GEC-targeted HO-1 expression reduces proteinuria in glomerular immune injury

Abstract

Induction of heme oxygenase (HO)-1 is a key defense mechanism against oxidative stress. Compared with tubules, glomeruli are refractory to HO-1 upregulation in response to injury. This can be a disadvantage as it may be associated with insufficient production of cytoprotective heme-degradation metabolites. We, therefore, explored whether 1) targeted HO-1 expression can be achieved in glomeruli without altering their physiological integrity and 2) this expression reduces proteinuria in immune injury induced by an anti-glomerular basement membrane (GBM) antibody (Ab). We employed a 4.125-kb fragment of a mouse nephrin promoter downstream to which a FLAG-tagged hHO-1 cDNA sequence was inserted and subsequently generated transgenic mice from the FVB/N parental strain. There was a 16-fold higher transgene expression in the kidney than nonspecific background (liver) while the transprotein immunolocalized in glomerular epithelial cells (GEC). There was no change in urinary protein excretion, indicating that GEC-targeted HO-1 expression had no effect on glomerular protein permeability. Urinary protein excretion in transgenic mice with anti-GBM Ab injury (days 3 and 6) was significantly lower compared with wild-type controls. There was no significant change in renal expression levels of profibrotic (TGF-beta1) or anti-inflammatory (IL-10) cytokines in transgenic mice with anti-GBM Ab injury. These observations indicate that GEC-targeted HO-1 expression does not alter glomerular physiological integrity and reduces proteinuria in glomerular immune injury.

Fig. 1.

Schematic plot of the pNeph-FLAG-human heme oxygenase 1 (hHO1) plasmid (9K). The FLAG-tagged human HO-1 was inserted between a murine nephrin promoter (4.125 kb) and human β-globin polyadenylation and intron sequences. Mlu1, XbaI, XhoI/SalI are key restriction sites used in ligation and construction. The fragment used for microinjection was released from the MluI to the EcoRV site of the plasmid. The thick band represents the EcoRI-EcoRI probe used for genotyping.

Fig. 2.

Schematic plot of the pNeph-FLAG-hHO1 plasmid construction strategy. Using a 2-step sequential PCR reaction, the tag sequence FLAG was linked in frame to the 5′-end of the hHO-1 coding sequence (A). The peptide sequence of FLAG and its 5′-joining region are shown in B. FLAG-hHO-1 was inserted to the 5′-end of the murine nephrin promoter (C). The polyadenylation and intron sequences of the human β-globin gene were obtained by PCR from pTRE2 and inserted at the 3′-end of the hHO-1-coding region (C). The final pNeph-FLAG-hHO1 plasmid construct is shown in D. A 6.5-kb MluI-EcoRV fragment (E) was released from the pNeph-FLAG-hHO1 plasmid for microinjection. P1-P5 are sequences of primers used in the PCR reactions shown. Underscoring indicates key restriction sites.

Fig. 3.

Antigenicity of FLAG-tagged hHO-1. Protein lysates extracted from HeLa cells transfected with pCMV-FLAG-hHO1 or pCMV-FLAG-BAP (bacterial alkaline phosphatase; BAP) were analyzed by Western blotting using an anti-FLAG antibody (top) or anti-HO1 antibody (bottom). The anti-FLAG antibody detected FLAG-tagged hHO-1 (33 kDa; white asterisk) and FLAG-tagged BAP (50 kDa). The anti-HO-1 antibody detected both endogenously expressed HO-1 (32 kDa, 288 amino acids) and FLAG-tagged HO-1 (33 kDa, 301 amino acids).

Fig. 4.

Summary of characteristics of mice that originated from one of the founders (founder 6). In the table, F2 hemizygotes from founder 6 generated 23 F3 pups, shown as 3 separate litter groups based on their dates of birth (DOB). The table summarizes gender, weight, genotype assessed by Southern blot analysis and PCR (+ or − indicates the presence or absence of the FLAG-hHO-1 transgene), and urine albumin (Ualb) excretion factored by that of urine creatinine (Uc). Zygosity was determined using real-time quantitative PCR (Q-PCR) and 2^−ΔΔCt method (described in materials and methods).

Fig. 5.

Differential expression of the transgene FLAG-hHO-1 in various organs of mNephrin-hHO1 transgenic mice. Total RNA was extracted from brain, lung, heart, liver, stomach, muscle, intestine and kidney, and analyzed by Q-PCR using GAPDH as an internal control. FLAG-hHO-1 mRNA level detected in the brain sample was arbitrarily set as 1 (B). Each data point in this panel is the average of 3 independent Q-PCR reactions. A: amplification plot of Q-PCR reaction in total RNA extracted from kidney and brain. Normalized reporter fluorescence (Rn) values of FLAG-hHO-1 mRNA levels in kidney and brain were determined by the comparative Ct (2^−ΔΔCt) method and plotted against the Q-PCR cycle number. There was a 15.6-fold higher transgene expression in kidney extract compared with that from the brain.

Fig. 6.

A: expression of transprotein FLAG-hHO-1. Protein lysates prepared from transgenic mouse kidney (top, lane 2), liver (top, lane 3), and wild-type mouse kidney (top, lane 1) were analyzed by Western blotting using an anti-FLAG antibody (top) or anti-β-actin antibody (bottom). B: immunolocalization of transprotein FLAG-hHO-1 in the glomerulus. The renal cortical section shown was obtained from a mNephrin-hHO1 transgenic mouse and was immunolabeled with FITC-conjugated sheep anti-FLAG antibody. Staining is apparent in glomerular epithelial cells.

Fig. 7.

Colocalization of transprotein FLAG-hHO1 with the podocyte marker WT1. A renal cortical section of a mNephrin-hHO1 transgenic mouse was double immunolabeled with FITC-conjugated sheep anti-FLAG antibody (A) and Cy3-conjugated anti-WT1 antibody (B). Superimposed FITC and Cy3 signals are shown in C.

Fig. 8.

Electron micrograph of glomeruli from a mNephrin-hHO1 transgenic mouse. Glomerular epithelial cell foot processes are clearly visible with no apparent effacement or microvillus transformation (A). Occasional “pockets” or “humps” (white arrow with asterisk) were present on the podocyte side of the glomerular basement membrane (GBM; B).

Fig. 9.

Up/Uc values (mg protein/mg creatinine) in individual wild-type (Wt, ⧫) and transgenic (Tg; ◊) mice. Mean values are also shown.

Fig. 10.

Proteinuria in mNephrin-hHO1 transgenic mice and their wild-type littermates in accelerated anti-GBM antibody-mediated injury. Shown are individual Up/Uc values (mg protein/mg creatinine) in Wt (⧫) and Tg (◊) mice 4 days before (−4) and on days 3 and 6 after administration of anti-GBM antibody. *P = 0.017 Tg vs. Wt on day 3. **P = 0.019 Tg vs. Wt on day 6.

Fig. 11.

IL-10 and transforming growth factor (TGF)-β1 mRNA levels in Tg or control mice with anti-GBM nephritis assessed by RT-PCR. Total RNA was extracted from kidneys of Tg (grey bars) or Wt (white bars) mice 4 days before and on days 1, 2, and 3 after injection of anti-GBM antibody. Values are means ± SE of 5 mice. *P = 0.336, **P = 0.367 compared with Wt control mice on day 3.