Sub-Saharan Africa (SSA) remains the epicenter of the HIV/AIDS pandemic, with more than 70% of the global HIV burden and 52% of HIV-related deaths (1). The widescale implementation of antiretroviral therapy (ART) has ameliorated the impact of HIV-associated morbidity and mortality, but not without ART-associated adverse drug events, some of which can be serious (2–4). Tenofovir disoproxil fumarate (TDF), a leading ART agent that is increasingly used for HIV prevention and chronic hepatitis B virus treatment, can uncommonly lead to nephrotoxicity that may manifest as acute kidney injury (AKI) associated with proximal tubular damage and can progress to chronic kidney disease (CKD) and/or end-stage kidney disease (ESKD) requiring dialysis (5, 6). AKI causes significant morbidity and mortality (7); it is a risk factor for a myriad of complications, including CKD, ESKD and cardiovascular disease (8, 9). The risk of these complications remains high, even following treatment discontinuation or dose modification of the causative agent (8–12). In SSA, the outcomes of ESKD are very poor; therefore, preventing TDF-related complications is of paramount importance.

TDF causes proximal tubular damage by drug-transporter interactions; specifically, the drug accumulates and inhibits mitochondrial DNA (mDNA) polymerase-γ, electron transport chain (ETC) complexes, depletes mitochondria, and reduces cell survival (13–16). Unfortunately, monitoring of kidney health entails measuring serum creatinine levels, which often detects kidney injury late in its course, often at a late stage when the damage is irreversible (17). Before creatinine levels rise, several blood and urine biomarkers may play a role in recognizing early kidney tubular injury, including interleukin-18 (IL-18), kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL) (18–21), N-acetyl-β-D-glycosaminidase (NAG) (22), liver-type fatty acid-binding protein (L-FABP) (23–25), retinol-binding protein-4 (RBP-4), and β2-microglobulin (26). This study assessed the value of urine KIM-1 and NGAL for the early detection of TDF-associated nephrotoxicity because the two biomarkers are produced in the proximal renal tubular cells which TDF nephrotoxicity primarily affects. For example, the blood/urine concentration of KIM1, a type 1 transmembrane protein of 60-90 KD molecular weight with an immunoglobulin and mucin domain, increases following kidney tubular injury (27). NGAL, in turn, is expressed in proximal tubular cells as a 135 KD barrel-shaped gelatinase heterodimer of the lipocalin family with the ability to bind lipophilic molecules and be sensitive to kidney injury (28).

There is a paucity of knowledge about the diagnostic value of KIM-1 and NGAL in detecting subclinical nephrotoxicity among Africans living with HIV within the early stages of treatment with TDF before overt manifestation as acute kidney disease and this study aims to close that knowledge gap. We hypothesized that the change in urine KIM-1 and urine NGAL levels can indicate subclinical nephrotoxicity after 2 weeks of exposure to TDF, since these two biomarkers are highly expressed in the proximal tubular cells, the primary site for TDF-associated kidney injury.

This was a prospective observational cohort study of 205 persons living with HIV (PLWH) who were recruited at the start of their TDF-based therapy and followed for a maximum period of 4 months at the Adult Infectious Disease Research Centre (AIDC), Zambia’s largest HIV referral and treatment center. Participants were ART-naïve adults (18+ years) who were initiating TDF-based ART under standard treatment conditions and had intact/preserved kidney function at enrollment [estimated glomerular filtration rate (eGFR)≥ 60 mL/min/1.73m² based on serum creatinine with eGFR calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation]. Clinicians initiated the participants’ treatment and treated them according to the Zambian National ART treatment guidelines (24) and their expert clinical discretion to minimize the risk of kidney injury. In addition to 300mg TDF, ART contained 300mg lamivudine (3TC) and 600mg efavirenz (EFV) in a fixed-dose combination pill taken once daily. After ART was started, eGFR was re-assessed at 2 weeks and 3 months. None of the participants were reported to have been taking any specific nephrotoxic drugs.

The primary outcome was the incidence of TDF-associated nephrotoxicity after 3 months of TDF-based ART defined according to the acute kidney disease and disorders (AKD) criteria (29) as having either 1) eGFR< 60ml/min/1.73m² at 3 months, 2) a reduction in eGFR of greater than 35% within 3 months, or 3) increase in serum creatinine of more than 50% from baseline at 3 months. In this study, participants were lost to follow-up (LTFU) if they did not attend their scheduled 3-month visit and were not traced during contact tracing. Contact tracing involved contacting the participants using the contact number they provided. If they were unreachable after four attempts weekly in the fourth month of follow-up and no kidney function data was found for the third visit in their electronic health record (EHR), they were deemed to be LTFU.

A total of 205 participants were studied of which two were not followed because they declined to provide blood and urine samples even though their demographics were collected, and the participants’ subsequent kidney outcomes were imputed in the analyses using Multiple Imputations by Chained Monte Carlo Equations (MICE). More details about the analyses can be found in our previous publication (32). Out of a total of 205 participants and 638 person-months, 45 (22%) developed nephrotoxicity during follow-up for an incidence rate of 70 cases per 1,000 person-months. The baseline clinical and laboratory characteristics of the participants stratified by TDF-associated nephrotoxicity are shown in Table 1 . There were no differences in nephrotoxicity by sex, cigarette smoking, alcohol use, herb intake, viremia, baseline eGFR, or baseline serum creatinine between those who did and did not develop nephrotoxicity. None of the participants self-reported or had a documented history of comorbidities including diabetes mellitus, kidney disease, chronic diarrhea, hypertension, hepatitis B, hepatitis C, and tuberculosis. Furthermore, there were no statistically significant differences between the two groups of participants in median age, sex-stratified BMI, mean arterial pressure (MAP), duration of follow-up, viral load, CD4+ count, serum creatinine, fasting blood glucose (FBG), eGFR, serum cholesterol, urine creatinine, urine albumin-to-creatinine ratio, Kidney Disease: Improving Global Outcomes (KDIGO) stage of kidney dysfunction, and urine phosphate-to-creatinine ratio ( Table 1 ).

Upon eliminating the four participants with high influential patterns (dbeta influence statistic > 0.2 and dx2 > 4) the model showed that every pg/mg increase in the difference between the baseline and 2-week concentration of urine KIM-1 (δKIM-1) was associated with a probability of 58% (95%CI 51, 69%) to develop TDF-associated nephrotoxicity. Furthermore, every 10 µmol/L increase in the baseline serum creatinine (creatinine_10pt) was associated with a 66% (95%CI 57, 74%) increase in the probability of developing TDF-associated nephrotoxicity. Furthermore, every 10 mL/min/1.73m² increase in the baseline eGFR (egfr_10pt) was associated with a 74% (95%CI 63, 83%) increase in the probability of developing TDF-associated nephrotoxicity while every 1-year increase in age of the participant was associated with a 53% (95%CI 52, 55%) increase in the probability of developing TDF-associated nephrotoxicity. Every kg/m² increase in BMI was associated with a 46% (95%CI 43, 49%) decrease in the probability of developing TDF-associated nephrotoxicity. Finally, compared to men, being a woman was associated with a 92% (95%CI 77, 98%) increase in the probability of developing TDF-associated nephrotoxicity ( Table 2 ). However, baseline MAP, urine albumin-to-creatinine ratio (lnuacr1), CD4 (CD4_100pt), and viremia were not associated with a risk of nephrotoxicity ( Table 2 ).

Fitting the model with δKIM-1 and δNGAL showed an improvement compared to another model we published previously (32) which did not have the biomarkers; a statistically significant improvement in the Akaike Information Criterion (AIC) was observed, p= 0.001. However, when the biomarkers δKIM-1 and δNGAL were introduced into the nested model one at a time, the improvement in model showed that only δKIM-1 improved the model, p= 0.001; δNGAL showed no significant change to the AIC, p= 0.15.

In a cohort of adults living with HIV in Zambia with no or minimal kidney dysfunction at the start of TDF-based ART according to standard treatment initiation criteria, we measured the incidence of TDF-associated nephrotoxicity within 3 months and sought to identify the associated urine, blood biomarker, and demographic factors. We found that the baseline serum creatinine, eGFR, BMI, age, and change in urine KIM-1 within 2 weeks of TDF initiation predicted the risk of TDF-associated nephrotoxicity, which built upon evidence from previous studies. These data support the use of change in the concentration of KIM-1 within 2 weeks of initiating a TDF-based regimen to predict subclinical TDF-associated nephrotoxicity before overt manifestation at 3 months and could be used in clinical care to stratify the patients’ risk before initiating treatment with TDF. This could be invaluable, especially in low- and middle-income countries (LMICs) where there are no readily available options for treating end-stage kidney dysfunction such as chronic dialysis and kidney transplant which may result from concomitant TDF-associated nephrotoxicity.

A plethora of studies have linked various factors to the risk of TDF-associated nephrotoxicity including female sex, advanced age, pre-existing kidney insufficiency, unsuppressed HIV viral load, protease inhibitor-containing regimens (32–34), co-infection with hepatitis B or C, concomitant TDF use, comorbidities such as hypertension and diabetes, and advanced HIV clinical stage (WHO stages 3 or 4) (35–37). Our previous study (32) showed that treatment with a TDF-based regimen caused nephrotoxicity that manifested within the first 4 months among PLWH who initiated treatment with intact/preserved kidney function. There is no prognostic model for Zambia and most LMICs to estimate the risk of PLWH developing TDF-associated nephrotoxicity if treated. Consequently, anecdotal clinical/laboratory assessments are used to decide who initiates TDF-based therapy. The ability of these assessments to stratify patients by risk of nephrotoxicity is questionable considering the high cases of kidney disease reported in this population (32). Our study derived a prognostic model using routine clinical/laboratory data from patients at TDF initiation and discovered that no significant difference existed between the clinical/laboratory characteristics of those who developed kidney injury and those who did not. Finding no significant difference in the clinical/laboratory characteristics on which the current treatment algorithm is based [initiating TDF for PLWH with eGFR> 60 mL/min/1.73m² (38)] exposed the futility of these assessments which are not based on found evidence. This algorithm based on clinical/laboratory assessments is pointless for kidney injury risk stratification as, for instance, it misclassifies approximately 87% of patients at risk of TDF-associated nephrotoxicity as being safe (32).

The primary aim of this study was to determine if including the change in the concentration of two biomarkers (KIM-1 and NGAL) within 2 weeks of initiating TDF-based therapy can predict subclinical nephrotoxicity before overt manifestation. The findings in this study showed that an increase in the difference between the baseline and the 2-week concentration of KIM-1-to-creatinine was associated with an increased probability of TDF-associated nephrotoxicity. This finding concurred with reports elsewhere that KIM-1 levels can be an early signal for kidney injury (19, 21, 25, 39). However, our finding that KIM-1 detected subclinical injury within 2 weeks of starting treatment with TDF was new considering that many studies measured the biomarker much later in the course of treatment, usually at a time when it has stabilized (18, 21, 40, 41). Furthermore, this study found that NGAL does not detect subclinical injury within 2 weeks of TDF use; this was also unique compared to reports by others that claimed NGAL could be used for the early detection of proximal tubular injury (42–44) but agreed with findings elsewhere (21) where the biomarker was assessed after 1 year of TDF therapy. The differences between our findings and other reports may be due to different times of assessing the change in the biomarker with our study assessing the change earlier in the treatment course.

The key findings of this study are that KIM-1 can predict subclinical TDF-associated nephrotoxicity within 2 weeks of initiating treatment for HIV infection while NGAL cannot. This suggests that using KIM-1 may augment risk stratification to prevent TDF-associated nephrotoxicity, especially among those predicted to have low/borderline risk of nephrotoxicity using the prognostic model we published previously (32). It should be underscored that most LMICs, such as Zambia, do not have options such as chronic dialysis and kidney transplant to effectively treat the ESKD that may result from TDF-associated nephrotoxicity, therefore, investing time in deriving and validating prognostic models that can predict and effectively discriminate patients by risk is crucial. Given that studies have linked nephrotoxicity to many kidney-related (45–49) and non-kidney-related complications (11, 48, 50–52) and that victims of kidney injury remain at high risk of complications even after removing the cause of injury (8, 9, 11), the findings of this study are important.

Finally, consistent with other reports (13, 32, 53, 54), this study also found that the baseline serum creatinine, eGFR, BMI, age, and being female were associated with the risk of developing nephrotoxicity. Advancing age increased the risk of TDF-associated nephrotoxicity, concurring with reports that an aging kidney suffers nephrosclerosis, which disturbs the transcriptomics, hemodynamics, and physiological behavior of the kidney, thereby impairing its response to injury (55, 56). The finding that women compared to men have a higher probability of developing TDF-associated nephrotoxicity is consistent with findings from South Africa and the USA (33, 57, 58); however, the pathophysiology of this difference remains unclear. Therefore, close monitoring of patients is crucial regardless of sex to mitigate the risk of kidney complications. Furthermore, on the one hand, an increase in serum creatinine (59, 60) was associated with an increased risk of TDF-associated nephrotoxicity consistent with findings elsewhere which showed that having pre-existing kidney impairment (61) increased the risk of TDF-associated nephrotoxicity (62, 63). On the other hand, paradoxically, an increase in baseline eGFR, which corresponds with better kidney function at baseline, seemed to be associated with a higher risk of TDF-associated nephrotoxicity (64, 65). This could be due to most participants initiating TDF with preserved kidney function entailing lower serum creatinine and high baseline eGFR with medians of 132 mL/min/1.73m2 and 140 mL/min/1.73m2, respectively. Extremely high and extremely low values are prone to regression to the mean. Additionally, since TDF typically significantly reduces kidney function within the first 3-6 months of therapy and the AKD criteria flagged nephrotoxicity when there is a 50% increase in creatinine, a 35% decrease in eGFR, or eGFR goes below 60mL/min/1.73m², it is more likely that those with an elevated baseline eGFR level fulfilled any of the above AKD criteria than those around the mean. This study also found that an increase in BMI was inversely associated with the risk of TDF-associated nephrotoxicity. This was contrary to other studies (66, 67) that found a direct association. This was probably because our study was conducted among PLWH who are more likely to have a low BMI.

The strengths of this study were that it leveraged routine clinical biomarkers that are done among PLWH and included an enzyme-linked immunosorbent assay-based biomarker which is not difficult to test for and can be determined within 3 hours to improve the prediction of subclinical TDF-associated nephrotoxicity. Furthermore, these findings are unique because to our knowledge, no study has attempted to assess the changes in these biomarkers following short-term exposure to TDF and determined how this would forecast the risk of adverse kidney outcomes downstream. The limitation of this study is that the prognostic model needs external validation before use and might need optimization for other excluded populations such as children and adolescents.

In conclusion, the change in the KIM-1-to-creatinine ratio that follows 2 weeks of initiating treatment with TDF-based therapy can predict subclinical nephrotoxicity before overt manifestation 4 months later as TDF-associated nephrotoxicity. Using a prognostic model is a realistic and cost-effective way of preventing TDF-associated nephrotoxicity among PLWH, and these results build upon our previous study (32) that built a model using routine clinical and laboratory data; including kidney injury sensitive biomarkers to improve prediction was the next realistic step. One may worry about the cost implication and complexity of including such biomarkers, however, KIM-1 is a convenient ELISA-based biomarker that can be tested within 3 hours for approximately $10 per test. Considering the huge cost of treating ESKD and the threat to life it poses, including such a biomarker may be more cost-effective. However, it must be emphasized that there is a need to externally validate these prognostic models before integration into clinical practice.