## 4 Discussions  
### Diagnosis and Prognosis of Mitochondrial Dysfunction in AAI-Induced Kidney Diseases  
Understanding the role of mitochondrial dysfunction in the etiology and pathogenesis of Aristolochic Acid Nephropathy (AAN) and Balkan Endemic Nephropathy (BEN), as well as diagnosing and treating these conditions, remains limited. Specific diagnostic tools for detecting mitochondrial impairment in AAN and BEN are lacking. Mitochondrial functional assays are typically performed on fresh renal tissue, posing a significant limitation for clinical diagnosis due to the invasive nature of obtaining these samples. Despite this, mitochondria represent a promising pharmacological target for patients with renal impairment, especially if novel and noninvasive diagnostic approaches are developed. Combining mitochondrial-targeted therapies with conventional treatments and appropriate lifestyle modifications could enhance patient outcomes.  
The scientific literature describes in vivo models of AAI-induced acute nephropathy in rats or mice and in vitro models where AAI cytotoxicity is tested on various cell lines and isolated mitochondria. These models reliably mimic the AAN condition observed in patients exposed to AAI through traditional medicines or contaminated foods. More than 90% of AAN patients consumed small amounts of AAI-containing medication for a median duration of 60 months. The nephrotoxic effects of AAI may manifest years after cessation, resulting in delayed diagnosis and treatment [10].  
A comprehensive set of investigations is required to confirm tubulointerstitial nephropathy. These include renal biopsy, evaluation of clinical parameters such as hemoglobin (Hb), serum creatinine (Scr), and urinalysis. Additionally, risk factors for renal dysfunction should be documented, including cumulative AAI intake, high blood pressure, diabetes, cardiovascular diseases, smoking, obesity, and family history of kidney diseases. Some  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Types of Samples** & **Imaging Investigations** & **Biochemical/ELISA** & **Western Blot** & \begin{tabular}{c} **Molecular Biology** \\ **Assays** \\ \end{tabular} & \begin{tabular}{c} **Immunology** \\ **Assays** \\ \end{tabular} & **Ref** \\ \hline C57 BL/6 mice & HE; IHC; TEM & PCr & - & AA-DNA & - & [36] \\ \hline \begin{tabular}{c} \begin{tabular}{c} \begin{tabular}{c} \begin{tabular}{c} \begin{tabular}{c} Immunol fluorescence \\ and fluorescence \\ microscopy \\ TEM \\ \end{tabular} \\ \end{tabular} \\ \end{tabular} & \
\begin{tabular}{c} - \\ - \\ \end{tabular} &
\begin{tabular}{c} Beclin-1, Atg5, PARP, \\ LC3 \\ \end{tabular} & - & Flow-cytometry & [37] \\ \hline \hline \end{tabular}
\end{table}
Table 1: _Cont._patients develop acute kidney injury (AKI) within three months of stopping AAI-containing herbs, leading to a diagnosis of acute AAN, with 50% progressing to chronic kidney disease (CKD) within four years of follow-up [10].  
Pathological features of different AAN subtypes are diagnosed through immunofluorescence staining of renal biopsy specimens from patients with acute, chronic, and incipient tubular dysfunction. In acute AAN cases, light microscopy reveals tubular epithelial cell vacuolization or granulation. Electron microscopy can reveal disruption of the tubular brush border, mitochondrial and endoplasmic reticulum swelling, rupture of mitochondrial cristae, and an increased number of lysosomes. Acute AAN involves less tubular necrosis compared to chronic AAN, which shows patchy or diffuse tubular epithelial cell atrophy. Acute AAN cases, characterized by a large cumulative AAI intake (1.04 g) over a short period (14 days), exhibit different features compared to chronic AAN. In chronic AAN, cumulative AAI intake and the duration from cessation of AAI-containing medication to follow-up onset are independent factors correlated with the rate of eGFR decline, depending on the length of the period the patient had stopped taking AAI-containing medication [10].