Macrophage Depletion Reduces Disease Pathology in Factor HDependent Immune Complex-Mediated Glomerulonephritis

Complement factor H (FH) is a key regulator of the alternative pathway of complement, in man and mouse. Earlier, our studies revealed that the absence of FH causes the C57BL6 mouse to become susceptible to chronic serum sickness (CSS) along with an increase in the renal infiltration of macrophages compared to controls. To understand if the increased recruitment of macrophages (Mϕs) to the kidney was driving inflammation and propagating injury, we examined the effect of Mϕ depletion with clodronate in FH knockout mice with CSS. Eight-week-old FHKO mice were treated with apoferritin (4 mg/mouse) for 5 weeks and with either vehicle (PBS) or clodronate (50 mg/kg ip, 3 times/wk for the last 3 weeks). The administration of clodronate decreased monocytes and Mϕs in the kidneys by >80%. 

Kidney function assessed by BUN and albumin remained closer to normal on depletion of Mϕs. Clodronate treatment prevented the alteration in cytokines, TNFα and IL-6, and increase in gene expression of connective tissue growth factor (CTGF), TGFβ-1, matrix metalloproteinase-9 (MMP9), fibronectin, laminin, and collagen in FHKO mice with CSS (P < 0:05). Clodronate treatment led to relative protection from the immune complex- (IC-) mediated disease pathology during CSS as assessed by the significantly reduced glomerular pathology (GN) and extracellular matrix. Our results suggest that complement activation is one of the mechanisms that regulate the macrophage landscape and thereby fibrosis. The exact mechanism remains to be deciphered. In brief, our data shows that Mϕs play a critical role in FH-dependent ICGN and Mϕ depletion reduces disease progression.

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1. Introduction

Chronic kidney disease (CKD) is a global health problem; 10% of which is immune complex- (IC-) mediated and occurs in serum sickness, infections, and autoimmune diseases. [1]. The USA Renal Data System Annual Report states that individuals with CKD "are clearly at a high risk of death" with a 10-fold increased incidence over three decades in which affected patients lost 70% of their lifespan. According to the NIDDK, approximately 14% of the adult population has been diagnosed with kidney disease, making it the 9th leading cause of death [2]. The kidney is highly susceptible to complement-mediated damage especially in autoimmune and inflammatory settings [3, 4].

The complement system participates in innate and adaptive immune responses and provides a first line of defense against microorganisms [5, 6]. It consists of three activation pathways, which are initiated in unique ways, and made up of more than 30 plasma and cell-associated proteins [7]. The alternative pathway is constantly on the alert and is regulated by proteins such as complement factor H (FH). FH is a circulating protein mostly synthesized by the liver [8]. FH-deficient (fh-/-) mice treated with apoferritin develop chronic serum sickness (CSS) leading to diffuse proliferative glomerulonephritis (GN) within 5 weeks of treatment, while littermate C57BL/6 mice that are factor H (FH) sufficient have little glomerular inflammation [9]. F4/80+ macrophage infiltration occurs in the kidney and is observed around IC deposits in the fh-/- mice. Complement activation generates anaphylatoxins, C3a and C5a, that participate in the trafficking of Mϕs to the site of inflammation. Inhibition of C5a/ C5aR1 signaling reduced the Mϕs in the kidney [10]. In this setting, kidney injury is associated with renal infiltration but the mechanisms that may be involved in causing renal injury remain unknown.

Figure 1: FH alters macrophage marker genes in the kidney of mice with FH-dependent ICGN. Real-time quantitative PCR for CD204 and CD206 using mRNA isolated from the kidneys after 5 weeks of saline or apoferritin. The expression is normalized to GAPDH (n = 6/group). ∗P < 0:01. Graphs depict the fold change of genes in apoferritin-treated fh-/- mice (n = 6) compared to fh-/- mice treated with saline (n = 6).

Monocytes (Mo) generated by hematopoietic cells [11] migrate through the endothelium into the tissues, where they differentiate into Mϕs. Markers such as F4/80, CD11b, CD68, and Fc receptors can identify the Mϕs. Mϕs are critically involved in renal injury, repair, and fibrosis in different experimental models of renal disease [12, 13]. Recently, our studies demonstrated that kidney injury in FH-dependent ICGN is associated with the renal infiltration of Mϕs. Moreover, previous studies have demonstrated that inhibition of renal Mϕ infiltration reduces arterial pressure and prevents glomerular injury and fibrosis in various animal models of nephropathy [14, 15]. However, to our knowledge, the impact of Mϕ depletion during renal injury associated with FH-dependent ICGN has not been addressed. Therefore, the present study examined the role of Mϕs on the renal injury associated with FH-dependent ICGN using liposome clodronate. Clodronate (clodronic acid) is a bisphosphonate that is metabolized to a nonhydrolyzable ATP analog [16]. Our experiments show that Mϕs play a key role in the loss of kidney function in a setting of unregulated complement activation. Therefore, reducing Mϕs or instructing Mϕs to assume a repair phenotype could reduce disease pathology and can be exploited therapeutically.

2. Materials and Methods

2.1. Experimental Animals.

The fh−/− mouse strain was generated and kindly provided by Drs. Matthew Pickering and Marina Botto (Imperial College of London) and continuously backcrossed onto the C57BL/6 strain in our laboratory for over 10 generations. Littermate FH-sufficient mice served as the controls (n = 8/group) Genotyping was performed using PCR-based approaches. Mice were maintained at a temperature of 20–22° C in 12 h light–12 h dark lighting cycle, in sterile, ventilated cages with food and water ad lib. The University of Chicago and the University at Buffalo Institutional Animal Care and Use Committees approved all studies.

2.2. Chronic Serum Sickness Protocol. 

CSS was induced in 6– 8 week fH−/− or wild-type mice with daily intraperitoneal administration of 4 mg horse spleen apoferritin (Calzyme Laboratories) without adjuvant [10, 17, 18] for 5 weeks. Littermate controls received intraperitoneal injections of saline vehicle contemporaneously 

2.3. Kidney Function Assessments. 

Blood urea nitrogen (BUN) concentrations were determined with a Beckman Autoanalyzer (Beckman Coulter, Fullerton, CA). Urinary albumin concentrations were measured with a mouse albumin ELISA kit (Bethyl Laboratories, Montgomery, TX) and normalized to urinary creatinine measured colorimetrically (Stanbio Laboratory, Boerne, TX) as described previously [19].

Figure 2: Clodronate increasingly reduces kidney macrophages in mice with FH-dependent ICGN. Given are the flow cytometry profiles of F4/80+ monocytes/Mϕs on day 0, 2, 4, and 7, representatively. The expression of F4/80+ cells was determined by flow cytometry. Data were analyzed using FlowJo V10 software. F4/80+ cells are significantly reduced at each time point reaching less than 10% by day 7. Each time point n = 6.

2.4. Histology. 

Kidneys were embedded in paraffin and formalin-fixed, and four-micrometer sections were generated. The sections were stained with Periodic Acid-Schiff and examined by a renal pathologist (A.C.) in a blinded manner. For each slide, proliferative glomerulonephritis, and glomerulo-sclerosis were graded from 0 to 4 as described previously [20]. For immunofluorescence microscopy, the kidney tissue was snap-frozen in 2-methylbutane precooled in dry ice. Four-micron cryostat sections were processed for immuno-fluorescence (IF) staining. Sections were fixed in 4% paraformaldehyde and stained with Alexa488-conjugated anti-mouse C3 (Cappel) and Alexa 555 anti-mouse IgG. A semi-quantitative score of staining intensity and distribution from 0 to 4 was provided in a blinded manner as described previously [19].

2.5. qRT-PCR. 

The kidneys were harvested, and total RNA was extracted from the tissues using TRIzol reagent according to the manufacturer's instructions (Invitrogen, USA). cDNA was synthesized using SuperScript III Reverse Transcriptase (Invitrogen). qRT-PCR was performed using Power SYBR Green PCR Master mix on the ABI 7700 sequence detector (Applied Biosystems). PCR amplification was performed in a total volume of 25 μl containing 12.5 μl of 2x TaqMan Universal PCR Master Mix (Applied Biosystems), 6.25 μl of nuclease-free water, 5 μl of cDNA, and 1.25 μl of the appropriate primers (IDT). All qPCR primers (Table 1) were designed using the Primer3 software (Whitehead Institute for Biomedical Research) to ensure specificity and sensitivity. To quantify the levels of mRNA, we normalized the expression of the target genes to glyceraldehyde-3- phosphate dehydrogenase. Relative mRNA expression levels were calculated using the 2 − ΔΔCt method and were normalized to the expression levels of GAPDH.

2.6. Statistics. 

All results are mean ± SD from at least 6-8 mice in each group. Either an unpaired two-tailed test (data with normal distribution) or the Mann–Whitney U test (data with no normal distribution) was used to compare 2 groups by GraphPad Prism 5.0. The statistical significance is expressed as follows: ∗P < 0:05; ∗∗P < 0:01; and n.s.: not significant.

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