Expression of the RNA-stabilizing protein HuR in ischemia-reperfusion injury of rat kidney

Abstract

The RNA-binding protein human antigen R (HuR) participates in the posttranscriptional regulation of mRNAs bearing 3' AU-rich and U-rich elements, which HuR can stabilize under conditions of cellular stress. Using the LLC-PK(1) proximal tubule cell line model, we recently suggested a role for HuR in protecting kidney epithelia from injury during ischemic stress (Jeyaraj S, Dakhlallah D, Hill SR, Lee BS. J Biol Chem 280: 37957-37964, 2005; Jeyaraj SC, Dakhlallah D, Hill SR, Lee BS. Am J Physiol Renal Physiol 291: F1255-F1263, 2006). Here, we have extended this work to show that small interfering RNA-mediated suppression of HuR in LLC-PK(1) cells increased apoptosis during energy depletion, while overexpression of HuR diminished apoptosis. Suppression of HuR also resulted in diminished levels of key cell survival proteins such as Bcl-2 and Hsp70. Furthermore, rat kidneys were subjected in vivo to transient ischemia followed by varying periods of reperfusion. Ischemia and reperfusion (I/R) affected intensity and distribution of HuR in a nephron segment-specific manner. Cells of the proximal tubule, which are most sensitive to I/R injury, demonstrated a transient shift of HuR to the cytoplasm immediately following ischemia. Over a 14-day period following the onset of reperfusion, nuclear and total HuR protein gradually increased in cortical and medullary proximal tubules, but not in non-proximal tubule cells. HuR mRNA was expressed in two forms with alternate transcriptional start sites that increased over a 14-day I/R period, and in vitro studies suggest selective translatability of these two mRNAs. Baseline and I/R-stimulated levels of HuR mRNA did not parallel those of HuR protein, suggesting translational control of HuR expression, particularly in medullary proximal tubules. These findings suggest that alterations in distribution and expression of the antiaptotic protein HuR specifically in cells of the proximal tubule effect a protective mechanism during and following I/R injury in kidney.

Fig. 1.

Antiapoptotic activity of human antigen R (HuR) in a proximal tubule cell line. Knockdown of HuR (average knockdown of ∼75%) in LLC-PK₁ cells with a specific small interfering (si)RNA (A) resulted in increased caspase 3/7 activity and condensation/fragmentation of nuclei (B). Conversely, overexpression of FLAG-tagged HuR by 2- to 3-fold (C) produced diminished caspase 3/7 activity and fewer apoptotic nuclei (D). A FLAG-tagged version of HuR was used to confirm that the exogenously expressed protein underwent appropriate nucleocytoplasmic trafficking and localization. In E, LLC-PK₁ cells with suppressed HuR levels showed diminished expression of Bcl-2 and Hsp70. In B and D, values are means ± SD; n = 3. β-Actin was included as a loading control in A, C, and E. *P < 0.05. **P < 0.01.

Fig. 2.

Serum creatinine levels of animals used in this study. Serum creatinine levels of ischemia-reperfusion (I/R)-injured rats were measured as described in materials and methods. At least 3 animals were used for each group. Values are means ± SD.

Fig. 3.

Immunolocalization of HuR in kidney sections of sham-operated rats. Using double label immunohistochemistry, HuR (shown in brown) nuclear localization was observed in all renal tubules identified by segment-specific markers (shown in purple). The proximal tubules and the thin descending limbs of Henle were identified as immunoreactive cells for aquaporin-1 (AQ1) in the cortex (A) and medulla (B), respectively. The thin ascending limb of Henle (C) was distinguished in the medulla by the presence of chloride channel-K1 (CLC-K1). The thick ascending limb was identified as Tamm-Horsfall glycoprotein (THP)-positive cells in the medulla (D). The thiazide-sensitive Na⁺-Cl⁺ transporter (TSC) was used as a marker of distal convoluted tubules in the cortex (E). Aquaporin-2 (AQ2) antibodies were used to visualize collecting tubules (F). Insets: marker-positive cells in higher magnification.

Fig. 4.

I/R injury induced changes in HuR protein expression and distribution in proximal tubules. Kidney sections from rats subjected to sham operation, ischemia only, and I/R injury for 1, 6, or 24 h or 14 days were stained with anti-HuR antibodies. Proximal tubules (P) were identified by staining of adjacent sections with antibodies against AQ1 (not shown). Arrowheads indicate non-proximal tubules. A–F: sections from the renal cortex. G–L: sections from the outer medulla.

Fig. 5.

Relative increase in levels of HuR in nuclei of proximal tubules following I/R injury. The intensity of HuR staining in nuclei of proximal and non-proximal tubules in kidney sections was measured using image-analysis software, and values were normalized as the ratio of nuclear HuR staining in proximal vs. non-proximal tubules. A: cortex. B: medulla. The level of HuR immunoreactivity in nuclei of non-proximal tubules was assigned a value of 1. At least 50 nuclei were assessed for each group. Values are means ± SE. *Statistically significant differences from sham-operated control (P < 0.01).

Fig. 6.

Expression of HuR protein in renal cortex and medulla following I/R injury. Fifteen micrograms of total protein from kidney cortex (A) or whole medulla (B) homogenates were assayed with anti-HuR antibody by Western blot analysis. Shown are homogenates obtained from 3 sham-operated and from 3 postischemic rats at indicated times post-I/R injury. Ponceau S staining of nitrocellulose membranes of corresponding blots was used as a loading control. C and D: data in A and B were quantified and charted. Values are means of HuR expression level ± SE. *Statistically significant differences from sham-operated control (P < 0.05).

Fig. 7.

In situ hybridization of kidney sections with rat-specific HuR probe. A: low-magnification images of sham-operated and 14-day I/R animals show changes in expression of HuR mRNA in the cortex and outer medulla. B: higher magnification images illustrate relative HuR mRNA expression levels in proximal tubule and non-proximal tubule cells (*). Scale bar = 50 μm.

Fig. 8.

Expression of alternate transcripts of HuR in rat kidney. RNAse protection assays were performed to quantify changes in levels of HuR mRNA in renal cortex (A) and whole medulla (B) following I/R injury. Lane 1, probe only; lane 2, probe only digested by RNAse; lane 3, yeast tRNA+probe; lane 4, yeast tRNA+probe digested by RNAse; lanes 5–7, RNAse-protected fragments of HuR mRNA from sham-operated animals; lanes 8–10, RNAse-protected fragments of HuR mRNA from 14-day I/R-injured animals. The 2 major bands are indicated by arrows and correspond to HuR mRNAs with 5′ untranslated regions (UTRs) of ∼150 and ∼20 bases. C, top: data along with results from 1-day I/R gels not shown were quantified and charted. Values are means relative HuR mRNA expression ± SE. *Statistically different from sham controls (P < 0.05). D: capped or uncapped HuR RNAs corresponding to the alternately transcribed forms are shown (left), along with an in vitro translation reaction showing the variable translatability of the 2 forms (right).