Autografting of Renal Progenitor Cells Ameliorates Kidney Damage in Experimental Model of Pyelonephritis

Abstract

Current therapies for pyelonephritic renal damage have severe limitations; stem cells may offer an exciting potential in regenerating nephrology. We aimed to investigate the feasibility of direct intrarenal injection of autologous renal progenitor cells (RPCs; originated from epithelial cells in Bowman’s capsule) in chronic pyelonephritis rat model. Twenty-seven rats were divided into three groups. The control group (GI, n = 3) underwent sham subcapsular injection of isotonic saline. Pyelonephritis was induced in the right kidney of the remaining 24 rats and isotonic saline (GII, n = 12) or labeled autologous RPCs, obtained from a biopsy of left kidney (GIII, n = 12), were injected into the subcapsular space 6 weeks later. At 7, 14, 28, and 60 days, dimercaptosuccinic acid scan was performed in three animals of each group at every interval and subsequently renal sections were obtained for the evaluation of tubular and glomerular regeneration and proliferation. Cell transplantation resulted in the reduction of tubular and glomerular atrophy after 2 weeks. The transplanted cells were observed in the reconstructed region of the kidneys as evidenced by the presence of fluorescently labeled cells both in tubules and glomeruli. We also observed significant decrease in interstitial fibrosis in the fourth week and there were higher amount of Ki-67-positive cells in GIII. Notably, the right renal tissue integrity was significantly improved in this group and revealed normal cortical function on day 60. Transplanting RPCs showed the potential for partial augmentation of kidney structure and function in pyelonephritis. Cellular repair was seen predominantly in the proximal tubule, the major site of injury in pyelonephritis. Our findings may pave the way toward the future regeneration of renal scarring of pyelonephritis in children.

INTRODUCTION

Pyelonephritis is characterized by severe inflammation, renal functional impairment, and ultimately glomerular and tubulointerstitial scarring. Currently, the mortality rate in children with pyelonephritis has dramatically decreased. Nevertheless, it still remains a major worldwide cause of end-stage renal failure in approximately 8–20% of affected children and young adults (25). Conventional strategy for prevention of scar formation was immediate initiation of antimicrobial agents (22). More recent approaches have involved radical scavengers and anti-inflammatory materials such as vitamin E, allopurinol, and prednisolone administration immediately after bacterial inoculation (3,10,19). However, they all mandate early intervention, which is often difficult in clinical practice due to initial nonspecific presentations of pyelonephritis and the delay may lead to irreversible renal scar and subsequently chronic renal disease. In this regard, despite the use of modern dialysis techniques and kidney transplantation, renal failure is still characterized by high mortality and morbidity.

On the other hand, current advancement in stem cell therapy and tissue engineering hold a great promise in regenerating nephrology (15,16). The extracorporeal renal tubule assist device that utilizes a bioreactor cartridge containing donors’ proximal tubule cells has improved survival in preclinical studies and clinical trial in patients with acute renal failure (ARF) in intensive care units (6). This short-term technique has significant drawbacks in terms of morbidity, availability, and cost. This situation warrants the development of effective treatment modalities. Stem cells as starting material offer powerful strategies for kidney repair and engineering with the capacity to replace a wider range of the kidney function.

Recently, attention has been focused on the role of kidney adult stem cells with the capability of homing and integration to the injured kidney. The technical availability of these cells as well as immunological acceptance and survival introduce them as an appropriate candidate for this purpose. However, the origin of renal progenitors has not yet been defined clearly. Intrarenal progenitor cells (RPCs), not bone marrow-derived stem cells (BMSCs), have been recommended for augmentation of kidney structure after ischemic ARF (14). Fully differentiated cells, tubular progenitors in renal intersti-tium, and progenitors within glomerular structures were reported as the cellular source in different studies (1,2). Sagrinati et al. demonstrated that a subset of parietal epithelial cells in Bowman’s capsule has the potential to generate functional tubular cells (21). Moreover, they showed a significant amelioration in renal morphology and function proposing a role for the transplanted cells in regeneration of different portions of the nephron in ARF (21).

These findings prompted us to investigate whether subcapsular injection of RPCs originated from parietal epithelial cells in Bowman’s capsule contribute to the kidney regeneration in a chronic pyelonephritis rat model. We evaluated kidney repair using histological parameters (alterations in renal tubules and glomeruli morphology and degree of interstitial fibrosis) and renal tissue integrity in renal scan.

MATERIALS AND METHODS

Experimental Design

The Animal Ethics Committee of the Tehran University of Medical Sciences, School of Medicine, Education Section of Basic Sciences approved the experimental protocol. All animals were treated in compliance with the principles of NIH guide for the care and use of laboratory animals. Twenty-seven juvenile male Sprague-Dawley rats (140–170 g) were allocated into three groups. In the control group (GI, n = 3), rats received subcapsular injection of isotonic saline. In the pyelonephritis group (GII, n = 12), pyelonephritis was induced in the right kidney and isotonic saline was injected into the subcapsular space after 6 weeks. In the cell transplanted group (GIII, n = 12), right-sided pyelonephritis was induced and rats underwent an open biopsy of the left kidney for glomeruli harvesting 4 weeks later. The isolated RPCs were autografted into the subcapsular space of the right kidneys after 2 weeks. At 7, 14, 28, and 60 days after subcapsular injection, renal scan was performed in three animals of GII and GIII at every interval; subsequently they were sacrificed and renal sections were obtained for immunofluorescence and immunohistochemistary.

Pyelonephritis Model

Under general anesthesia right kidneys were exposed by a dorsal retroperitoneal approach and an inoculum (1 × 10⁹ colony-forming units) of E. coli (ATCC 25922) was injected gradually through the cortex down to the medulla in the superior and inferior poles. The left kidneys were intact in all animals. Kaye first described this procedure to induce a constant and severe pyelonephritis in the affected right kidney with extensive inflammation and subsequent scarring (11). All of the animals were given intraperitoneal injection of ceftriaxon, 30 mg/kg once daily, for 5 consecutive days beginning on the third day after bacterial inoculation.

Isolation and Culture of RPCs Originated From Parietal Epithelial Cells in the Bowman’s Capsule

Four weeks after pyelonephritis induction a renal biopsy from the contralateral kidneys was performed in GIII through a dorsal incision. Renal progenitor cells were isolated exploiting glomerular explant technique according to Sagrinati et al. (21). Briefly, after mincing the cortex, the glomeruli were isolated using a graded sieving technique. The glomeruli were suspended in proliferating medium (EGM-MV; Lonza) and cultured in fibronectin-coated dishes (Sigma-Aldrich).

To further evaluate the differentiation capacity of the isolated RPCs, tubulogenic differentiation was induced by culturing the clones of expanded cell in differentiation media (EGM-MV, hydrocortisone, epidermal growth factor, insulin, triiodothyronine, transferrin, hepatocyte growth factor) for 25–30 days.

Autologous RPC Transplantation

The outgrowing RPCs were labeled with PKH-26 red fluorescent cell-linker kit (Sigma-Aldrich) and used for cell implantation process after 2 weeks of culture. The right kidney was exposed as described for modeling and 1.7 × 10⁶ cells were injected into the subcapsular space.

Immunofluorescence and Immunocytochemistry

Expression of PAX-2 and CD24 (Santa-Cruz Biotechnology) by immunofluorescence, and CD34 and CD45 (Dako Cytomation) using Immunocytochemistry were used for characterization of the isolated cells. The tubulogenicity was determined using antirenal cell carcinoma antigen (RCC, Abcam).

After sacrificing the animals the right kidneys were harvested and cut sagittally; one for evaluation of PKH-26, another for embedding in paraffin, which was used for all staining process. Histopathological assessment was performed by an experienced renal pathologist who was unaware of the treatment protocol. Microscopic examination consisted of three main histological criteria: inflammation, cell proliferation, and fibrosis. Hematoxylin and eosin sections were evaluated for inflammation (scored as 0, none; 1, mild; 2, moderate; 3, severe/focal; 4, multifocal) and Masson’s trichrome sections for interstitial fibrosis (scored as 0, none; 1, mild; 2, moderate, 3 severe/multifocal) using a semiquantitative comparison between three groups. Periodic acid-schiff stained kidney sections were assessed for the alterations in renal tubules and glomeruli morphology representing tissue reconstruction. Moreover, all of the specimens were examined using immunohistochemical methods for Ki-67, CD31, and CD34 (DakoCytomation).

Donkey anti-goat IgG-FITC (Santa-Cruz Biotechnology) was used as a secondary antibody when needed.

Dimercaptosuccinic Acid Scan Findings (DMSA)

Ten MBq of ⁹⁹mTc DMSA was injected in the tail vein. After 3 h, anterior and posterior static images were obtained over 15 min using a high-resolution collimator. Renal scars were graded as described previously (18). Briefly, the extent of injury was considered according to the following criteria: 0, no damage; 1, less than 33% damage; 2, 33–66%; 3, more than 66% damage.

Statistical Analysis

Statistical analyses were performed using SPSS statisticalsoftware (version 17.0). Comparison between groups was performed by the Kruskal-Wallis test. The significant Kruskal-Wallis test was followed by Mann-Whitney U-test for comparisons of several groups with the pyelonephritis group to determine which of the groups are different. The results were considered significant only with a value of p < 0.01.

RESULTS

Establishment of Pyelonephritis Model

The kidneys in the pyelonephritis group showed characteristic depressed linear scars in all time courses. Moderate-to-severe inflammation, glomerular atrophy, and tubular degeneration, which all characterized the development of scar tissue, were found in all the rats of this group. Renal scan and pathological parameters did not show any change during different time points of the study in this group.

Characterization of RPCs

The isolated capsulated glomeruli were adhered to the fibronectin-coated surface after 2 days of culture, and subsequently the cellular outgrowth was noted at the 7th day (Fig. 1A). The positive CD24 staining confirmed that the outgrowing cells are multipotent progenitors with high cell purity (Fig. 1B). Additionally, they expressed PAX-2, an embryonic renal marker, suggesting their renal origin; immunofluorescence study revealed the typical nuclear staining for Pax-2 (Fig. 1C). They lacked the expression of hematopoietic markers (CD34, CD45), and also were negative for RCC antigen, indicating the absence of tubular cells (Fig. 1D–F).

Figure 1.

Characterization of progenitor cells originated from parietal epithelial cells in the Bowman’s capsule (scale bar: 100 μm). (A) Phase contrast microscopy image of outgrowing cells from attached glomeruli at day 7 (original magnification 400×). (B) Immunofluorescence examination reveals that all the outgrowing cells are CD24 positive at day 7. (C) Immunofluorescence study using anti Pax-2 antibody revealing typical nuclear staining at day 5. (D, E, F) Negative immunocytochemistry analysis for CD34, CD45 (hematopoietic markers), and renal cell carcinoma antigen (RCC; tubular marker) at day 7, respectively. (G) Positive RCC staining on differentiated cells at day 28.

To further investigate the capacity of the RPCs to differentiate to the kidney structure, we exclusively conducted the tubular differentiation procedure (see Materials and Methods). Consequently, RCC, a marker of renal tubules, appeared on the cells after 28 days (Fig. 1G).

Contribution of RPCs in Kidney Regeneration

Red fluorescent-labeled cells were present both in tubules and glomeruli in GIII at all the time points. Seven days after injection, PKH-positive cells populated the renal cortex and migrated toward the medulla. Quantification of the number of fluorescently labeled cells revealed that 43.7% of PKH-positive cells were detected in reconstructed tubules, 15.1% in the glomeruli, and the remaining in the interstitium in the 28th day (Fig. 2A).

Figure 2.

Contribution of renal progenitor cells in kidney regeneration (scale bar: 100 μm). (A) PKH-26 red fluorescent cell linker for localization of the engrafted cells. (B) Hematoxylin and eosin staining to evaluate inflammatory cells infiltration. (C) Periodic acid-schiff staining for structural study. (D) Masson’s trichrome staining for fibrosis. (E) Ki-67 expression. (F) CD34 staining. (G) CD31-positive microvessels.

Light microscopic examination showed reduced tubular and glomerular atrophy in GIII after 2 weeks when compared to GII, while moderate-to-severe local inflammation still persisted (Fig. 2B, C). In trichrome study we observed lower interstitial fibrosis in GIII in comparison with GII in the 4th and 8th week after cell injection (Fig. 2D).

Evaluation of periodic acid-schiff sections showed significant decrease in the percentage of abnormal scarred glomeruli and tubules along with the normal morphology of the existing structure in GIII in comparison with GII after 4 weeks.

Interestingly, measurement of cell proliferation marker Ki-67 revealed early tubular and glomerular regeneration 1 week after cell injection. There were strikingly higher amounts of Ki-67-positive cells in GIII than in the pyelonephritic group. In this group the proliferation rate remained elevated after 8 weeks, although lower than the initial levels (Fig. 2E).

The universal marker for hematopoietic stem cell, CD34, was used to evaluate any probable role of peripheral blood stem cell in renal reconstruction. Less than 1.8% of cells were positive for CD34 in GII and GIII in the first week and it reached control levels after 1 month (Fig. 2F). Neovascularization was determined using CD31 for microvessels. Initially, CD31-positive microvessels were detectable in pyelonephritic rats as well as the cell grafted kidneys. This value was still elevated in the cell-grafted kidneys after 2 months (Fig. 2G). The microscopic findings are summarized in Table 1.

Table 1.

Inflammation and Fibrosis Grade, Proliferation Rate (Number of Ki-67-Positive Cells per High Power Field), and Renal Integrity Using Dimercaptosuccinic Acid (DMSA) Scan of the Right Kidneys in the Study Groups

Parameter	Control	Pyelonephritis	Cell Transplant 1 Week	Cell Transplant 2 Weeks	Cell Transplant 4 Weeks	Cell Transplant 8 Weeks	p-Value*
Inflammation	2.17	15.67	14.50	10.17	8.00	6.50	0.011
p-Value†	p < 0.001		p = 3.00	p = 1.50	p < 0.001	p < 0.001
Fibrosis	3.50	15.50	14.00	12.50	6.50	5.00	0.011
p-value†	p < 0.001		p = 3.00	p = 1.50	p < 0.005	p < 0.001
Proliferation	2.50	4.50	17.00	14.00	11.00	8.00	0.005
p-value†	p = 1.50		p < 0.001	p < 0.001	p < 0.001	p < 0.005
Renal damage	3.00	16.00	13.00	13.00	7.50	4.50	0.008
p-value†	p < 0.001		p = 1.50	p < 0.005	p < 0.001	p < 0.001

Data expressed in mean rank of the parameter for each group.

^*Kruskal-Wallis test.

^†Mann-Whitney U-test in comparison with pyelonephritis group.

Dimercaptosuccinic Acid Scan Finding

In pyelonephritic group renal scarring was higher than controls (Fig. 3A). The engraftment of renal progenitor cells resulted in marginally better right renal integrity compared with pyelonephritic kidney after 1 week. Notably, the renal tissue integrity in the cell-implanted rats was noticeably improved in 4 and 8 weeks after transplantation and revealed normal cortical function on day 60 (Fig. 3C).

Figure 3.

Right renal tissue integrity on dimercaptosuccinic acid (DMSA) scan and macroscopic examination. (A, B) Pyelonephritic rats treated with antibiotics. (C, D) Cell-engrafted kidney after 8 weeks; note the normal cortical function.

DISCUSSION

Compensation for the damaged glomeruli and tubules along with the capability for the reconstruction of functional kidney structure in chronic pyelonephritis remains a challenging task. Renal scarring after acute pyelonephritis in children has been reported with incidence of 10–65% (18,23). Current management of pyelonephritis consists of early intervention to ameliorate the acute inflammatory processes and subsequently renal scarring. Anti-inflammatory and antimicrobial agents are the integral part of the standard treatment with varying degree of success (10,19).

Renal replacement therapies such as dialysis and allogenic transplantation remain the most common treatment option despite their significant complications and cost.

The extracorporeal renal tubule assist has shown promising result in the setting of dialysis-dependent ARF (6). However, the leading advantages of this bioartificial kidney are replacing filtration, transport, metabolic, and endocrine functions of the kidney in acutely uremic patients. Obviously, an extension of this approach to chronic renal failure is largely limited by high cost, availability, and long-term dependency to this device.

The concept of exploiting novel renal replacement modality with the capacity to replace a wider range of kidney functions has led us to evaluate the contribution of subcapsular implantation of autologous RPCs in chronic pyelonephritis. The present study demonstrated significant kidney tissue repair 2 weeks after transplantation of RPCs. We used a highly specific population of cells that demonstrated stem cell features and excellent immunologic acceptance. The PKH-labeled cells were evident in the reconstructed regions of the kidneys during follow-up, indicating role of the injected cells as well as a reasonable cell survival. We demonstrated that the cells were integrated in the damaged tubules and glomeruli and replaced structures that have been destroyed following pyelonephritis induction. Considering the trend of improvement in renal tissue integrity, we propose that the kidney augmentation was mainly due to functional tissue regeneration following cellular transplantation.

The regenerative potential of different stem cells has been explored in ARF (12,16). In this regard, the possibility for participation of BMSCs in kidney repair has been widely postulated. Early experimental studies of BMSCs in ARF have shown that they can contribute to renal remodeling (5,7,9). Later, it was demonstrated that although infusion of BMSCs might limit the initial rise in BUN levels following ARF, improvement in renal function to greater than controls was not documented (8). Togel et al. revealed that BMSCs partially reversed the increase in creatinine after ischemic injury but no direct engraftment to renal tubules was reported (24). They proposed paracrine and/or endocrine effects for BMSCs. Moreover, they implicated that the cells might promote RPCs differentiation into kidney structure (17). It is worthy to note that Kunter et al. provided evidence regarding mal-differentiation of BMSCs into adipocytes, which may lead to glomerular sclerosis in the long term (13). However, recent studies demonstrated that intrare-nal cells, not BMSCs, seem to be responsible for cellular reconstitution of the nephron following ARF (4,14).

Considering the growing body of evidence, it is imperative to note that BMSCs may be beneficial in recovery from ARF they seem to be unproductive in chronic renal failure. In this setting, renal insufficiency mainly is the consequence of substantial irreversible nephron loss, compromised by tissue fibrosis.

All these obviate the need for introducing appropriate cell type for kidney repair. Kidney-specific stem/progenitor cells might be the most appropriate candidate because of their inherent organ-specific differentiation and the possible capacity to modulate tissue remodeling in chronic nephropathies. The proposed mechanism could be homing to the injured kidney, proliferation, and differentiation or integration in the damaged structure to physically replace the destroyed structure.

In the present work we used the kidney progenitor cells originated from parietal epithelial cells in the Bowman’s capsule. Bussolati et al. demonstrated the existence of this population of resident multipotent cells in adult human glomeruli. They showed the regeneration of tubular structures of different portions of nephron following injection of this cell type in a model of ARF. Injection of these cells to glycerol-induced acute tubular necrosis model led to BUN levels that were not significantly different from normal animals (9).

Interestingly, in present work we observed RPCs both in tubules and glomeruli, though mainly in tubules, indicating a possible role for these cells to contribute to cellular reconstitution of the damaged nephrons. The large amount of Ki-67-positve cells, a cell cycle regulator protein strictly associated with cell proliferation, confirmed the ongoing intensive proliferating activity in the reconstituted regions of the transplanted kidneys. Our follow-up revealed a declining trend in Ki-67 expression.

Considering this point that progression of renal fibrosis is the common ultimate pathway leading to end-stage renal disease (26), amelioration of the fibrosis might be of major clinical relevance in the setting of chronic pyelonephritis. In our study, severity of chronic renal scarring was ameliorated in the cell-treated group. Although minor neovascularization was reported in the pyelonephritis group (GII), the ongoing microvessel formation in cell-engrafted kidneys (GIII) might indicate the promoting role of RPCs for endothelial differentiation. Moreover, DMSA scan findings were consistent with the histopathological results proposing satisfying functional kidney regeneration. It was observed that engraftment of RPCs led to normal cortical function on day 60 while no significant change in cortical function was noted in the pyelonephritis group during the time course. This finding demonstrated the essential role of RPCs for renal repair rather than the natural tendency of pyelonephritic kidneys to recover spontaneously.

Although the regenerative potential of different stem cells has been explored widely in animal models of ARF (16), limited preclinical and clinical application of cell-based extracorporeal renal tubule assist device has been reported in patients with ARF in intensive care unit. On the other hand, no report of stem cell-based management exists in chronic renal failure in patients. The important perquisites for introduction of this method in management of patients with pyelonephritis are the easy accessible autologous source of the cells with acceptable plasticity and proliferation capability, which roots in the natural healing process of the kidney. The cells should have the minimal risk of mal-differentiation in the kidney environment and this might be provided by the tissue adult stem cells. In this regard, we used autologous RPCs that were obtained from a renal biopsy to assess the feasibility of this method in management of patients with pyelonephritis. We propose that the tissue can be obtained via laparoscopic or percutaneous biopsies. The probable clinical application might be in patients with renal damage due to previous pyelonephritis leading to deteriorated renal function. While the biopsy should be obtained from a scarred kidney, availability of areas with reliable kidney structure is possible; although their clearance capacity might be below the threshold for maintaining normal kidney function. The capacity for efficient in vitro expansion of the isolated autologous RPCs may contribute to acceptable clinical outcomes. Additionally, subcapsular implant technique for cell transplantation was chosen, which provides a relatively simple approach. One important character of such a technique is the easy access in human kidneys, which warrants its clinical applicability in the setting of kidney augmentation. Cell-to-cell and cell-to-matrix interactions may play roles for integration of RPCs to the suitable niche.

In the current study the change in creatinine as an index of renal function has not been considered. For including creatinine as a parameter there was a need for the complete elimination of the renal function, which would mandate application of a renal replacement modality in the rats and in our setting this was not practical. Additionally, determination of kidney clearance function in laboratory animals (i.e., inulin infusion, etc.) is limited by technical difficulty and complexity (20).

It is worth noting that studies concerning stem cell application tend to have small sample sizes, especially when animals are used as subjects. In the current study this limitation makes meaningful statistical comparisons difficult, though possible.

The current study was performed in a rodent model. Further studies in larger mammalian models, including sheep and monkey, may confer more clinically reliable outcomes. They offer more similarities to humans in the process of nephrogenesis and the kidney structure than rodents and consequently they might be better suited to correlate changes in histology and renal function.

Although we have demonstrated engraftment of renal progenitors in the injured kidney, it remains to be determined whether they would continue to survive and differentiate over time. Additionally, the ability of the regenerated nephrons as functional units of kidney should be elucidated in the future via a long-term study, though the trend of improvement may ensure a promising functional outcome.

CONCLUSION

These results provide proof-of-principle evidence that autologous RPCs have the capacity to contribute to cellular reconstitution in chronic pyelonephritis. Cellular repair was documented predominantly in the proximal tubule, a major site of cellular injury in pyelonephritis. This modality might be clinically applicable with the potential for organization into a functional kidney structure and may afford a proper renal replacement therapy with a notable impact on the treatment of renal failure.