Histopathological and Immunohistochemical Study of Acute Tubular Injury in Native Kidney Biopsy

Introduction

The clinical syndrome of acute kidney injury (AKI) encompasses prerenal, intrinsic renal and post-renal causes with the categories based on dynamics of serum creatinine and urine output.^1,2 AKI is a serious condition which not only affects the kidney function leading to significant morbidity and mortality, but also has long-term detrimental effects on kidney function which is determined by the cause, duration and severity of injury, associated comorbidities and renal reserve.^3-5

Acute tubular injury (ATI) is likely to be responsible for majority of the cases of AKI requiring renal replacement therapy.⁶ The term ATI should be reserved for the clinicopathologic entity of intrinsic renal failure resulting from ischemic or toxic insult to the kidney with evidence of tubular injury and altered specific tubular injury markers.⁶

There are a multitude of tubular morphologic changes associated with ATI. There are very few studies correlating the histopathological features with clinical parameters. In one such study by Kudose et al.,⁷ a semiquantitative scoring system using features of tubular epithelial simplification, cell sloughing, and mitosis was developed. In the present study, we have used the same semiquantitative scoring system and correlated the scores and grades of tubular injury with serum creatinine, recovery status, and time to recovery. Comorbid conditions like age, hypertension, and diabetes, known to cause irreversible chronic injury to the kidney, were also correlated with the recovery profile.

Macrophages are highly heterogeneous and plastic inflammatory cells. When activated, they are polarized into different phenotypes (M1-proinflammatory and M2-pro repair) in response to injury. The polarized macrophages carry out specific functions by secretion of different cytokines and growth factors.⁸ The imbalance between M1 and M2 response or the persistence of M2 response is thought be to profibrotic.^9,10 In this study, densities of the different types of macrophages, CD68 representing all macrophages, HLA-DR representing the M1 and CD163, the M2 phenotype, were correlated with serum creatinine, grades of injury, and the recovery status.

Materials and Methods

This is a prospective study conducted at Apollo Hospital, Hyderabad. A total of 58 native kidney biopsies, in the study period of 6 months from July 2021 to December 2021, in patients presenting with AKI and with pure histological features of ATI on histology were included. Biopsies with coexisting features of glomerulonephritis, overt interstitial nephritis, malignant hypertension/ thrombotic microangiopathy, etc., and biopsies showing background diabetic nephropathy were excluded.

Light microscopy

Each biopsy was studied with the help of Hematoxylin eosin, Periodic acid Schiff, Jones methenamine silver, and Masson trichrome stains. Three morphological features of ATI were evaluated and scored according to the definitions and scoring system proposed by Kudose et al. as described below.⁷ Total injury scores were calculated and cases were graded as mild/equivocal/severe depending on the total scores obtained in each biopsy [Figure 1].

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Figure 1:

Top left: PAS Arrow showing simplification of tubular epithelial cell with loss of brush borders, flattened lining and relatively dilated tubular lumina. Top middle: PAS -Arrow showing cell sloughing with detached tubular epithelial cells within the tubular lumina. Top right: H and E-Arrow showing two mitoses in a tubular epithelial cell. Bottom left: IHC for CD68 -Cytoplasmic CD68 positivity in interstitial macrophages. Bottom middle: IHC for CD163 -Cytoplasmic CD163 positivity in interstitial macrophages. Note the CD163 positive cells lying close to and surrounding the tubular basement membrane. Bottom right: IHC for HLA-DR -Microphotograph showing HLA-DR positive interstitial macrophage. PAS: Periodic acid-schiff; IHC: Immunohistochemistry; HLA: Human leucocyte antigen; H&E: Hematoxylin and eosin.

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Results

Baseline demographic and clinical characteristics and the recovery profile of the patients are shown in Table 1. Only 6 cases were under the age of 6 years.

Discussion

A systematic review of clinical characteristics and histologic descriptions of ATI by Wen et al. emphasized the diverse histological features seen in ATI and a need for standardizing pathology reporting to objectively quantify the severity of tubular injury, to establish the markers for renal recovery, to stimulate research, and eventually to search for therapeutic options.¹² There are few studies which have tried to develop a scoring system based on the histological findings in ATI to look for clinical correlation and prognosis in native and transplant biopsies.^7,13-15 However, these scoring systems have not been widely assessed in large cohorts of native kidney biopsies, and the prognostic value of each histologic description remains unclear.

In the present study, we found that the severity of tubular epithelial simplification, cell sloughing, and mitosis in tubular epithelial cells positively correlate independently with the serum creatinine level. Similarly, the grades of tubular injury calculated using the same parameters also correlate positively with the serum creatinine values.⁷ This result, if validated in larger studies, could lead to the clinician looking for other causes of a raised serum creatinine where the tubular injury scores are low. Moreover, in the present study only biopsies with pure ATI were included and those with glomerular, interstitial or other causes of AKI are excluded. The results, therefore, can be attributed to the component of tubular injury and the contribution by glomerular and other causes to ATI are considered nullified. In a study of histological features of ATI with delayed graft function (DGF) in transplant kidney biopsies, Pieters et al. evaluated morphological characteristics like edema, casts, vacuolization, and dilatation with a scoring system. They found features of edema and casts to significantly correlate with prognosis. However, whether these results hold true for native biopsies is uncertain. They also found number of Ki67 positive cells correlated with severity of tubular damage.¹⁴ On the similar ground, we also found the number of mitosis in tubular epithelial cells had a positive correlation with the severity of injury. Pizov and Friedlaender also concluded that proliferative activity in transplant kidney biopsies did correlate positively with the tubular damage.¹⁶

In literature, there has been no single histologic feature that could predict recovery from ATI. In our study too, there was no significant correlation between the three histological parameters studied as also the derived grades of ATI, with the recovery status of the patients or with the time to recovery. In a small study of native kidney biopsies of patients with acute renal failure (ARF), Abdulkader et al. analysed four histological features semi-quantitatively (tubular atrophy, interstitial inflammatory infiltrate, interstitial fibrosis, and acute tubular necrosis [ATN]). They also found that no single histological parameter was associated with partial recovery, but the sum of all four was when expressed as an injury index.¹³ Note that chronicity indices (IFTA) and inflammatory response were included in their scoring system. Pieters et al. also quantified regenerative potential of tubular cells using Ki67 and CD133 index, but none of them correlated with renal functional recovery. This was attributed to maladaptive regenerative response of kidney tubules.¹⁴

Recovery from ATI depends on multiple factors like severity and duration of insult, inflammatory response to injury, background chronicity, and the patient-related risk factors like high age and comorbidities such as hypertension and diabetes mellitus.^5,17-20 Some of them indicate a reduced renal reserve. In consonance with these studies, our study also had an association of background chronicity, older age, hypertension, and diabetes with partial and delayed recovery from renal dysfunction.

With regard to the etiology, the numbers in each group were small with no single attributable cause in 17 cases. Besides, there was a significant overlap of the injury patterns in each group, and hence, further analysis etiology-wise was not pursued.

The limitation of the present study is that serum creatinine levels at the time of biopsy was considered and this may not be the best reflection of renal function. Biopsies are indicated in clinically severe AKI leading to skewed distribution of the studied cohort towards severe grade of ATI. Baseline creatinine and urine output were not known in all cases which are essential for staging clinical AKI. And lastly interobserver variability in scoring was not assessed.

Many studies have evaluated the role of macrophages in ATI and in progression to chronic kidney disease (CKD).^8,10,21-24 Macrophages are known for their plasticity. In response to injury and dominant Th1 response (Interferon-IFN-γ), there is polarization of macrophages to M1 type (HLA-DR +). M1 macrophages secrete proinflammatory cytokines like tumor necrosis factor alpha (TNF-α) and various interleukins. This is thought to be the first-line response. As the injury progresses, usually after 48 h, there is a predominant Th2 response and macrophages get polarized to M2 phenotype (CD 163+). M2 macrophages can be a “double-edged sword.” On one side, the anti-inflammatory phenotype [mediated by transforming growth factor-β1 (TGF-β1) and interleukin-10 (IL-10)] is essential for adequate tissue repair; on the other side, it is a potential mediator of fibrosis and scarring (mediated by TGF-β1).¹⁰ If the injury persists or is severe enough, and the nephron has reached a point of no return, M2 macrophage shows profibrotic phenotype and the potential to transdifferentiate into myofibroblasts. These events in turn lead to a profibrotic milieu and the disease progresses to fibrosis.¹⁷ Current pharmacological treatments that present beneficial effects in retarding progression of CKD diminish macrophages infiltration in the kidney including the SGLT2 inhibitors.²⁵

In response to injury, macrophages are recruited in the kidney from circulation. In the present study, we found that densities of CD68+, CD163+, and HLA-DR+ macrophages significantly increase in response to ATI as compared to MCD. Similar findings were noted by Palmer et al. evaluating interstitial macrophages in ATI. In addition, they also demonstrated M2 macrophages localized close to the tubular basement membrane of injured proximal tubule cell ultrastructurally.²⁴

Kim et al. performed the first human study demonstrating the possible role of macrophages in the injury and repair phases of AKI.²¹ They also found that the infiltration of both CD 68+ and CD163+ macrophages increased significantly in ATI, with the density of the CD68+ macrophages being significantly higher in advanced-stage AKI. We also found strong and positive correlation between severity of tubular injury and densities of CD 68+ and CD163+ macrophages.

There was no significant correlation with density of HLA-DR + macrophages and the severity of ATI in our study, probably because the biopsies were done late in the course of the disease, where M1 macrophages are thought to be the first responders to injury.¹⁰

In the present study, we noted that the patients with complete recovery had a slightly higher densities of CD68+ and CD163+ macrophages when compared with the partial recovery group; however, this difference was not statistically significant. These findings reinforce the fact that inflammatory or wounding response to injury is essential for recovery in the first place. Injuries which do not show adequate inflammatory response do not heal. Later, the imbalance between M1 and M2 macrophages is believed to decide the fate of the healing process to recovery or fibrosis.¹⁷ Kim et al. found that persistence of CD163+ macrophages predicted nonrecovery.²¹ In the present study, we did not find any association between the densities of CD68+ and CD 163+ macrophages and the recovery profile. This can be explained by the longer duration of follow-up and the difference in defining the recovery in the study by Kim et al.²¹

Conclusion

Presently, there is no objective grading system of ATI. The system we propose is simple and can be used, as it correlates with severity of injury, namely serum creatinine. Though our study has not shown a correlation of these scores and the macrophage response to recovery patterns, which is driven by multiple factors, we believe that similar studies with larger numbers and the study of more patterns of injury could help evolve scoring systems for prediction of recovery. Age >60 years, hypertension, diabetes, and chronicity score on biopsy correlated with partial and delayed recovery.