Along with the disease spectrum that evolved around the world over the past 30 years, diabetic kidney disease (DKD) has become the leading cause of end-stage kidney disease (ESKD) at daunting rates in both developed and developing countries (1, 2). Due to the increased risk of morbidity and mortality of DKD, the number of DKD related studies rapidly increased over the past two decades, with more than 27,500 papers published from 2000 to 2017 (3). Growing evidence suggests the underlying pathogenesis of DKD involves the renal proximal tubular epithelial cell dysfunction in a high glucose environment, oxidative stress, inflammation, fibrosis, and apoptosis (4). In addition, a large number of tubular biomarkers for DKD screening, diagnosis, and prognosis are tightly associated with the prognosis of the kidney in DKD, providing evidence for potential shifting of the paradigm from glomerulocentric to tubulocentric theory (5). It has been repeatedly shown that compared with the glomerular lesions, the extent of tubulointerstitial lesions correlates well with renal function, and the associated biomarkers have been identified (6). Urinary tubular injury markers may increase in patients with diabetes even before the onset of microalbuminuria (7, 8). Plasma tubular markers, which may reflect inflammatory and fibrotic responses, oxidative stress, and capacity of reabsorption in DKD, were also reported to be associated with early renal function decline and DKD progression (9, 10). Moreover, the biomarkers of tubulointerstitial function and structural changes were ultimately proven to be better predictors of disease progression and long-term prognosis than the current markers (11). Current prognostic markers of DKD have certain limitations. Estimated glomerular filtration rate (eGFR) and albuminuria are only modestly useful for risk prediction in type 2 diabetes mellitus (T2DM) patients with preserved renal function, and DKD progresses even in the absence of albuminuria (12, 13). Most importantly, inhibition of proximal tubule glucose transport via sodium-glucose cotransporter-2 (SGLT-2) has shown nephroprotective effects in a variety of large-sample, multicenter, placebo-controlled, and randomized clinical trials. By investigating the mechanism of the newest disease-modifying treatments for DKD, an accumulating body of research had documented the vital role of tubule function in regulating glomerular filtration through tubuloglomerular feedback. Moreover, the growth of the proximal tubule in the diabetic context supplies muscular strength to the established status of renal tubules in DKD (14, 15). The tubuloglomerular feedback mechanism begins with the theory that diabetic hyperfiltration and glomerular capillary hypertension are significant treatable stressors contributing to the progression of DKD (16–19). In diabetic conditions, the increased filtered load of glucose results in an increase in sodium-coupled glucose reabsorption by the proximal tubule. Decreased sodium delivery to the macula densa subsequently inhibits adenosine-triphosphate (ATP) conversion into adenosine, which results in the vasodilation of the afferent arteriole and the intrarenal activation of the renin–angiotensin–aldosterone system (RAAS), ultimately inducing glomerular hypertension and kidney hyperfiltration (15, 16). Hence, hyperreabsorption of water and solutes has a central role in the regulation of eGFR, highlighting the importance of alteration in the tubuloglomerular feedback for the development of DKD.

This review aimed to summarize the latest updates on the pathogenesis of renal tubular dysfunction in DKD, potential applications of tubular biomarkers, and renal tubule-targeting therapeutics based on evidence from recent trials in DKD.

In clinical practice, therapeutic strategies for early identification of the kidney lesions in diabetic conditions and consequent slowing of the progression of DKD are still limited and currently mostly rely upon conventional biomarkers. The urine albumin-to-creatinine ratio (uACR) and eGFR are well-standardized and widely used biomarkers for evaluating kidney function and determining different stages of kidney disease in clinical practice. Although carrying prognostic information, eGFR is subject to variation owing to the analytical error of the creatinine measurement and biological variation derived from serum creatinine, patient’s age, and gender (65, 66). ACR, a tubuloglomerular-centric marker, has been recognized as the hallmark of DKD and precedes renal function loss in years. It not only reflects the capacity of glomerular permeability but is also a valuable indicator of tubular damage or dysfunction. The increase in albuminuria followed by glomerular hyperfiltration places a burden on the proximal tubule and elicits an inflammatory response leading to tubulointerstitial damage (67). Nevertheless, a substantial proportion of patients with T1DM or T2DM have renal function impairment without proteinuria, which is known as non-proteinuric DKD (68–70). The data on clinicopathological characteristics, renal prognosis, and all-cause mortality are limited to a handful of clinical trials and longitudinal studies focused on this phenotype. In 2018, the Chronic Renal Insufficiency Cohort (CRIC) study showed that the absence of albuminuria or proteinuria was common and carried a much lower risk for ESKD, chronic kidney disease (CKD) progression, or rapid decline in eGFR than those with albuminuria or proteinuria did (71). In line with this, another propensity score-matched analysis of a nationwide, biopsy-based cohort reported that non-proteinuric DKD patients presented better-controlled blood pressure and fewer typical morphological changes. They were also at a lower risk of CKD progression and all-cause mortality (72). The possible mechanism of developing non-proteinuric DKD may rely on racial/ethnic differences, aging, and response to RAAS inhibitors or other glomerulus-protective drugs before the diagnosis of DKD (68, 73). Therefore, there is still a compelling need to discover potential novel biomarkers for early diagnosis and timely risk stratification in DKD.

Recent advancements in omics-based biomarkers including proteomics, metabolomics, genome, transcriptome, or lipidome and the integration of these different approaches continue to unveil new potential biomarkers (74). Urinary novel proteomics, peptidomics markers may be associated with impaired proximal tubular reabsorption that almost all of these filtered proteins are reabsorbed into the proximal tubules through megalin/cubilin-mediated endocytosis (75). One study also demonstrated that empagliflozin, the SGLT2i, significantly impacts urinary peptides (76). However, their detection is relatively expensive and still needs time to promote clinical use. Rigorous technical and clinical validation studies are demanded to clarify the specific role and the underlying mechanism. Future research in DKD should attempt to explain how the novel biomarkers can be combined with traditional clinical and biochemical biomarkers in clinical practice to guide screening programs, improve risk stratification, predict response to treatment, and provide a method of monitoring response to treatment. The tubular biomarkers in DKD are summarized in Figure 2 , which outlines three main classes of the principal tubular biomarkers that may be helpful in early detection and risk-stratification of DKD. The potential applications of these biomarkers in DKD were shown in Table 1 .

In addition to the new tubulocentric insights for DKD mentioned above, the emergence of new anti-hyperglycemic agents has considerably altered the therapeutic landscape of DKD. For decades, the cornerstone of DKD therapeutics relied on lifestyle interventions, strategies for hyperglycemia and hypertension in combination with the use of angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin receptor blockers (ARBs) (117). Recent advances in studies on novel glucose-lowering agents promote the new era in the advanced glycemic control and concurrently promise cardiorenal protection in DKD management. Figure 3 depicts the current high-profile classes of potential novel anti-hyperglycemic agents for DKD, mainly grouped into renal tubule-targeting therapies, incretin therapies, and energy pathways-targeting therapies (117, 118). The tubule-targeting medicine, SGLT2i also affects the energy pathway associated with enhanced sirtuin1 and hypoxia-inducible factor (HIF)-2a signaling (119). In addition to SGLT2i, incretin drugs include glucagon-like peptide 1 receptor (GLP1R) agonists and dipeptidyl peptidase 4 (DPP4) inhibitors, which also have the potential to improve tubulointerstitial function. GLP1R expression was detected in macrophages, endothelial cells, juxtaglomerular cells, and proximal tubules within the kidney in various animal models and human tissue (117). Endogenous GLP1R signaling exerts a natriuretic action in DKD. Direct GLP1R-stimulation induces diuresis and natriuresis by increasing GFR and inhibiting the activity of the sodium-hydrogen exchanger isoform 3 (NHE3) in the proximal tubule (120, 121). Nevertheless, DPP4 inhibitors demonstrate modest kidney-protective effects. Compared with the GLP1R agonists, they mainly attenuate albuminuria without an impact on eGFR decline. DPP4 inhibitors indirectly modulate glucose-dependent insulin secretion and suppress glucagon secretion from pancreatic α-cells by elevating endogenous GLP1 levels (122). Linagliptin, the only available DPP4 inhibitor, showed a significant improvement in albuminuria progression but not in kidney outcomes in the Cardiovascular and Renal Microvascular Outcome Study with Linagliptin (CARMELINA) trial (123). No significant placebo-adjusted changes in eGFR or albuminuria with linagliptin therapy were observed in the Modification of Albuminuria in T2D and CKD with the LINAgliptin (MARLINA-T2D™) study (124).

Among diabetic medications, SGLT2i attracts considerable attention for their pleiotropic effects on glycemic control, renal protection, cardiovascular benefits, blood pressure control, and attenuation of lipid levels. SGLT2 is a low-capacity and high-affinity glucose transporter with 1:1 Na+/glucose stoichiometry. It is located in the S1–2 segment of the proximal convoluted tubules and is responsible for reabsorption of 90% of glucose filtered through the glomerulus (125). Multiple mechanisms are explored involving the kidney protection of SGLT2 inhibition, mainly characterized into (1) attenuation of proximal tubular oxidative stress, mitochondrial morphology, modulation of key metabolism and reabsorptive proteins, pro-inflammatory and profibrotic cytokines, and improvement of tubulointerstitial fibrosis; (2) through activation of tubuloglomerular feedback to regulate glomerular hemodynamic stability and metabolic effects (126, 127). Table 2 summarizes the underlying mechanism of kidney protection by SGLT2 inhibition in DM reported in recent years.

There is increasing evidence suggesting that SGLT2i has renal protective effects in addition to cardiovascular protection, as reported by diverse clinical trials (summarized in Table 3 ). The first clues involving the potential nephroprotection with SGLT-2 inhibitors originated from glucose-lowering trials that set albuminuria as a secondary outcome (167). In the Empagliflozin Cardiovascular Outcome Event (EMPA-REG OUTCOME) trial, the treatment of empagliflozin significantly reduced the primary end points which were defined as progression to macroalbuminuria, doubling of the serum creatinine level (D-Scr), initiation of kidney replacement therapy, or renal death, and incident albuminuria (159) ( Table 3 ). In addition, all individual renal end points showed notable attenuation (31, 168). In the subsequent published Canagliflozin Cardiovascular Assessment Study (CANVAS) and CANVAS-Renal (R) program studies, clear renal protective effects were also noted (160, 161). Kidney function declined in a relatively stable manner, and urine albumin loss decreased in participants who received canagliflozin vs. placebo. Regarding the Dapagliflozin Effect on Cardiovascular Events-Thrombolysis in Myocardial Infarction 58 (DECLARE-TIMI 58) trials, although treatment with dapagliflozin showed a non-inferior rate of major adverse cardiovascular events (MACEs) than placebo, a possible lower rate of adverse renal outcomes in the dapagliflozin group than in the placebo group was observed (163). Although the above cardiovascular trials indicated nephroprotective effects of SGLT2i, it should be noted that the recruitment of participants was biased, considering that the selected patients had a high risk of cardiovascular events and mostly normal kidney function (169). Canagliflozin and Renal Events in Diabetes with Established Nephropathy Clinical Evaluation(CREDENCE) was the first dedicated renal outcomes trial of an SGLT2i canagliflozin, the recruitment of which was randomized in 4,401 T2DM patients with CKD, severely elevated albuminuria, and already ACEIs or ARBs receivers (162). The incidence rates of primary composite outcomes (D-Scr, ESKD or renal/CV death) and the renal-specific composite outcomes (D-Scr, ESKD or renal death) were significantly lower in the canagliflozin group than in the placebo group. Subsequently, two trials embarked on investigating the kidney effects of SGLT-2 inhibitors in CKD patients with or without DM (169). The Dapagliflozin and Prevention of Adverse Outcomes in Chronic Kidney Disease (DAPA-CKD) trial enrolled 4,304 CKD patients with an eGFR ranging from 25 to 75 ml/min/1.73 m², and uACR range from 200 to 5,000 mg/g (164). The trial aimed to evaluate the effect of dapagliflozin 10 mg once daily compared with placebo in addition to a maximum tolerated labeled dose of an ACEI or ARB. Reductions of the same magnitude in the primary outcomes (a composite of a sustained decline in the estimated GFR of at least 50%, ESKD, or renal/CV death) and renal-specific composite outcomes (D-Scr, ESKD, or renal death) were noted. The benefit was comparable for patients with diabetic and non-diabetic CKD. The Heart and Kidney Protection with Empagliflozin (EMPA-KIDNEY) trial commenced in November 2018, with a plan to recruit 5,000 participants and to be completed in June 2022 (170). The empagliflozin on estimated extracellular volume, estimated plasma volume, and measured glomerular filtration rate in patients with heart failure (Empire HF Renal) trial focused on the effects of empagliflozin in both heart failure and CKD patients. It enrolled 391 patients with left ventricular ejection fraction (LVEF) ≤40% and eGFR >30 ml/min/1.73 m². The results showed that empagliflozin reduced estimated extracellular volume, estimated plasma volume, and measured GFR after 12 weeks, implying that fluid volume changes might be an important mechanism underlying the beneficial clinical effects of SGLT2i (165). However, the recent Evaluation of Ertugliflozin Efficacy and Safety Cardiovascular Outcomes Trial (VERTIS CV) reported no significant benefit of ertugliflozin for the renal composite outcomes (death from renal causes, renal replacement therapy, or D-Scr) (166). Further analyses in the trial using renal different end points are underway and may give more clues. To sum up, both in the cardiovascular outcomes trials, which set different definitions of renal outcomes as secondary end points, and in the dedicated trials in CKD patients in which cardiorenal composite outcomes were primary end points, SGLT2i mostly displayed a convincing significant hindering of kidney progression.

These impressive clinical trials and mechanistic studies of SGLT2i promoted the clinical guidelines and recommendations to update the optimal approaches for the prevention and management of DKD. In 2019, the American Diabetes Association (ADA), European Association for the Study of Diabetes (EASD), and European Society of Cardiology (ESC) published updated recommendations for the management of patients with T2DM and a high cardiovascular risk, highlighting the cardiorenal benefits of SGLT2i and glucagon-like peptide-1 receptor agonists (GLP-1 RA) (171–174). The ESC guidelines suggest that SGLT2i or GLP­1 receptor agonists should have priority when patients coexist with cardiovascular disease and those at high or very high cardiovascular risk. Likewise, the ADA-EASD consensus report indicates that patients at high risk of cardiorenal disease are recommended to be treated with SGLT2i or GLP­1 receptor agonists, independent of glycosylated hemoglobin (HbA1c) levels. Additionally, SGLT2i, as well as metformin, was recommended as first-line glycemic management for patients with T2D and CKD according to the 2020 Kidney Disease Improving Global Outcomes (KDIGO) guideline for diabetes management in CKD, in light of the kidney benefits for most patients with eGFR ≥30 ml/min per 1.73 m² (175). Empagliflozin and canagliflozin are FDA-approved for use in patients with eGFR ≥45 ml/min/1.73 m², and ertugliflozin and dapagliflozin are used for those with eGFR ≥60 ml/min/1.73 m² (166, 176).

Great research progress in understanding the pathogenesis of tubular damage and novel biomarkers and treatments has been made, promoting us the transition into a new era of personalized diagnosis and therapy in DKD. As a complex and major complication of metabolism disease, diabetic tubular dysfunction should be regarded with close interconnection with glomerular changes and compact interrelation with systemic metabolic changes. The major current challenges in discovered biomarkers in DKD include the integration of clinical and biochemical biomarkers and omic biomarkers and translation into the pathophysiology, differential diagnosis, risk stratification, prognosis, and individual therapy in clinical practice. The ongoing progress with new anti-hyperglycemic agents provides invaluable and novel insights into the pathophysiology and potential biomarkers of renal tubules in DKD, the combination of which will shed light on better clinical management of DKD.

SD drafted the manuscript, designed the figures and tables. FL and DS corrected the figures and tables. CZ and BZ reviewed the draft. CX was responsible for the final substance. YY was the guarantor and supervised the review and edited the review. All authors contributed to the article and approved the submitted version.

This work was supported by grants from the National Natural Science Foundation of China (No. 81870469, 81670628, 81300573), the Natural Science Foundation of Jiangsu Province (No. BK20131030 to Yanggang Yuan, BK20191075 to Suyan Duan), the China Scholarship Council (CSC, File No. 201608320124), Chinese Society of Nephrology (17010060675 to Yanggang Yuan, 17010090678 to Suyan Duan), the Clinic Research Center of Jiangsu Province (No. BL2014080) and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.