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: Of the 48 RTX patients, ten showed HCMV copies in their urine sediment cells. Within this group, three recipients had negative HCMV serology and received an allograft from an HCMV-seropositive donor. In addition, all three RTX patients on a belatacept-based immunosuppressive regimen had HCMV transcripts in their urine. Of the 42 nRTX patients, only two had detectable HCMV transcripts in urine sediment cells and both were under immunosuppression. CONCLUSIONS: Ten immunosuppressed renal allograft recipients and two immunosuppressed non-transplant patients with AKI showed HCMV copies in urine sediment. Thus, HCMV positivity in urinary sediment appears to be associated with immunosuppression. This study describes a novel noninvasive method for detection of HCMV in urinary sediment. Whether all HCMV infections can be detected or only those with viral replication warrants further investigation.

A Novel Inside-out Access Approach for Hemodialysis Catheter Placement in Patients With Thoracic Central Venous Occlusion.

RATIONALE & OBJECTIVE: Left-sided internal jugular and all subclavian central venous catheters (CVCs) cause thoracic central vein occlusions (TCVOs) more often than right-sided internal jugular catheters. To enable right-sided CVC placement in patients with TCVO, an inside-out access (IOA) approach was established at 3 vascular access centers in Europe involving use of a novel IOA device advanced from the right femoral vein. In the current analysis, we assessed the eligibility and success rate of this IOA approach in a cohort of patients with TCVO requiring a tunneled dialysis catheter. STUDY DESIGN: Retrospective multicenter observational study. SETTING & PARTICIPANTS: 36 patients with TCVO treated in Vienna, Austria; Oxford, England; or Cologne, Germany, who required hemodialysis access between July 2016 and June 2018. EXPOSURE: Application of the IOA approach to gain vascular access. OUTCOME: The primary end point was the success rate of passing the TCVO to gain dialysis access using the IOA approach. Secondary end points were catheter patency at 3 months and procedure-related complications (early infections, bleeding, hematoma, and pericardial effusions). ANALYTICAL APPROACH: Descriptive statistics to characterize eligibility, success rate, and complications of the IOA approach. RESULTS: 36 patients with TCVO and history of multiple CVCs and arteriovenous fistulas were referred to the participating centers for vascular access. 32 (89%) patients were eligible for the IOA approach. 39 treatments were performed, with 7 patients undergoing the IOA procedure a second time more than 3 months after initial CVC placement. Dialysis access was established successfully in 38 of 39 (97%) implementations of the IOA procedure. Median intervention time was 43 minutes. No complications occurred. LIMITATIONS: No comparison to other methods to place CVCs and the observational study design. CONCLUSIONS: The IOA approach is a promising method to enable rapid access to the right jugular vein in the setting of pre-existing TCVO. Additional experience is needed to understand the generalizability of these observations.

Correction to: The urinary microbiome shows different bacterial genera in renal transplant recipients and non-transplant patients at time of acute kidney injury - a pilot study.

An amendment to this paper has been published and can be accessed via the original article.

The urinary microbiome shows different bacterial genera in renal transplant recipients and non-transplant patients at time of acute kidney injury - a pilot study.

BACKGROUND: In the past urine was considered sterile. Through the introduction of next generation sequencing, it has become clear that a urinary microbiome exists. Acute kidney injury (AKI) represents a major threat to kidney transplant recipients. Remarkable changes in the urinary metabolome occur during AKI, which may influence the urinary microbiome. To our knowledge, this is the first study that examines the urinary microbiome in renal transplant recipients (RTX) and non-transplant recipients (nRTX) at time of AKI. METHODS: In this cross-sectional pilot-study the urinary microbiome of 21 RTX and 9 nRTX with AKI was examined. Clean catch morning urine samples were obtained from all patients on the first day of AKI diagnosis. AKI was defined according to KDIGO guidelines. Urinary microbiota and the urinary metabolome during AKI were assessed in one patient. 16S rRNA sequencing was performed. Sequences were processed using UPARSE-pipeline for operational taxonomic units (OTU) and taxon finding. RESULTS: We successfully extracted and sequenced bacterial DNA from 100% of the urine samples. All 30 patients revealed at least 106,138 reads. 319 OTU and 211 different genera were identified. The microbiotic diversity richness in the RTX group was no different from the nRTX group. Eighteen genera were solely present in nRTX and 7 in RTX. CONCLUSIONS: The urinary microbiome at time of AKI showed different bacterial genera in RTX compared to nRTX. The nRTX group exhibited no different diversity to the RTX group. Irrespective of the status of a previous renal transplantation, the urinary microbiome comprised > 210 different genera. An intraindividual change in microbiota diversity and richness was observed in one study patient during recovery from AKI.

Association of Body Mass Index and Outcome in Acutely Ill Patients With Chronic Kidney Disease Requiring Intensive Care Therapy.

OBJECTIVE: An association of body mass index (BMI) and outcome, the "obesity paradox," has been described in patients with chronic kidney disease (CKD) and end-stage renal disease. We sought to assess whether a potential beneficial effect of a high body mass is also seen in CKD patients with critical illness. METHODS: In a retrospective analysis of a prospectively collected database of 123,416 patients from 107 Austrian intensive care units (ICUs) in whom BMI was available, the association of 6 groups of BMI and hospital mortality was assessed in 12,206 patients with CKD 3-5 by univariate and multivariate logistic regression analyses. RESULTS: Patients with CKD were sicker, had a longer ICU stay, and had a higher ICU and hospital mortality than those without. The association of BMI and outcome in CKD patients indicated a U-shaped curve with the highest mortality in patients with BMI <20 and ≥40, and the lowest with a BMI between ≥25 and <40. This relationship was also significant in a multivariate analysis adjusted for severity of illness assessed by Simplified Acute Physiology Score III score, age, gender, admission diagnosis, and pre-existing comorbidities. It was not found in patients with CKD 5 on renal replacement therapy, in patients below 60 years of age, and those with diabetes mellitus requiring insulin treatment. CONCLUSIONS: BMI is associated with better outcomes in CKD 3-5 patients who have acquired acute intermittent diseases and are admitted to an ICU, but not those requiring renal replacement therapy. This higher tolerance to acute disease processes may in part explain the "obesity paradox" observed in CKD patients.

An integrative approach for the assessment of peritubular capillaritis extent and score in low-grade microvascular inflammation-associations with transplant glomerulopathy and graft loss.

Background: Peritubular capillaritis (ptc), reported by the ptc score, is a major feature of kidney allograft rejection and microvascular inflammation (MVI). MVI sum scores (ptc + glomerulitis score ≥2) are accepted diagnostic surrogates of human leucocyte antigen (HLA)-antibody interaction. However, low-grade inflammation is common and ptc scores (number of leucocytes/capillary) may not mirror all aspects of ptc morphology. Recently we observed a relationship of the diffuse extent of ptc (inflammation of >50% of the renal cortex) with graft loss and significantly higher donor-specific antibody levels, suggesting potential inclusion of diffuse ptc as an additional surrogate of antibody-antigen interaction. Methods: We sought to assess how a combination of ptc score and extent in low-grade inflammation (ptc1) affects transplant glomerulopathy (TG) and graft loss risk. Patients (n = 616) were assessed for MVI in first indication biopsies. Cases with a ptc score of 1 but diffuse extent (ptc1diffuse, g-score = 0, n = 26) were considered additional surrogates of HLA-antibody interaction and compared with MVI ≥2 and MVI <2. Results: The ptc1diffuse and MVI score ≥2 subjects had worse graft survival (42% and 59%) compared with an MVI score <2 (70%) (P = 0.002). The incorporation of ptc1diffuse in the MVI score ≥2 increased the receiver operating characteristics curve for TG [area under the curve (AUC) 0.602; P = 0.008] compared with a Banff MVI score ≥2 (AUC 0.56; P = 0.12); cases with baseline TG were excluded. In multivariate analysis, ptc1diffuse remained independently related to TG (odds ratio 3.89; P = 0.008) and graft loss (hazard ratio 2.64; P = 0.001) even after inclusion of all rejection episodes. Conclusion: An integrated view of ptc morphology including diffuse ptc in MVI is superior for TG and graft loss risk assessment.

Urinary nephrospheres indicate recovery from acute kidney injury in renal allograft recipients - a pilot study.

BACKGROUND: Acute kidney injury represents a major threat to the transplanted kidney. Nevertheless, these kidneys have the potential to fully recover. Tubular regeneration following acute kidney injury is driven by the regenerative potential of tubular cells originating from a tubular stem cell pool. We investigated urinary sediments of acute kidney injury transplanted patients and compared it to those of non-transplanted patients. Thereby we discovered tubular cell agglomerates, which have not been described in vivo. We hypothesized that these so-called nephrospheres were associated with recovery from acute kidney injury. METHODS: Urine sediment of 45 kidney-transplanted and 19 non-transplanted individuals was investigated. Nephrospheres were isolated and stained for several molecular markers including aquaporin 1 (AQP1) and calcium sensing receptor (CASR). Nephrospheres were cultured to examine their growth behavior in vitro. In addition, quantitative PCR for CASR, AQP1, and podocin (NPHS2) was performed. RESULTS: Nephrospheres were excreted in the urine of 17 kidney-transplant recipients 7 days after onset of acute kidney injury and were detectable over several days until kidney function was recovered to baseline creatinine levels. None were found in the urine of non-transplanted individuals. Nephrospheres were either AQP1+/CASR+ or AQP1-/CASR+ and could be cultured for 27 days. Mitotic cells could still be visualized after 17 days in culture. Quantitative PCR detected AQP1 in both kidney-transplanted and non-transplanted individuals during the phase of creatinine decline. As a limitation qPCR was only performed for the entire urinary sediment. CONCLUSIONS: Nephrospheres are three dimensional tubular cell agglomerates which appeared in urine of kidney transplant recipients recovering from acute kidney injury. Appearance of nephrospheres in urine was independent of the duration after kidney transplantation. Nephrospheres proliferated in cell culture and kept expressing kidney specific marker. Presence of nephrospheres in urine showed a specificity of 100% and a sensitivity of 60.71% for recovery.

Taurolidine-based catheter lock regimen significantly reduces overall costs, infection, and dysfunction rates of tunneled hemodialysis catheters.

Catheter-related infections and dysfunction are the main catheter complications causing morbidity and mortality in hemodialysis patients. However, there are no consistent data for the choice of catheter lock solutions for tunneled hemodialysis lines. In this prospective, multicenter, randomized, controlled trial, two lock regimens using three commercial catheter lock solutions were compared in 106 hemodialysis patients with a newly inserted tunneled central catheter. In the taurolidine group, TauroLock™-Hep500 was used twice per week and TauroLock™-U25,000 once a week. In the citrate group, a four percent citrate solution was used after each dialysis. Both groups were compared regarding catheter-related infections, catheter dysfunction, and costs. Over a period of 15,690 catheter days, six catheter-related infections occurred in six of 52 patients in the taurolidine group, but 18 occurred in 13 of 54 patients in the citrate group, corresponding to 0.67 and 2.7 episodes of catheter-related infections per 1000 catheter days, respectively (Incidence Rate Ratio 0.25, 95% confidence interval, 0.09 to 0.63). Catheter dysfunction rates were significantly lower in the taurolidine group (18.7 vs. 44.3/1000 catheter days) and alteplase rescue significantly more frequent in the citrate group (9.8 vs. 3.8/1000 catheter days). These differences provided significant catheter-related cost savings of 43% in the taurolidine group vs. citrate group when overall expenses per patient and year were compared. Thus, use of taurolidine-based catheter lock solutions containing heparin and urokinase significantly reduced complications related to tunneled hemodialysis catheters when compared to four percent citrate solution and was overall more cost-efficient.

A SAGE based approach to human glomerular endothelium: defining the transcriptome, finding a novel molecule and highlighting endothelial diversity.

BACKGROUND: Large scale transcript analysis of human glomerular microvascular endothelial cells (HGMEC) has never been accomplished. We designed this study to define the transcriptome of HGMEC and facilitate a better characterization of these endothelial cells with unique features. Serial analysis of gene expression (SAGE) was used for its unbiased approach to quantitative acquisition of transcripts. RESULTS: We generated a HGMEC SAGE library consisting of 68,987 transcript tags. Then taking advantage of large public databases and advanced bioinformatics we compared the HGMEC SAGE library with a SAGE library of non-cultured ex vivo human glomeruli (44,334 tags) which contained endothelial cells. The 823 tags common to both which would have the potential to be expressed in vivo were subsequently checked against 822,008 tags from 16 non-glomerular endothelial SAGE libraries. This resulted in 268 transcript tags differentially overexpressed in HGMEC compared to non-glomerular endothelia. These tags were filtered using a set of criteria: never before shown in kidney or any type of endothelial cell, absent in all nephron regions except the glomerulus, more highly expressed than statistically expected in HGMEC. Neurogranin, a direct target of thyroid hormone action which had been thought to be brain specific and never shown in endothelial cells before, fulfilled these criteria. Its expression in glomerular endothelium in vitro and in vivo was then verified by real-time-PCR, sequencing and immunohistochemistry. CONCLUSIONS: Our results represent an extensive molecular characterization of HGMEC beyond a mere database, underline the endothelial heterogeneity, and propose neurogranin as a potential link in the kidney-thyroid axis.

Lessons from Polyomavirus Immunofluorescence Staining of Urinary Decoy Cells.

Decoy cells that can be detected in the urine sediment of immunosuppressed patients are often caused by the uncontrolled replication of polyomaviruses, such as BK-Virus (BKV) and John Cunningham (JC)-Virus (JCV), within the upper urinary tract. Due to the wide availability of highly sensitive BKV and JCV PCR, the diagnostic utility of screening for decoy cells in urine as an indicator of polyomavirus-associated nephropathy (PyVAN) has been questioned by some institutions. We hypothesize that specific staining of different infection time-dependent BKV-specific antigens in urine sediment could allow cell-specific mapping of antigen expression during decoy cell development. Urine sediment cells from six kidney transplant recipients (five males, one female) were stained for the presence of the early BKV gene transcript lTag and the major viral capsid protein VP1 using monospecific antibodies, monoclonal antibodies and confocal microscopy. For this purpose, cyto-preparations were prepared and the BK polyoma genotype was determined by sequencing the PCR-amplified coding region of the VP1 protein. lTag staining began at specific sites in the nucleus and spread across the nucleus in a cobweb-like pattern as the size of the nucleus increased. It spread into the cytosol as soon as the nuclear membrane was fragmented or dissolved, as in apoptosis or in the metaphase of the cell cycle. In comparison, we observed that VP1 staining started in the nuclear region and accumulated at the nuclear edge in 6-32% of VP1+ cells. The staining traveled through the cytosol of the proximal tubule cell and reached high intensities at the cytosol before spreading to the surrounding area in the form of exosome-like particles. The spreading virus-containing particles adhered to surrounding cells, including erythrocytes. VP1-positive proximal tubule cells contain apoptotic bodies, with 68-94% of them losing parts of their DNA and exhibiting membrane damage, appearing as "ghost cells" but still VP1+. Specific polyoma staining of urine sediment cells can help determine and enumerate exfoliation of BKV-positive cells based on VP1 staining, which exceeds single-face decoy staining in terms of accuracy. Furthermore, our staining approaches might serve as an early readout in primary diagnostics and for the evaluation of treatment responses in the setting of reduced immunosuppression.

Urinary Collectrin as Promising Biomarker for Acute Kidney Injury in Patients Undergoing Cardiac Surgery.

BACKGROUND: Early detection of acute kidney injury (AKI) is crucial for timely intervention and improved patient outcomes after cardiac surgery. This study aimed to evaluate the potential of urinary collectrin as a novel biomarker for AKI in this patient population. METHODS: In this prospective, observational cohort study, 63 patients undergoing elective cardiac surgery with cardiopulmonary bypass (CPB) were studied at the Medical University of Vienna between 2016 and 2018. We collected urine samples prospectively at four perioperative time points, and urinary collectrin was measured using an enzyme-linked immunosorbent assay. Patients were divided into two groups, AKI and non-AKI, defined by Kidney Disease: Improving Global Outcomes Guidelines, and differences between groups were analyzed. RESULTS: Postoperative AKI was found in 19 (30%) patients. Urine sample analysis revealed an inverse correlation between urinary collectrin and creatinine and AKI stages, as well as significant changes in collectrin levels during the perioperative course. Baseline collectrin levels were 5050 ± 3294 pg/mL, decreased after the start of CPB, reached their nadir at the end of surgery, and began to recover slightly on postoperative day (POD) 1. The most effective timepoint for distinguishing between AKI and non-AKI patients based on collectrin levels was POD 1, with collectrin levels of 2190 ± 3728 pg/mL in AKI patients and 3768 ± 3435 pg/mL in non-AKI patients (p = 0.01). CONCLUSIONS: Urinary collectrin shows promise as a novel biomarker for the early detection of AKI in patients undergoing cardiac surgery on CPB. Its dynamic changes throughout the perioperative period, especially on POD 1, provide valuable insights for timely diagnosis and intervention. Further research and validation studies are needed to confirm its clinical usefulness and potential impact on patient outcomes.

Monitoring of Sotrovimab-Levels as Pre-Exposure Prophylaxis in Kidney Transplant Recipients Not Responding to SARS-CoV-2 Vaccines.

Background Sotrovimab, a monoclonal antibody against SARS-CoV-2, is used as a pre-exposition prophylaxis (PrEP) against COVID-19, but monitoring strategies using routine test systems have not been defined. Methods Twenty kidney transplant recipients without antibodies after vaccination received 500 mg Sotrovimab. Antibody levels were quantified over eight weeks using live-virus neutralization (BA1 and BA2), antibody binding assays (TrimericS, Elecsys, QuantiVAC) and surrogate virus neutralization tests (sVNTs; TECOmedical, cPass and NeutraLISA). Results Sotrovimab neutralized both Omicron subvariants (BA1 NT titer 90 (+-50) > BA2 NT titer 33 (+-15) one hour post infusion). Sotrovimab was measurable on all used immunoassays, although a prior 1:100 dilution was necessary for Elecsys due to a presumed prozone effect. The best correlation with live-virus neutralization titers was found for QuantiVAC and TrimericS, with a respective R2 of 0.65/0.59 and 0.76/0.57 against BA1/BA2. Elecsys showed an R2 of 0.56/0.54 for BA1/BA2, respectively. sVNT values increased after infusion but had only a poor correlation with live-virus neutralization titers (TECOmedical and cPass) or did not reach positivity thresholds (NeutraLISA). Conclusion Antibody measurements by the used immunoassays showed differences in antibody levels and only a limited correlation with neutralization capacity. We do not recommend sVNTs for monitoring SARS-CoV-2 neutralization by Sotrovimab.

Lisinopril pharmacokinetics and erythropoietin requirement in haemodialysis patients.

BACKGROUND: There is ongoing controversy whether angiotensin-converting enzyme inhibitors (ACE-I) contribute to anaemia by causing hyporesponsiveness to erythropoiesis-stimulating agents (ESA). However, it is unknown whether or not plasma levels or area under the curve (AUC) of ACE-I are associated with responsiveness to ESA therapy. MATERIALS AND METHODS: We examined the association between lisinopril AUC, lisinopril plasma levels and ESA requirements that was assessed using an ESA index [(ESA IU/week/body weight kg)/(haemoglobin g/dL)]. After screening 184 haemodialysis patients, 14 fulfilled the inclusion criteria, mainly long-term use of oral lisinopril in the upper end of dosage range for this population with stable haemoglobin levels and intravenous ESA therapy. Lisinopril plasma levels were measured at eight different time points (predialysis, immediate post-dialysis and hourly for 6h thereafter; AUC1), and the seven post-dialysis lisinopril plasma levels were used for calculation of AUC2. RESULTS: The mean ESA index of all patients was 27·90±25·84 (IU/week/kg)/(g/dL). Average lisinopril AUC1 was 1212·48±1209·75 [mg*h/L], whereas AUC2 averaged 947·67±977·07 [mg*h/L]. Two patients (14%) had no detectable lisinopril plasma levels, indicating their noncompliance. There was no association between ESA index and AUC or plasma levels of lisinopril at any time point for all 14 or for the 12 compliant patients. CONCLUSIONS: Our study shows that long-term, high-dose lisinopril therapy has no effect on ESA responsiveness. Thus, avoidance or a dose reduction of ACE-I in dialysis patients will not necessarily lead to reduced ESA requirements and costs.

Rapid T-cell-based immunodiagnosis of tuberculous peritonitis in a peritoneal dialysis patient.

Tuberculous peritonitis is a rare complication during peritoneal dialysis (PD). This report presents the case of a patient with clinical signs and symptoms indicative of bacterial peritonitis, but without culture growth of conventional bacteria or fungi. Cytokine flow cytometry after overnight stimulation of cells from peripheral blood and the peritoneal dialysate with Mycobacterium tuberculosis (MTB)-specific antigens revealed a 40-fold increase in MTB-specific CD4 + T cells expressing interferon-γ (IFN-γ) in peritoneal fluid compared with blood, which was indicative of active tuberculosis (TB). The presence of TB was later confirmed by polymerase chain reaction and growth of MTB in culture of the dialysate. The case illustrates the usefulness of MTB-specific immunodiagnosis for the rapid identification of peritoneal TB in PD patients.

Urinary Collectrin (TMEM27) as Novel Marker for Acute Kidney Injury.

Acute kidney injury (AKI) is a leading complication in hospitalized patients of different disciplines due to various aetiologies and is associated with the risk of chronic kidney disease, the need for dialysis and death. Since nephrons are not supplied with pain signals, kidney injury is mostly diagnosed by serum creatinine with a time delay. Recent work has shown that certain urinary biomarkers are available for early detection of AKI. In total, 155 subjects, including 102 patients with AKI at various stages and 53 subjects without AKI, were enrolled, and their course and laboratory data were recorded. Urinary collectrin (TMEM27) was measured by a commercially available ELISA assay. Changes in serum creatinine were used to determine AKI stage. Patients with AKI presented with significantly lower levels of urinary collectrin compared to patients without AKI (1597 ± 1827 pg/mL vs. 2855 ± 2073; p = 0.001). Collectrin was found to inversely correlate with serum creatinine and stages of AKI. Collectrin levels were lowest in AKI stage III (1576 ± 1686 pg/mL; p = 0.001) and also significantly lower in stage II (1616 ± 2148 pg/mL; p = 0.021) and stage I (1630 ± 1956 pg/mL; p = 0.019) compared to subjects without AKI. An optimal minimum collectrin cut-off value of 1606 [95% CI 1258 to 1954] pg/mL was determined to detect AKI. In conclusion, urinary collectrin represents an indicator of AKI that, unlike all other established AKI biomarkers, decreases with stage of AKI and thus may be associated with a novel pathogenic pathway.

Tubular epithelial progenitors are excreted in urine during recovery from severe acute kidney injury and are able to expand and differentiate in vitro.

Background: Acute kidney injury (AKI) is a serious condition associated with chronic kidney disease, dialysis requirement and a high risk of death. However, there are specialized repair mechanisms for the nephron, and migrated committed progenitor cells are the key players. Previous work has described a positive association between renal recovery and the excretion of tubular progenitor cells in the urine of kidney transplant recipients. The aim of this work was to describe such structures in non-transplanted AKI patients and to focus on their differentiation. Methods: Morning urine was obtained from four patients with AKI stage 3 and need for RRT on a consecutive basis. Urine sediment gene expression was performed to assess which part of the tubular or glomerular segment was affected by injury, along with measurement of neprilysin. Urine output and sediment morphology were monitored, viable hyperplastic tubular epithelial clusters were isolated and characterized by antibody or cultured in vitro. These cells were monitored by phase contrast microscopy, gene, and protein expression over 9 days by qPCR and confocal immunofluorescence. Furthermore, UMOD secretion into the supernatant was quantitatively measured. Results: Urinary neprilysin decreased rapidly with increasing urinary volume in ischemic, toxic, nephritic, and infection-associated AKI, whereas the decrease in sCr required at least 2 weeks. While urine output increased, dead cells were present in the sediment along with debris followed by hyperplastic agglomerates. Monitoring of urine sediment for tubular cell-specific gene transcript levels NPHS2 (podocyte), AQP1 and AQP6 (proximal tubule), and SLC12A1 (distal tubule) by qPCR revealed different components depending on the cause of AKI. Confocal immunofluorescence staining confirmed the presence of intact nephron-specific epithelial cells, some of which appeared in clusters expressing AQP1 and PAX8 and were 53% positive for the stem cell marker PROM1. Isolated tubule epithelial progenitor cells were grown in vitro, expanded, and reached confluence within 5-7 days, while the expression of AQP1 and UMOD increased, whereas PROM1 and Ki67 decreased. This was accompanied by a change in cell morphology from a disproportionately high nuclear/cytoplasmic ratio at day 2-7 with mitotic figures. In contrast, an apoptotic morphology of approximately 30% was found at day 9 with the appearance of multinucleated cells that were associable with different regions of the nephron tubule by marker proteins. At the same time, UMOD was detected in the culture supernatant. Conclusion: During renal recovery, a high replicatory potential of tubular epithelial progenitor cells is found in urine. In vitro expansion and gene expression show differentiation into tubular cells with marker proteins specific for different nephron regions.

Individualization of Mycophenolic Acid Therapy through Pharmacogenetic, Pharmacokinetic and Pharmacodynamic Testing.

Mycophenolic acid (MPA) is a widely used immunosuppressive agent and exerts its effect by inhibiting inosine 5'-monophosphate dehydrogenase (IMPDH), the main regulating enzyme of purine metabolism. However, significant unexplained differences in the efficacy and tolerability of MPA therapy pose a clinical challenge. Therefore, broad pharmacogenetic, pharmacokinetic, and pharmacodynamic approaches are needed to individualize MPA therapy. In this prospective cohort study including 277 renal transplant recipients, IMPDH2 rs11706052 SNP status was assessed by genetic sequencing, and plasma MPA trough levels were determined by HPLC and IMPDH enzyme activity in peripheral blood mononuclear cells (PBMCs) by liquid chromatography-mass spectrometry. Among the 277 patients, 84 were identified with episodes of biopsy-proven rejection (BPR). No association was found between rs11706052 SNP status and graft rejection (OR 1.808, and 95% CI, 0.939 to 3.479; p = 0.076). Furthermore, there was no association between MPA plasma levels and BPR (p = 0.69). However, the patients with graft rejection had a significantly higher predose IMPDH activity in PBMCs compared to the controls without rejection at the time of biopsy (110.1 ± 50.2 vs. 95.2 ± 45.4 pmol/h; p = 0.001), and relative to the baseline IMPDH activity before transplantation (p = 0.042). Our results suggest that individualization of MPA therapy, particularly through pharmacodynamic monitoring of IMPDH activity in PBMCs, has the potential to improve the clinical outcomes of transplant patients.

Preclinical Establishment of a Divalent Vaccine against SARS-CoV-2.

First-generation vaccines against SARS-CoV-2 do not provide adequate immune protection. Therefore, we engineered a divalent gene construct combining the receptor-binding domain (RBD) of the spike protein and the immunodominant region of the viral nucleocapsid. This fusion protein was produced in either E. coli or a recombinant baculovirus system. Subsequently, the fusion protein was mixed with adjuvant and administered to mice in a prime-booster mode. Mice (72%) produced an IgG response against both proteins (titer: 10-4-10-5) 14 days after the first booster injection, which was increased to 100% by a second booster. Comparable IgG responses were detected against the delta, gamma and omicron variants of the RBD region. Durability testing revealed IgGs beyond 90 days. In addition, cytolytic effector cell molecules were increased in lymphocytes isolated from peripheral blood. Ex vivo stimulation of T cells by nucleocapsid and RBD peptides showed antigen-specific upregulation of CD44 among the CD4+ and CD8+ T cells of vaccinated mice. No side effect was documented in the central nervous system. Cumulatively, these data represent a proof-of-principle approach alternative to existing mRNA vaccination strategies.