**2. Kidney Tubular Stress Test Assessment**

Human organ systems have developed the capability to increase their workload in stressful situations. The analysis of this reserve capacity is a useful tool to uncover subclinical disease [16]. Reserve capacity tests are widely applied to study other pathologies, such as coronary artery disease (dobutamine stress echocardiography or exercise electrocardiogram tests). The kidney reserve capacity is built upon two main components, glomerular and tubular (Figure 1). The degree of injury of each component in AKI or CKD can present great variability and be completely independent. Therefore, testing both components could help describe the underlying pathophysiological process with much greater accuracy.

Glomerular reserve testing has been described in detail, but is sparingly used in day-to-day clinical practice. In brief, GFR, which is commonly used as a surrogate of kidney function, depends on age, sex, weight, or diet and presents great fluctuation among individuals. In healthy subjects, a protein load of 1–1.2 g/kg can induce a considerable increase of GFR above its baseline in 60–120 min. The difference between baseline and maximum GFR is considered the renal glomerular function reserve and is directly associated with stress-associated nephron recruitment and increased renal blood flow [16]. However, the lack of large cohort studies that may help describe the population variability of renal glomerular function reserve is an important limitation of this test.

The study of tubular reserve capacity is a relatively new diagnostic tool whose clinical application holds great potential [16,29]. The tubules and tubulointerstitium occupy a significant portion of the kidney and are responsible for a wide variety of functions, such as water and electrolyte handling, secretion of endogenous and exogenous acids, and protein catabolism. Tubular epithelial cells (TECs) regulate tubulointerstitial inflammation and are key mediators of tissue repair and fibrosis processes due to their capability to release cytokines, chemokines, and reactive-oxygen species [16,30]. Damaged TECs facilitate tubulointerstitial inflammation due to their capability to modulate the immune response through the formation of several proinflammatory cytokines such as interleukin-6,

interleukin-34, or tumor necrosis factor alpha [30,31]. The consequent macrophage infiltration and neutrophil recruitment can aggravate tubular cell injury and perpetuate the pro-inflammatory response to AKI [32,33]. Furthermore, TECs can endure adaptative changes after kidney injury and modify their structure and phenotype, increasing the production of profibrotic factors that stimulate fibroblast proliferation, tubular cell de-differentiation, and epithelial–mesenchymal transition such as connective tissue growth factor, tubular growth factor beta, or renin-angiotensin components [34–36]. The epithelial–mesenchymal transition of TECs is a potential key point in the progression of renal fibrosis. This phenomenon involves the replacement of epithelial-type markers, such as E-cadherin or cytokeratin, for mesenchymal-type markers such as vimentin, fibronectin, or type I collagen [30,37].

**Figure 1.** Renal reserve capacity testing.

Recent studies have also described the role of incomplete healing of tubulointerstitial damage as a possible link between AKI and CKD. Although TECs have several repair mechanisms to facilitate full recovery after injury, the repair process can be halted in an intermediate phase, inducing cell atrophy and fibrosis [38]. Mitochondria also play a significant part in AKI associated cell death. The use of SS-31, a drug with mitochondria-protecting effects, has been shown to diminish the development of ischemic damage and interstitial fibrosis in the kidney [39]. Additionally, disturbances of TEC energy-producing pathways, fundamentally fatty acid oxidation, can induce cell death, fibrogenesis, and inflammation [40].

The main tool to assess tubular function is to study tubular secretion of an endogenous or exogenous substance, such as creatinine or furosemide. Salt or acid loading can be used to analyze the efficiency of the tubule to eliminate sodium or acid, while water deprivation or the administration of desmopressin would serve as tests of the concentrating capabilities of the tubule [16].
