Toll-Like Receptor Signaling and SIGIRR in Renal Fibrosis upon Unilateral Ureteral Obstruction

Innate immune activation via IL-1R or Toll-like receptors (TLR) contibutes to acute kidney injury but its role in tissue remodeling during chronic kidney disease is unclear. SIGIRR is an inhibitor of TLR-induced cytokine and chemokine expression in intrarenal immune cells, therefore, we hypothesized that Sigirr-deficiency would aggravate postobstructive renal fibrosis. The expression of TLRs as well as endogenous TLR agonists increased within six days after UUO in obstructed compared to unobstructed kidneys while SIGIRR itself was downregulated by day 10. However, lack of SIGIRR did not affect the intrarenal mRNA expression of proinflammatory and profibrotic mediators as well as the numbers of intrarenal macrophages and T cells or morphometric markers of tubular atrophy and interstitial fibrosis. Because SIGIRR is known to block TLR/IL-1R signaling at the level of the intracellular adaptor molecule MyD88 UUO experiments were also performed in mice deficient for either MyD88, TLR2 or TLR9. After UUO there was no significant change of tubular interstitial damage and interstitial fibrosis in neither of these mice compared to wildtype counterparts. Additional in-vitro studies with CD90+ renal fibroblasts revealed that TLR agonists induce the expression of IL-6 and MCP-1/CCL2 but not of TGF-β, collagen-1α or smooth muscle actin. Together, postobstructive renal interstitial fibrosis and tubular atrophy develop independent of SIGIRR, TLR2, TLR9, and MyD88. These data argue against a significant role of these molecules in renal fibrosis

Does Stepwise Voltage Ramping Protect the Kidney from Injury During Extracorporeal Shockwave Lithotripsy? Results of a Prospective Randomized Trial

BACKGROUND Renal damage is more frequent with new-generation lithotripters. However, animal studies suggest that voltage ramping minimizes the risk of complications following extracorporeal shock wave lithotripsy (SWL). In the clinical setting, the optimal voltage strategy remains unclear. OBJECTIVE To evaluate whether stepwise voltage ramping can protect the kidney from damage during SWL. DESIGN, SETTING, AND PARTICIPANTS A total of 418 patients with solitary or multiple unilateral kidney stones were randomized to receive SWL using a Modulith SLX-F2 lithotripter with either stepwise voltage ramping (n=213) or a fixed maximal voltage (n=205). INTERVENTION SWL. OUTCOMES MEASUREMENTS AND STATISTICAL ANALYSIS The primary outcome was sonographic evidence of renal hematomas. Secondary outcomes included levels of urinary markers of renal damage, stone disintegration, stone-free rate, and rates of secondary interventions within 3 mo of SWL. Descriptive statistics were used to compare clinical outcomes between the two groups. A logistic regression model was generated to assess predictors of hematomas. RESULTS AND LIMITATIONS Significantly fewer hematomas occurred in the ramping group(12/213, 5.6%) than in the fixed group (27/205, 13%; p=0.008). There was some evidence that the fixed group had higher urinary β2-microglobulin levels after SWL compared to the ramping group (p=0.06). Urinary microalbumin levels, stone disintegration, stone-free rate, and rates of secondary interventions did not significantly differ between the groups. The logistic regression model showed a significantly higher risk of renal hematomas in older patients (odds ratio [OR] 1.03, 95% confidence interval [CI] 1.00-1.05; p=0.04). Stepwise voltage ramping was associated with a lower risk of hematomas (OR 0.39, 95% CI 0.19-0.80; p=0.01). The study was limited by the use of ultrasound to detect hematomas. CONCLUSIONS In this prospective randomized study, stepwise voltage ramping during SWL was associated with a lower risk of renal damage compared to a fixed maximal voltage without compromising treatment effectiveness. PATIENT SUMMARY Lithotripsy is a noninvasive technique for urinary stone disintegration using ultrasonic energy. In this study, two voltage strategies are compared. The results show that a progressive increase in voltage during lithotripsy decreases the risk of renal hematomas while maintaining excellent outcomes. TRIAL REGISTRATION ISRCTN95762080

Bcl-2 predicts response to neoadjuvant chemotherapy and is overexpressed in lymph node metastases of urothelial cancer of the bladder

PURPOSE To assess whether Bcl-2, an inhibitor of the apoptotic cascade, can predict response to neoadjuvant chemotherapy in patients with urothelial cancer of the bladder (UCB). METHODS Bcl-2 expression was analyzed in 2 different tissue microarrays (TMAs). One TMA was constructed of primary tumors and their corresponding lymph node (LN) metastases from 152 patients with chemotherapy-naive UCB treated by cystectomy and pelvic lymphadenectomy (chemotherapy-naive TMA cohort). The other TMA was constructed of tumor samples obtained from 55 patients with UCB before neoadjuvant chemotherapy (transurethral resection of the bladder cancer) and after cystectomy with pelvic lymphadenectomy (residual primary tumor [ypT+], n = 38); residual LN metastases [ypN+], n = 24) (prechemotherapy/postchemotherapy TMA cohort). Bcl-2 overexpression was defined as 10% or more cancer cells showing cytoplasmic immunoreactivity. RESULTS In both TMA cohorts, Bcl-2 overexpression was significantly (P<0.05) more frequent in LN metastases than in primary tumors (chemotherapy-naive TMA group: 18/148 [12%] in primary tumors vs. 39/143 [27%] in metastases; postchemotherapy TMA: ypT+7/35 [20%] vs. ypN+11/19 [58%]). In the neoadjuvant setting, patients with Bcl-2 overexpression in transurethral resection of the bladder cancer specimens showed significantly (P = 0.04) higher ypT stages and less regression in their cystectomy specimens than did the control group, and only one-eighth (13%) had complete tumor regression (ypT0 ypN0). In survival analyses, only histopathological parameters added significant prognostic information. CONCLUSIONS Bcl-2 overexpression in chemotherapy-naive primary bladder cancer is related to poor chemotherapy response and might help to select likely nonresponders

SIGIRR and the activation of renal immune and non-immune cells.

<p>Renal cell suspensions from wildtype and <i>Sigirr</i>-deficient mice were separated into CD45 positive immune cells and CD45 negative non-immune cells by magnetic bead isolation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019204#s4" target="_blank">methods</a>. Cells were stimulated with 1 µg/ml Pam3Cys lipopeptide (TLR2), LPS (TLR4) or CpG-DNA (TLR9) and mRNA expression levels were determined by real-time RT-PCR for several genes after 6 hours of stimulation as indicated. Results were related to the respective 18S rRNA expression and are expressed as means ± SD. * p<0.05 versus wildtype.</p

TLR and TLR agonist mRNA expression after UUO.

<p>Wildtype mice underwent surgery and UUO kidneys (black bars) or contralateral kidneys (Co, grey bars) were harvested at different time points after surgery as indicated. mRNA expression levels were determined by real-time RT-PCR and related to the respective 18S rRNA expression. Data are expressed as means ± SD. * p<0.05 versus wildtype of the same time point, # p<0.05 versus time point day 2.</p

Renal SIGIRR expression after UUO.

<p>A. Wildtype mice underwent surgery and UUO kidneys (black bars) or contralateral kidneys (Co, grey bars) were harvested at different time points after surgery as indicated. mRNA expression levels were determined by real-time RT-PCR and related to the respective 18S rRNA expression. Data are expressed as means ± SD. * p<0.05 versus wildtype of the same time point, # p<0.05 versus time point day 2. B. Protein samples were obtained from the same kidney for SIGIRR Western blot. A kidney from <i>Sigirr</i>-deficient mouse served as negative control for the specificity of the SIGIRR antibody. β-tubulin staining is given as a loading control. The quantitative analysis is shown below. *p<0.05 versus contralateral kidney.</p

TLR-induced expression of cytokines and profibrotic mediators in renal fibroblasts.

<p>CD90+ fibroblasts were prepared from mice and cultured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019204#s4" target="_blank">methods</a>. The cells were stimulated with 1 ug/ml of either Pam3Cys lipopeptide (TLR2 agonist), LPS (TLR4 agonist), CpG-DNA (TLR9 agonist) or 5 ng/ml TNFα. A: After 24 hours of stimulation cell mRNA was isolated and quantified by real-time PCR and refered to the respective 18S rRNA level. Data represent means ± SEM from three independent experiments. * p<0.05 versus medium. B: Renal fibroblasts were stimulated as before and cell culture supernatants were obtained after 24 (white bars) and 48 hours of stimulation (black bars). MCP-1 and TGF-β levels were measured by ELISA. Data represent means ± SEM from three independent experiments. * p<0.05 versus medium.</p

TLRs and morphometric analysis of tissue remodeling after UUO.

<p>A: Cortical renal sections were stained with silver. Images illustrate representative sections of UUO kidneys 10 days after UUO in mice of the respective group as indicated (original magnification 200×). B: The indices for tubular cell damage, tubular dilatation, interstitial volume were determined by quantitative morphometry in obstructed kidneys at 10 days as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019204#s4" target="_blank">methods</a>. C: Interstitial fibrosis was quantified by digital morphometry on Masson Trichrom-stained sections as percentage of entire high power field. Values represent means ± SD from 10–15 high power fields.</p