HIV-transgenic mice are not predisposed to LPS-induced AKI.

<p>Blood was drawn from Tg26 mice and wild-type littermates prior to injection with LPS. (A) Comparison of baseline SUN demonstrates no statistical difference between the two groups (P = 0.29). (B)Twenty-four hours after LPS injection, similar increases in SUN were seen in both groups. (C) The rise of SUN in Tg26 and WT mice, 85.4 and 75.1 mg/dL, respectively, was not statistically significant (P = 0.41).</p

Autophagy Limits Endotoxemic Acute Kidney Injury and Alters Renal Tubular Epithelial Cell Cytokine Expression - Fig 1

LPS Induces Autophagy in RTEC: Western Blot for LC3 in HPT1b cells incubated over increasing time with 1 microgram /mL LPS in the absence (A and B) or presence of 100nM Baf A1 (C and D). LPS-induced autophagy was confirmed by immunofluorescence staining of LC3 in HPT1b cells before (E) and after (F) 24-hour treatment with 1 microgram /mL LPS.</p

qPCR analysis of fold change compared to wild type control injected mice.

<p>qPCR analysis of fold change compared to wild type control injected mice.</p

Cytokine analysis of saline and LPS injected animals.

<p>qPCR analysis was performed using RNA from kidneys of Tg26 and wild type mice injected with LPS or saline. The expression of several mediators of inflammation and modifiers of viral transcription were examined. CXCL5, ICAM, MCP, and IL-6 transcripts were similarly increased in LPS injected Tg26 and wild type mice compared to saline controls. TNFa, IL-10, Interferon-a and b were not increased amongst any of the groups.</p

LPS exposure increases IL6 expression and STAT3 activation in ATG7KO RTEC cells.

<p>qPCR analysis demonstrated increased LPS-induced IL-6 expression in cortex of ATG7KO kidneys compared to LPS injected controls (A) which was accompanied by (B) decreased LC3-II and increased STAT3 phosphorylation. (C) Densitometric analysis of the expression of phospho-STAT3 relative to total STAT3. (D)Western blot of FKT- and FKT+ cells indicated that the ablation of Atg7 impaired accumulation of the LC3-II isoform in response to LPS (D). At the same time, qPCR analyses showed that the LPS induction of IL-6 was enhanced in the ATG7 deficient FKT- cell line (E) and in RTEC isolated from wild type mice co-incubated in the absence or presence of 5mM 3MA (F).</p

Autophagy Limits Endotoxemic Acute Kidney Injury and Alters Renal Tubular Epithelial Cell Cytokine Expression - Fig 3

LPS-induced autophagy is TLR4 dependent: Western blot for LC3 and corresponding quantification in lysates from renal cortex of C57BL/10ScN mice 24hr after injection with 15mg/kg LPS or control sterile saline demonstrated no increased autophagy (A, C). Immunodetection of LC3 in primary RTEC isolated from C57BL/10ScN mice grown in the absence or presence of 1 microgram /mL LPS for 24 hours (B, D) revealed no differences in the abundance or distribution of LC3 isoforms.</p

LPS causes similar tubular injury in HIV transgenic and wild type mice.

<p>(A) Hematoxylin and eosin stained sections from Tg26 and wild type (WT) mice demonstrated features of acute tubular cell necrosis (ATN) including nuclear condensation (*), cytoplasmic swelling and hypereosinophilia, and focal loss of nuclei (arrow) only in LPS injected samples (bottom panels, x400) and not in control injected mice (top panels, x400). Proteinaceous casts (P), a feature of murine HIVAN, were seen in the tubules of both control and LPS injected Tg26 mice. (B) Tubular damage in LPS injected mice resulted in significantly higher ATN scores compared to control mice (p<.001) but not when compared to each other (p = .60).</p

LPS induces a minimal increase in HIV transcription in an infected human RTEC cell line.

<p>(A) qPCR analysis of RT3 RNA demonstrated expression of the epithelial markers cytokeratin ande-cadherin and the proximal tubular marker aquaporin-1. RT3 cells did not express distal tubular marker Tamm-Horsfall protein or von Willebrand's factor (endothelial marker). (B) The tubular cell line RT3 displays tubular cell morphology, with polygonal/refractile appearance, and tendency to form characteristic domes when overgrown. (C) The human RT3 cell line was transduced with a <i>gag/pol</i>-deleted HIV lentiviral vector and incubated with LPS four days later. LPS induced a minimal, but significant (*), increase in HIV RNA that was 1.32 and 1.47-fold after 24 and 48 hours, respectively (p<0.05).</p

<i>In vitro</i> differentiation of mouse bladder mesenchymal stem cells.

<p>(A) Sca-1⁺/CD34⁺/lin^- cells grown in osteogenic medium, two weeks post-sort, stained with alizarin red for calcium deposition. (B) Quantification of alizarin red by absorbance at 556nm. Red hatched bar represents Sca-1⁺/CD34⁺/lin^- cells grown in osteogenic induction medium, blue hatched bars represent Sca-1⁺/CD34⁺/lin^- grown in non-induction medium (α-MEM) and green hatched bars represent Sca-1^-/CD34^-/lin^- cells grown in osteogenic induction medium. (C) Sca-1⁺/CD34⁺/lin^- cells grown in osteogenic induction medium two weeks post-sort, stained for endogenous alkaline phosphatase. (D) Quantification of alkaline phosphatase staining in cultures by absorbance at 420nm. (E) Sca-1⁺/CD34⁺/lin^- cells grown in adipogenic medium, two weeks post-sort, stained with Oil Red O. Lipid droplets are stained red. (F) Quantification of Oil Red O stain by absorbance at 519nm. (G) Sca-1⁺/CD34⁺/lin^- cells grown in chondrogenic induction medium formed pellets two weeks post sorting whereas Sca-1⁺/CD34⁺/lin^- cells grown in α-MEM and Sca-1^-/CD34^-/lin^- cells grown in osteogenic induction medium did not form pellets (H, J). (I) Cross section of chondrogenic pellet such as in (G) stained with toluidine blue. Asterisks in (B), (D) and (F) represent significance values of P < 0.05 *, P < 0.01 ** and P < 0.001 *** after Student’s T-Tests. Scale bars are as shown.</p

Analysis of gene and protein expression in FACS sorted bladder cells.

<p>(A) qPCR analysis of Sca-1 and CD34 mRNA expression levels in lin^- cells that were FACS sorted based on expression of Sca-1 and CD34. Expression level is normalized to Sca-1^-/CD34^-/lin^- Sca-1 expression levels. (B) qPCR analysis of smooth muscle myosin (SMM) and smooth muscle alpha <b>α</b> actin (ACTA2) of lin^- cells that were FACS sorted based on Sca-1 and CD34 expression levels. Expression level is normalized to Sca-1⁺/CD34⁺/lin^- SMM expression levels. (A, B) Asterisks represent significance values of P < 0.05 * and P < 0.01 ** after 1 Way ANOVA. Graphs represent expression averages from 4 separate sorts with 3–4 CD1 mice pooled per sort. (C, D) Confocal micrographs of Sca-1⁺/CD34⁺/lin^- sorted cells cultured on a glass coverslips after incubation with EdU for the first 24h (D) Two cells that are Sca-1⁺ (green), EdU⁺ (white) but SMM^- (red) at 48h in culture in α-MEM media. (E) Two cells that are Sca-1⁺ (green), EdU⁺ (white) and SMM⁺ (red) at 4d in culture in α-MEM media.</p