Blind Spot in the Radar of MEST-C Score: Type and Severity of Tubulointerstitial Nephritis in IgA Nephropathy

Background The updated version of predictive classification for immunoglobulin A nephropathy (IgAN) prognosis “The Oxford Classification” identifies five histopathological features including mesangial hypercellularity (M), endocapillary proliferation (E), segmental glomerulosclerosis (S), tubular atrophy/interstitial fibrosis (T) and crescents (C), the MEST-C. However, few studies suggest that tubulointerstitial inflammation, which is not included in the MEST-C, is also linked to disease progression and is, consequently, a neglected determinant of prognosis among others. Therefore, there is a need to evaluate this histopathological parameter in patients with IgA nephropathy. Materials and Methods This cross-sectional descriptive study was conducted at Shaukat Khanum Memorial Cancer Hospital and Research Center, Lahore, Pakistan. Data of histopathological and immunofluorescence proven renal biopsies (300) of IgA nephropathy patients from January 2016 through May 2022 were extracted using a convenient sampling technique. Biopsies were histologically reviewed for type and severity of tubulointerstitial inflammation, in addition to the MEST-C score. Renal biopsies of patients who had a history of transplant, autolyzed tissue, no glomeruli on histological examination, and/or a tubular atrophy/interstitial fibrosis score of 2 (T2) in MEST-C scoring were excluded. Data were analyzed using SPSS 20. An association between the variables was analyzed using the chi-square and Fischer exact tests. A p value less than 0.05 was considered statistically significant. Results A total of 247/300 biopsies were eligible for inclusion. The mean age at the time of biopsy was 31.90 ± 12.48 with 63.6% in the age group between 21 and 40 years, and 69.6% were male. Tubulointerstitial inflammation was observed in 90.2% cases with 49.4% showing moderate while 4.5% showing severe degree of inflammation. A strong association of both the type and severity of tubulointerstitial inflammation was found with M, E, T, and C scores (p value < 0.05). Conclusion The high-frequency and strong statistical association of tubulointerstitial inflammation with the M, E, T, and C scores in our study elucidate its prognostic role in the progression and management of IgA nephropathy.


Introduction
Immunoglobulin A nephropathy (IgAN), frst described by Jean Berger in 1968, is the most commonly recognized primary glomerulonephritis worldwide. It has heterogeneous pathological and clinical presentations, leading variably to end-stage renal disease. It has been known to occur in all age groups and ethnicities [1,2]. In order to determine and implement an earlier precise treatment intervention, it is imperative to accurately recognize the clinical and pathological fndings and predict the prognosis. For this purpose, various classifcation systems have been suggested over the years. One such predictive histopathological classifcation for IgAN prognosis was published in 2009: Te Oxford Classifcation. Tis classifcation has been validated in different ethnicities to predict prognosis independent of the clinical features, and the updated version identifes fve histopathological features including mesangial hypercellularity (M), endocapillary proliferation (E), segmental glomerulosclerosis (S), tubular atrophy/interstitial fbrosis (T), crescents (C), the MEST-C. Te MEST-C score has been studied and authenticated in both the pediatric and adult study groups and has assisted nephrologists to better evaluate the course of end-stage renal disease. It was established that the Oxford classifcation may also help in risk stratifcation and recruitment of patients into clinical trials for immunosuppression and other specifc treatments based on the identifcation of more specifc pathological features [3][4][5][6].
In this context, few studies suggest that tubulointerstitial infammation, which is not included in the MEST-C score, is a specifc pathological feature that is linked to disease progression and is, consequently, a neglected determinant of prognosis among other well-known histological features. It is suggested that parameters related to tubulointerstitial infammation predict a decline in renal function in IgA nephropathy patients [7,8]. In this regard, the immunohistochemical expression of tubulointerstitial LCA, CD3+Tlymphocytes, CD68-positive macrophages, and IL-1β has been studied and associated with the outcome. By establishing the MEST-C score in addition to the type and severity of tubulointerstitial infammation in renal biopsies of patients with IgAN, we can alarm and guide the clinician to accurately target the medical treatment to obtain signifcant beneft for such patients [8]. Terefore, there is a need to evaluate this histopathological parameter in renal biopsies of diferent populations with IgA nephropathy. In this article, we report the frequency and types of tubulointerstitial infammation in addition to the MEST-C score that we encounter in patients with IgA nephropathy and discuss their clinical relevance in conjunction with the established MEST-C score.

Materials and Methods
Patients with a renal biopsy diagnosis of IgAN between January 2016 and May 2022 were identifed from the archives of Shaukat Khanum Cancer Hospital and Research Center. Tese patients were diagnosed on the basis of histopathological and immunofuorescence features. Tis hospital has been serving a wide population of all provinces of Pakistan as well as a small proportion of Afghanis. Te available clinicopathological data was recorded from the biopsy reports including age and gender and reviewed for cumulative MEST-C scores, in addition to the type and severity of tubulointerstitial infammation. Te relevant clinical laboratory and follow-up data for most of the patients were unavailable or incomplete and therefore not included in the study. Patients with a history of renal transplant, autolyzed tissue biopsies, and biopsies without glomeruli were excluded. In addition, patients with a T2 score were also excluded so that tubulointerstitial infammation could be evaluated in an area of viable cortex. Te biopsies were reported by two pathologists. Te MEST-C score was evaluated according to the international guidelines (Table 1) [2,4,9]. Tubulointerstitial infammation was estimated in the viable cortex by the microscopic presence of acute and chronic infammatory cells (determined at 10x) in the interstitium and was categorized as absent, acute, acute and chronic, and chronic. Te severity of infammation was considered as mild, moderate, and severe by using the Banf criteria (mild 0-25%, moderate 26-50%, and severe >50%). A viable cortical area was obtained by subtracting the atrophic cortex from the total surface area of the biopsy with tubulointerstitial infammation.
After collection, the data were arranged and entered for analysis into computerized software called Statistical Package for Social Sciences (SPSS 20.0). Frequencies and percentages were presented as tables for both qualitative and quantitative variables. Clinicopathological parameters were associated with the type and degree of tubulointerstitial infammation using the chi-square test and Fischer's exact test. A p value <0.05 was considered statistically signifcant.

Results
A total of 300 cases of biopsy-proven IgA nephropathy were identifed. Of these, 53 cases having tubular atrophy/interstitial fbrosis (T) involving more than 50% cortical area (T2) were excluded. Of the remaining 247 cases, the mean age at the time of biopsy was 31.90 ± 12.48 with 63.6% in the age group between 21 and 40 years and 69.6% were male. Global sclerosis of the glomeruli was observed in 157 cases. Fibrous crescents were observed in 14, cellular crescents in 41, and fbrocellular crescents in 43 cases. Te frequencies of MEST-C score along with the type and degree of tubulointerstitial infammation are given in Table 2. Te relationship between the type and degree of tubulointerstitial infammation and clinicopathological parameters is shown in Tables 3-5, respectively. Tubulointerstitial infammation was found in 90.2% of cases. It was found that a signifcant percentage of patients (49.4%) showed a moderate degree of tubulointerstitial infammation at the time of diagnosis. A strong association was found between the type and degree of tubulointerstitial infammation with the M, E, T, and C scores (p value less than 0.05). Te severity of infammation and IgA immunofuorescence is depicted in Figures 1(a)-1(d).

Discussion
IgA nephropathy is the most common form of glomerulonephritis worldwide, yet the management of this disease remains challenging due to extensive variation in clinical practice and conficting data from published literature regarding the use of immunosuppressive therapy. Recognizing the pathological correlates of IgA nephropathy is critical for directing and planning prospective therapies so that maximum patients can obtain beneft. IgA nephropathy has a heterogeneous pathogenesis based on various clinical and pathological factors. It is credible to divulge that virtually no single pathway has a dominant role during disease progression, and diversity is also partly attributable to the genetics of various ethnicities. A few review articles summarize clinicopathological prognostic factors for IgAN. Histopathological parameters like widespread global and/or segmental glomerulosclerosis and marked tubulointerstitial lesions are strong predictors of disease progression [2,10]. Te Oxford Classifcation has proved to be a remarkable achievement for laying down the foundation to predict the prognosis of this diverse disease and the eforts of researchers are admirable that they continue to evolve it. Table  1: MEST-C criteria of IgA nephropathy [2,4,9]. Tubulointerstitial infammation is not included in the MEST-C score in Oxford classifcation. However, it has been studied to be associated with disease progression. A review of the published literature reveals that this was the frst study in Pakistan which was conducted on tubulointerstitial infammation in patients with IgA nephropathy until present.
In this study, we specifcally observed tubulointerstitial infammation in addition to the MEST-C score to observe the status of these parameters in IgAN patients in our population.
In addition to glomerular lesions, tubulointerstitial changes such as infammation were also seen in IgAN renal biopsies. Infammatory cell infltrates are considered to refect an active disease process. However, the type and severity of tubulointerstitial infammation have not been contemplated much as compared to the MEST-C score [8]. It was previously not included in the classifcation on the basis of the fact that infammation in the total cortex was closely correlated with the degree of interstitial fbrosis. In our study, 223 of 247 cases (90.2%) revealed tubulointerstitial infammation. Of these, 122 (49.4%) cases revealed moderate and 11 (4.5%) cases showed severe degree of infammation.
Te infammatory cell infltrate comprised of both acute and chronic types. Infammation was exclusively studied in an area of nonscarred/viable cortex, similar to the reported pattern of transplant biopsies. In this regard, patients with a T2 score were specifcally excluded. Frees et al. reported cellular infltrates in 89% of cases of IgAN, which were described as strong in 8 of these cases and predominantly comprised of lymphocytes alone or mixed with plasma cells. Tey found an association between these infltrates with an increased risk and a shorter progression period to renal failure in such patients [11]. Myllymäki et al. studied the parameters refecting tubulointerstitial infammation. Tey reported interstitial infammation in 25% of the cases and graded infammation as normal, mild, or marked. Tey studied the quantitative assessment of infammation using immunohistochemical expression of LCA, CD3, CD68, and IL-β in 204 patients and concluded that evaluation of the level of infammation and the grade of infammation can predict the renal outcome [8]. Both of the above-mentioned studies did not describe the area of cortex where infammation was reported. However, Rankin et al. demonstrated the assessment of active tubulointerstitial  infammation (ATIN) in nonscarred cortex by subtracting the cortex with tubular atrophy from the total area of the cortex harboring interstitial infammation. Tis overcame the correlation with interstitial fbrosis. Tey reported ATIN in 49% cases. Tey concluded that the presence of more than 10% of active tubulointerstitial infammation in nonscarred cortex was signifcantly associated with disease prognosis and correlated independently with prognosis as equal, or even better than the MEST-C score [7]. MEST-C parameters in the Oxford classifcation have been studied and validated previously for their prognostic role in IgA nephropathy. Mesangial hypercellularity, segmental glomerulosclerosis, tubulointerstitial lesions, particularly T1 and T2, and crescents are associated strongly with disease progression independent of laboratory and clinical parameters, and the predictive role of endocapillary lesions has been linked to immunosuppressive therapy [3,6,12]. Haas et al. reported that using corticosteroids can reverse the active lesions and improve the clinical picture [13]. However, the tubulointerstitial lesions (tubular atrophy and interstitial fbrosis) are not improved after immunosuppressive therapy [14]. Results of our histopathological   [7]. Infammation was more pronounced in our renal biopsies. Tis can be attributed to the lower socioeconomic status, higher illiteracy rate of our population, drug abuse and lack of awareness that these patients present late during disease. Tis may also be related to the fndings of a study conducted by Barbour et al. (2013) that Asian population has a higher risk of progression to end-stage renal disease [15]. However, for unexplainable reasons, no association was found with segmental glomerulosclerosis (p value >0.05), similar to the results shown by Rankin et al.
Tis is contrary to the results of other studies which validate the role of segmental glomerulosclerosis in renal outcome [2,3]. Te association of tubulointerstitial infammation with M, E, T, and C scores suggests that tubulointerstitial infammation is also linked to disease progression and may help in prediction of the disease progression and survival and hence guide therapy. Te major limitation of our study was a defciency of clinical laboratory data and follow-up of IgA patients.
Terefore, only histopathological parameters were studied. Unfortunately, the capacity for risk stratifcation into prognostic groups based on known predictive and prognostic factors in our health care system is too bare at present and therefore remains a therapeutic challenge. Tere is a lack of education and counselling among the patients who are also lost to follow-up. Patients destined to do well without immunosuppressive therapy might receive toxic therapies, and many other patients eventually develop end-stage kidney disease regardless of meticulous therapy. Research studies committed thus far have been impeded by variable evaluation techniques, small sample sizes, and a lack of clinical data or follow-up. For this purpose, it is imperative to create awareness among the patients as well as the clinicians in developing countries. In subsequent years, it is suggested for the clinicians to work in collaboration with the pathologists and formulate an improved standardized approach to evaluate potentially useful factors. A comprehensive approach to disease evolution will lead to robust management of this disease.
Heterogeneity in IgA nephropathy has both spatial and temporal ingenious facets that may be more diverse than is understood at present. Considering the vigorous studies conducted to validate the Oxford classifcation system, it is disappointing that an important histological parameter like tubulointertstitial infammation remains a blind spot. It is reasonable to evaluate it further in risk stratifcation, clinical trials, and follow-up studies to guide management.  International Journal of Nephrology Tubulointerstitial infammation in addition to the MEST-C score may behold future refulgence in the management of IgA patients.

Data Availability
Te patient data used to support the fndings of this study are restricted by the Institutional Review Board of Shaukat Khanum Memorial Cancer Hospital and Research Center in order to protect patient privacy. Te data are available from the corresponding author upon request, for researchers who meet the criteria for access to confdential data.