Kidney injury in the murine models of hematopoietic stem cell transplantation

Abstract

Acute graft-versus-host disease (GVHD) affects different organs, including the skin, liver and gastrointestinal tract. Although kidneys are not among the organs commonly known to be the target of acute GVHD, kidney damage is frequently reported after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We have studied the effect of bone marrow transplant (BMT) on the kidneys in different murine models of GVHD. We found that glomerular damage in the kidneys is a common pathologic finding in mice after BMT. The histopathologic features of glomeruli damage included mesengiolysis, mesangial proliferation and edema, subendothelial and endothelial thickening, splitting of capillary walls in glomeruli, narrowing and collapsing of capillary lumens, fibrinoid necrosis of afferent arterioles, intimal hyperplasia, and microthrombi. These pathologic features are similar to those detected in kidneys of patients with thrombotic microangiopathy (TMA) after allo-HSCT. We have previously shown that activation of the complement system plays a role in the GVHD-induced tissue injury in mice. In the current study, we showed the presence of complement activation products in the kidney specimens of mice after BMT. We also showed that complement deficiency reduced the extent and severity of post-BMT glomerular damage in mice. We concluded that BMT in mice is associated with glomerular injury and tubulointerstitial nephritis, and that kidney damage is at least partially mediated by activation of the complement system.

Introduction

Allogenic hematopoietic stem cell transplantation (allo-HSCT) can cause an acute graft-versus-host disease (GVHD) ¹. Acute GVHD is an alloimmune response elicited by donor T-cells against epithelial cells, resulting in tissue injury in different organs of the recipient. Organs commonly targeted by acute GVHD are the skin, liver, and gastrointestinal tract. However, tissue injury in other organs, including kidneys, are frequently detected after allo-HSCT ². Acute kidney injury is reported in 50%−73% of patients undergoing allo-HSCT ². Although infection, total body irradiation, and chemotoxicity contribute to kidney injury, whether acute GVHD damages the kidneys is not well characterized. We investigated the histologic changes in kidneys in murine models of bone marrow transplant (BMT). We found that in addition to interstitial nephritis and tubulitis ^3,4, the glomerular injury is a common manifestation of kidney injury after BMT in mice. Many of the pathologic changes in the kidneys after BMT were similar to the histopathologic changes detected in the kidneys of patients with post-BMT thrombotic microangiopathy (TMA) ^5,6. Previously, we have shown that the complement system has a role in the GVHD-induced organ injury after BMT in mice ^7,8. In the current study, we investigated whether the complement system is also involved in the BMT-induced damage to the kidneys. We detected deposition of the complement proteins in kidney glomeruli after BMT. The extent of glomerular injury in the kidneys was less in C3 deficient mice as compared to wild-type recipient mice. We concluded that kidney damage is common after BMT and the complement system plays a role in the kidney injury after BMT.

Material and Methods

Murine models of acute GVHD

The animal experiments are approved by the Institutional Animal Care and Use Committee at the University of Texas M.D. Anderson Cancer Center.

All recipient mice were age-matched females at 2–6 months of age at the time of BMT. We used two different murine models of GVHD for our studies. In the first model, GVHD was induced by the disparity in MHC class I and II antigens between BALB/c (H-2^d) donor C57BL/6 (H-2^b) recipient mice. To generate BMT chimeras, recipient wild-type (WT) B6 or C3^−/− (C57BL/6 background, Jackson Lab) mice received 12 Gy TBI (¹³⁷Cs source split into 2 doses) on day −1, and received 10×10⁶ T cell depleted (TCD) bone marrow (BM) cells plus 15×10⁶ splenocytes from BALB/c (NCI-Frederick) mice on day 0 ⁹. The WT control mice received only T-cell depleted BM cells. Mice were monitored for clinical signs of GVHD (hair loss, hunched back and diarrhea) and weighed twice a week. Serum concentration of blood urea nitrogen (BUN) and creatinine was measured in blood samples collected by retro-orbital route on days 7 and 10 post-transplant. For histopathological studies, tissue samples were collected from the skin, liver, intestine, lung, and kidney, and fixed in 10% formalin. The tissue samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Tissue slides were graded by a pathologist according to the published GVHD scoring system ¹⁰.

In the second model GVHD model (Parent to F1), 50 × 10⁶ splenocytes from female wild-type C5BL/6 (H-2b) donors were infused to B6D2F1 (H-2b/d) recipient mice. In this model, GVHD was manifested in the recipient mice in 2 weeks. The blood and tissue samples from GVHD and control mice were assessed at 2, 4 and 8 weeks post-transplant.

Tissue Preparation and Histological Staining

Fixed tissues were processed and embedded in paraffin using routine protocols. Tissues were sectioned at 4-μm thickness for routine staining with hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), and immunohistochemistry. Thin sections (2-μm thickness) were used for periodic acid methenamine silver stain (PAM). Morphometric analysis was performed by using the Image-Pro Plus software (Media Cybernetics, Silver Spring, MD, USA). Fifteen random glomerular cross-sections per experimental animal were photographed using a digital camera (Olympus DP11) (Olympus America, Melville, NY, USA). Hematoxylin and eosin–stained sections were used for the assessment of glomerular cellularity while the amount of extracellular matrix was evaluated using PAM silver stains. Quantification of mesangial area, glomerular area, and capillary wall thickness was performed using Image J analysis ¹¹ of silver stained slides.

Statistical Analysis

Statistical analysis was performed using the SPSS program (SPSS Inc., Chicago, IL). Means between groups were compared using t-test without assuming equal variances. A P value of <0.05 was considered statistically significant. All data are expressed as mean ± SEM.

Results

Kidney pathology after transplant in mice

WT B6 and C3^−/− mice were lethally irradiated and infused with BM cells and splenocytes from BALB/c donors to induce GVHD. Within 8 weeks post-transplant, 75% of C3 deficient recipients survived, compared to only 20% of WT mice. Whereas WT recipients had severe GVHD in the skin, intestine, liver, and lung; C3 deficient mice exhibited only mild changes in these organs, reflected in their significantly lower GVHD scores ¹².

We examined histology of kidneys resected from recipient mice, using hematoxylin-eosin, silver staining, and Periodic acid–Schiff (PAS) staining. We detected widespread tubulointerstitial nephritis and widespread glomerular damage (Fig. 1). Tubulitis and interstitial nephritis have previously been reported in murine and rat models of bone marrow transplant ^3,4, but we found that more than 60% of glomeruli in kidneys of recipient mice showed morphological evidence of injury.

Figure 1. Glomerular injury after bone marrow transplant in mice.

Irradiated recipient C57BL/6 mice received 10×10⁶ T cell depleted bone marrow (BM) cells plus 15×10⁶ splenocytes from BALB/c mice. Kidney specimens were collected 7 days after BMT, were embedded in paraffin, sectioned, and stained with periodic acid-Schiff (PAS) and silver stains. (A) Normal glomerulus in mice (silver stain). * shows a glomerular capillary lumen. Arrowhead shows mesangial cells. (B) Normal glomerulus in mice (hematoxylin-eosin stain). (C) Subendothelial and mesangial swelling resulted in narrowing and collapsing capillary lumens (silver stain). (D) Mesangial proliferation and swelling (PAS stain). (E) Thickening and splitting (arrowhead) of capillary walls in glomeruli. * Occlusion of capillary lumen. (F) Thickening and splitting of capillary walls (arrowhead) and * occlusion of afferent arteriole lumen by a hyaline microthrombus (PAS stain). (G) Mesangial proliferation (silver stain). (H) mesengiolysis (PAS stain).

Serum concentration of blood urea nitrogen (BUN) and creatinine in blood samples collected on days 7 and 10 post-transplant remained within normal limits. Seven days after BMT, BUN was 19.6 ± 1.7 mg/dl in C3 deficient mice and 27.5 ± 9.6 mg/dl in WT mice (p = 0.30, n = 8 mice, t-test) and creatinine was ≤ 0.2 mg/dl in both groups. Ten days after BMT, BUN was 19.1 ± 1.2 mg/dl in C3 deficient mice and 19.9 ± 1 mg/dl in WT mice (p = 0.64, n=6 mice, t-test) and creatinine was ≤ 0.2 mg/dl in both groups.

The histopathologic features of glomeruli damage included mesengiolysis, mesangial proliferation and edema, subendothelial and endothelial thickening, splitting of capillary walls in glomeruli, narrowing and collapsing of capillary lumens, fibrinoid necrosis of afferent arterioles, intimal hyperplasia, and microthrombi (Fig. 1). In addition to tubilitis, inflammatory cells also infiltrated subendothelium in glomeruli, mainly macrophages (Supplemental Figure). Many of the pathologic findings in the kidneys after BMT in our murine models were similar to those identified in patients with thrombotic microangiopathy after BMT ^13,14.

We also examined deposition of complement proteins in murine kidneys after BMT. Immunostaining for kidney specimens for C3 and its degradation products showed evidence of an increase in complement deposition in the majority of kidney glomeruli after BMT (Fig. 2).

Figure 2. Deposition of complement in kidney glomeruli after bone marrow transplant (Parent to F1) in mice.

Splenocytes (50 × 10⁶) from female C57BL/6 (H-2b) donors were infused to B6D2F1 (H-2b/d) recipient mice (GVHD group) or to C57BL/6 (H-2b) mice (Control group). The kidney samples from GVHD and control mice were collected at 2, 4 and 8 weeks post-transplant. (A) Immunostaining of kidney specimens from control and GVHD mice using C3 antibody showed a progressive deposition of C3 in glomeruli. Arrowheads point to glomeruli in kidney sections. (x 10 magnification) (B) C3 deposition in kidney glomerulus examined at a higher magnification. (x 40 magnification).

Complement deficiency reduces the severity of kidney injury after transplant

We have previously shown that complement deficiency reduces GVHD-associated injury in the skin, liver, and intestine of recipient ⁷. To investigate the role of complement activation in the kidney injury after BALB/c to C57BL/6 BMT, we compared the kidneys of wild-type and C3 deficient recipient mice. Lack of C3 in the recipient mice significantly reduced the severity of kidney injury after BMT. While histopathologic evidence of glomerular damage (as summarized above) was present in 65% of glomeruli in wild-type C57BL/6, the percentage of damaged glomeruli in C3 deficient mice reduced to 46% (PAS stain, p=0.01, n=4 mice per group, and 70–80 glomeruli examined per mice, t-test) (Figure 3A). Silver staining showed a similar reduction in the percentage of damaged glomeruli in C3 deficient mice as compared to wild-type mice (62% vs 45% respectively, P=0.001, t-test).

Figure 3. Complement and kidney injury after bone marrow transplant in mice.

(A) The kidney specimens collected from recipient mice and stained with periodic acid–Schiff (PAS) or silver stain were examined for the evidence of glomerular injury (n=4 mice/ group). From each specimen, 70–90 glomeruli examined and the percentage of injured glomeruli was compared between wild-type (n=4) and C3 deficient recipient mice (n=4). Statistical significance was calculated using a t-test. (B) Mesangial area, glomerular area, and capillary wall thickness were quantified in kidneys of WT mice and C3 deficient post-transplant mice. At least 10 glomeruli from each silver stained kidney slide were imaged with 60 x magnification, and 5 areas of each glomerulus were selected for Image J analysis. Area statistics were calculated in square pixels.

Further quantification of the pathologic changes by image analysis of the silver-stained slides from kidney specimens demonstrated that the mesangial area was significantly more in WT glomeruli compared to that in C3 deficient glomeruli (mesangial area/glomerular area of 0.16 ± 0.04 in WT mice and 0.12 ± 0.03 in C3 deficient mice). The average capillary wall in the WT glomeruli was significantly thicker than that of C3 KO mice (16.5 ± 3.1 pixels in WT mice and14.5 ± 3.5 pixels in C3 deficient mice)(Figure 3B).

Discussion

A large percentage of patients undergoing allo-HSCT develop acute kidney injury within 100 days after transplant ². This percentage can be as high as 73% in patients who received myeloablative conditioning prior to allo-HSCT. Patients who develop acute kidney injury early after transplant are at a higher risk of progression to chronic kidney failure later in their post-transplant course ^15,16. Although clinical and laboratory abnormalities consistent with acute kidney injury may be related to the toxicity of the preparatory regimen (chemotherapy or total body irradiation), calcineurin inhibitors, infections, and hepatic sinusoidal obstruction syndrome; but the occurrence of acute GVHD is also an important risk factor for acute kidney failure ^2,17–19. We used murine models of BMT ²⁰ to investigate kidney injury after allo-HSCT. Studying kidney injury in different murine models of BMT provided us the opportunity to monitor kidney histopathology over time after BMT and to dissect the pathogenesis of BMT-associated acute kidney injury. Two previous studies reported kidney injury after bone marrow transplant in mice and rats ^3,4. Both of these studies identified tubulitis and interstitial nephritis, evident by infiltration of T cells, as the main pathologic findings in the kidneys after BMT. In our study, in addition to tubulointerstitial nephritis, we detected extensive glomerular injury including mesengiolysis, the proliferation of mesangial cells, endothelial and subendothelial swelling, narrowing and collapsing of capillaries and microthrombi in kidneys of recipient mice after bone marrow transplant. Many of these findings are similar to the findings in thrombotic microangiopathy in kidneys. Despite the pathologic changes in kidneys after BMT in mice, serum markers of kidney function remained within normal limits, consistent with the resistance of mice to immune and non-immune injuries to their kidneys ²¹.

We have previously shown that the complement system has a role in the pathogenesis of GVHD in mice and complement deficiency reduced the mortality and severity of GVHD ⁷. In the mixed lymphocyte reaction assays, a complement inhibitor reduced the proliferation and differentiation of alloreactive T-cells ⁸. In the current study, we examined whether complement activation also plays a role in kidney injury after BMT. We found the progressive deposition of complement proteins in kidneys after BMT in both C5BL/6 to B6D2F1 (Parent to F1) and BALB-c to C5BL/6 models of hematopoietic stem cell transplantation.

There are three complement pathways: the classical, alternative, and lectin pathways. These pathways differ in their initiation steps but converge on the formation of a C3 convertase that propagates complement activation. The C3 convertase complex cleaves plasma C3 to generate C3b that is deposited on the target cell membrane. The membrane-bound C3b participates in the serial activation of complement proteins (C5, C6, C7, C8, and C9) that eventually forms the C5b-9 complex (membrane attack complex). Other active end-products of complement activation are the anaphylatoxins (C3a and C5a) that mediate several biological functions. Unregulated activation of the complement system in atypical hemolytic uremic syndrome (aHUS) results in kidney injury. There are many clinical and pathologic similarities between aHUS and transplant-associated thrombotic microangiopathy (TA-TMA) ^13,14 and even mutations in genes encoding complement protein have been reported in TA-TMA patients ²². Pathologic findings in the kidneys in our murine models were similar to those reported in kidneys of patients with BMT-associated thrombotic microangiopathy.

From our studies, we concluded that the complement system is involved in the pathogenesis of kidney injury after BMT, and lack of a functional complement system (C3 deficiency in recipient mice) reduced the severity of kidney injury. Whether inhibition of the complement system reduces the severity or frequency of acute kidney failure after bone marrow transplant needed to be investigated in future studies.

Highlight.

• Kidney injury after bone marrow transplant (BMT) in mice is associated with a widespread glomeruli injury.

• Pathology findings in the kidney after BMT in mice are similar to those in patients with transplant-associated thrombotic microangiopathy.

• Kidney injury after BMT in mice is at least partially dependent on the activation of the complement system.