ABSTRACT
The complement system, and specifically C5a, is involved in renal ischemia‐reperfusion (IR) injury. The 2 receptors for complement anaphylatoxin C5a (C5aR1 and C5aR2) are expressed on ...leukocytes as well as on renal epithelium. Extensive evidence shows that C5aR1 inhibition protects kidneys from IR injury; however, the role of C5aR2 in IR injury is less clear as initial studies proposed the hypothesis that C5aR2 functions as a decoy receptor. By Using wild‐type, C5aR1‐/‐, and C5aR2‐/‐ mice in a model of renal IR injury, we found that a deficiency of either of these receptors protected mice from renal IR injury. Surprisingly, C5aR2‐/‐ mice were most protected and had lower creatinine levels and reduced acute tubular necrosis. Next, an in vivo migration study demonstrated that leukocyte chemotaxis was unaffected in C5aR2‐/‐ mice, whereas neutrophil activation was reduced by C5aR2 deficiency. To further investigate the contribution of renal cell‐expressed C5aR2 vs. leukocyte‐expressed C5aR2 to renal IR injury, bone marrow chimeras were created. Our data show that both renal cell‐expressed C5aR2 and leukocyte‐expressed C5aR2 mediate IR‐induced renal dysfunction. These studies reveal the importance of C5aR2 in renal IR injury. They further show that C5aR2 is a functional receptor, rather than a decoy receptor, and may provide a new target for intervention.—Poppelaars, F., van Werkhoven, M. B., Kotimaa, J., Veldhuis, Z. J., Ausema, A., Broeren, S. G. M., Damman, J., Hempel, J. C., Leuvenink, H. G. D., Daha, M. R., van Son, W. J., van Kooten, C., van Os, R. P., Hillebrands, J.‐L., Seelen, M. A. Critical role for complement receptor C5aR2 in the pathogenesis of renal ischemia‐reperfusion injury. FASEB J. 31, 3193–3204 (2017). www.fasebj.org
Abstract SET domain-containing 2 ( SETD2 ) is responsible for the trimethylation of histone H3 lysine36 (H3K36me3) and is one of the genes most frequently mutated in clear cell renal cell carcinoma ...(ccRCC). It is located at 3p21, one copy of which is lost in the majority of ccRCC tumors, suggesting that SETD2 might function as a tumor suppressor gene. However, the manner in which loss of SETD2 contributes to ccRCC development has not been studied in renal primary tubular epithelial cells (PTECs). Therefore, we studied the consequences of SETD2 knockdown through lentiviral shRNA in human PTECs. Consistent with its known function, SETD2 knockdown (SETD-KD) led to loss of H3K36me3 in PTECs. In contrast to SETD2 wild-type PTECs, which have a limited proliferation capacity; the SETD2-KD PTECs continued to proliferate. The expression profiles of SETD2-KD PTECs showed a large overlap with the expression profile of early-passage, proliferating PTECs, whereas nonproliferating PTECs showed a significantly different expression profile. Gene set enrichment analysis revealed a significant enrichment of E2F targets in SETD2-KD and proliferating PTECs as compared with nonproliferating PTECs and in proliferating PTEC compared with SETD2-KD. The SETD2-KD PTECs maintained low expression of CDKN2A and high expression of E2F1 , whereas their levels changed with continuing passages in untreated PTECs. In contrast to the nonproliferating PTECs, SETD2-KD PTECs showed no β-galactosidase staining, confirming the protection against senescence. Our results indicate that SETD2 inactivation enables PTECs to bypass the senescence barrier, facilitating a malignant transformation toward ccRCC.
The complement system is an essential component of our innate immunity, both for the protection against infections and for proper handling of dying cells. However, the complement system can also ...contribute to tissue injury and inflammatory responses. In view of novel therapeutic possibilities, there is an increasing interest in measurement of the complement system activation in the systemic compartment, both in the clinical setting as well as in experimental models. Here we describe in parallel a sensitive and specific sandwich ELISA detecting mouse C3 activation fragments C3b/C3c/iC3b, as well as functional complement ELISAs detecting specific activities of the three complement pathways at the level of C3 and at the level of C9 activation. In a murine model of renal ischaemia/reperfusion injury (IRI) we found transient complement activation as shown by generation of C3b/C3c/iC3b fragments at 24h following reperfusion, which returned to base-line at 3 and 7days post reperfusion. When the pathway specific complement activities were measured at the level of C3 activation, we found no significant reduction in any of the pathways. However, the functional complement activity of all three pathways was significantly reduced when measured at the level of C9, with the strongest reduction being observed in the alternative pathway. For all three pathways there was a strong correlation between the amount of C3 fragments and the reduction in functional complement activity. Moreover, at 24h both C3 fragments and the functional complement activities showed a correlation with the rise in serum creatinine. Together our results show that determination of the systemic pathway specific complement activity is feasible in experimental mouse models and that they are useful in understanding complement activation and inhibition in vivo.
OBJECTIVE:Variations in the HIV-1 gp120 Env variable loop sequences correlate with virus phenotypes associated with transmission and/or disease progression. We aimed to identify whether signature ...sequences could be identified in the gp120 Env between acute infection and chronic infection viruses obtained from a group of individuals infected with closely related viruses.
METHODS:To analyse acute infection versus chronic infection viruses, we studied a transmission cluster of 11 individuals, in which six presented during acute infection and five during chronic infection. Multiple HIV-1 gp120 Env clones were sequenced from each patient with predicted amino acid sequences compared between the groups.
RESULTS:Cluster analysis of V1V5 Env sequences (n = 215) identified that acute infection viruses had lower potential N-linked glycosylation site (PNGS) densities than viruses from chronic infection, with a higher amino acid length/PNGS ratio. We found a negative correlation between the V1V2 and V4V5 regions for both amino acid length (Pearson P < 0.01) and PNGS numbers (Pearson P < 0.01) during HIV-1 transmission. This association was lost following seroconversion. These findings were confirmed by analysing sequences from the Los Alamos database that were selected and grouped according to timing of transmission. This included acute infection sequences collected 0–10 days (n = 400) and chronic infection sequences 0.5–3 years postseroconversion (n = 394).
CONCLUSION:Our observations are consistent with a structural association between the V1V2 and V4V5 gp120 regions that is lost following viral transmission. These structural considerations should be taken into consideration when devising HIV-1 immunogens aimed at inducing protective antibody responses targeting transmitted viruses.
•TLR2 and TLR4 expression dynamics in several human post-transplant injury biopsies.•Unique opportunity to stain healthy human living donor biopsies for TLR2 and TLR4.•Human acute rejection biopsies ...were characterized by TLR4+ infiltrating cells.•A tubular staining was found in mouse and rat but not in human biopsies.•Results from this study have an impact on future TLR inhibitor studies.
Activation of the innate immunity through Toll-like receptors (TLRs) has been postulated to play an important role in the pathophysiology of renal allograft dysfunction. TLR2 and TLR4 dynamics in different human post-transplant pathological entities has never been studied. Therefore, we evaluated pre- and post-transplantation protein expression of TLR2 and TLR4 in human kidney biopsies.
Human kidney biopsies obtained from living kidney donors and patients with acute tubular necrosis, acute cellular and vascular rejection and interstitial fibrosis/tubular atrophy (IF/TA) were used. Translating results from animal studies to the clinical situation is highly important considering the upcoming clinical studies with TLR inhibitors in human renal transplantation. Hence, the TLR2 and TLR4 expression in healthy mouse and rat kidneys was analyzed and compared with human kidneys. In healthy human kidneys, TLR2 is expressed on the endothelium and Bowman's capsule, while TLR4 is expressed on the endothelium only. No tubular staining was found for both receptors in human kidneys. In contrast to human biopsies, TLR2 and TLR4 expression in rodents was observed on tubular epithelial cells. In all acute rejection human biopsies, increased infiltration of TLR4+ leukocytes was observed. In conclusion, a discrepancy exists between human and rodent renal TLR expression, which suggests careful attention when translating results from rodent studies to the human situation. Additionally, this study revealed human TLR2 and TLR4 expression dynamics in human biopsies pre- and post-transplantation.
► Both C5aR and C5L2 expression are expressed in the human kidney. ► Renal C5aR and C5L2 are expressed in the distal tubule, but in different cell types. ► C5aR is de novo expressed in proximal ...tubuli under inflammatory conditions. ► Distal tubular C5aR and C5L2 are persistently expressed after renal transplantation. ► Distinct expression of C5aR and C5L2 implicates a functional role for C5L2.
The complement system, and especially C5a, plays an important role in the pathophysiology of renal diseases and post-transplant renal injury. The two receptors for C5a are C5a receptor (C5aR) and C5a-like-receptor-2 (C5L2). Only renal C5aR expression has been reported, although exact localization and alterations in expression after transplantation are unknown.
Renal C5aR and C5L2 expression and localization were analyzed immunohistochemically. C5aR and C5L2 expression was analyzed in human kidney biopsies obtained from living donors and patients suffering from acute tubular necrosis, acute cellular and vascular rejection or IF/TA.
C5aR was expressed in the thick ascending limb of Henle's loop and first part of the distal convoluted tubule (DCT). Under inflammatory conditions, C5aR was de novo expressed in proximal tubuli. C5L2 was expressed in the kidney and localized to DCT1, DCT2 and connecting tubule. Persistent distal tubular expression of both receptors was demonstrated after renal transplantation.
This study shows distinct renal expression patterns for C5aR and C5L2. Our findings suggest a functional role for renal C5L2 rather than being a C5a decoy receptor. Future studies focusing on renal C5a–C5aR interaction should take differential C5aR and C5L2 expression into account, alongside abundant C5aR expression on infiltrating cells.
The concept of transmission bottlenecks in HIV-1 infection is well established. Coinfections and superinfections have been increasingly documented and provide a founding cause for the expansion of ...viral diversity through recombination. It is still relatively unclear how HIV-1 will propagate and evolve in individuals infected with more than one viral strain. Here we report on the parallel transmission of genetically distant viral strains cocirculating in one individual over many years to a single recipient.
SET domain-containing 2 (
SETD2
) is responsible for the trimethylation of histone H3 lysine36 (H3K36me3) and is one of the genes most frequently mutated in clear cell renal cell carcinoma (ccRCC). ...It is located at 3p21, one copy of which is lost in the majority of ccRCC tumors, suggesting that
SETD2
might function as a tumor suppressor gene. However, the manner in which loss of SETD2 contributes to ccRCC development has not been studied in renal primary tubular epithelial cells (PTECs). Therefore, we studied the consequences of SETD2 knockdown through lentiviral shRNA in human PTECs. Consistent with its known function, SETD2 knockdown (SETD-KD) led to loss of H3K36me3 in PTECs. In contrast to SETD2 wild-type PTECs, which have a limited proliferation capacity; the SETD2-KD PTECs continued to proliferate. The expression profiles of SETD2-KD PTECs showed a large overlap with the expression profile of early-passage, proliferating PTECs, whereas nonproliferating PTECs showed a significantly different expression profile. Gene set enrichment analysis revealed a significant enrichment of E2F targets in SETD2-KD and proliferating PTECs as compared with nonproliferating PTECs and in proliferating PTEC compared with SETD2-KD. The SETD2-KD PTECs maintained low expression of
CDKN2A
and high expression of
E2F1
, whereas their levels changed with continuing passages in untreated PTECs. In contrast to the nonproliferating PTECs, SETD2-KD PTECs showed no β-galactosidase staining, confirming the protection against senescence. Our results indicate that
SETD2
inactivation enables PTECs to bypass the senescence barrier, facilitating a malignant transformation toward ccRCC.
OBJECTIVE:To quantify the impact of individual complications on mortality, organ failure, hospital stay, and readmission after pancreatoduodenectomy.
SUMMARY OF BACKGROUND DATA:An initial ...complication may provoke a sequence of adverse events potentially leading to mortality after pancreatoduodenectomy. This study was conducted to aid prioritization of quality improvement initiatives.
METHODS:Data from consecutive patients undergoing pancreatoduodenectomy (2014–2017) were extracted from the Dutch Pancreatic Cancer Audit. Population attributable fractions (PAF) were calculated for the association of each complication (ie, postoperative pancreatic fistula, postpancreatectomy hemorrhage, bile leakage, delayed gastric emptying, wound infection, and pneumonia) with each unfavorable outcome ie, in-hospital mortality, organ failure, prolonged hospital stay (>75th percentile), and unplanned readmission), whereas adjusting for confounders and other complications. The PAF represents the proportion of an outcome that could be prevented if a complication would be eliminated completely.
RESULTS:Overall, 2620 patients were analyzed. In-hospital mortality occurred in 95 patients (3.6%), organ failure in 198 patients (7.6%), and readmission in 427 patients (16.2%). Postoperative pancreatic fistula and postpancreatectomy hemorrhage had the greatest independent impact on mortality PAF 25.7% (95% CI 13.4–37.9) and 32.8% (21.9–43.8), respectively and organ failure PAF 21.8% (95% CI 12.9–30.6) and 22.1% (15.0–29.1), respectively. Delayed gastric emptying had the greatest independent impact on prolonged hospital stay PAF 27.6% (95% CI 23.5–31.8). The impact of individual complications on unplanned readmission was smaller than 11%.
CONCLUSION:Interventions focusing on postoperative pancreatic fistula and postpancreatectomy hemorrhage may have the greatest impact on in-hospital mortality and organ failure. To prevent prolonged hospital stay, initiatives should in addition focus on delayed gastric emptying.
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