Effect of anti-retroviral regimen on proximal tubular function in Zambian adolescents and young adults living with HIV: A cross sectional study

Introduction

Combination antiretroviral therapy (cART) has mortality and morbidity benefits in both adolescents and young adults^1,2. An ART regimen containing the nucleotide reverse transcriptase inhibitor (NtRTI) tenofovir disoproxil fumarate (TDF) is preferred globally^3,4

In Zambia, the use of TDF in children above five years of age, and adolescents was approved in 2010 but guidelines were revised in 2016 to include only adolescents principally due to TDF’s unknown effects on growth, kidney function and bone integrity in children^5–9. Despite these concerns, TDF regimens remain the preferred formulation in the Zambian HIV guidelines for adolescents and young adults in line with global guidelines¹⁰. However, some adolescents and young adults are maintained on abacavir (ABC) regimens rather than switching to TDF by clinicians. In adults, TDF may increase the risk of proximal tubular dysfunction (PTD) among susceptible individuals^4,11–13. Fractional Excretion of Phosphate (FePO4) is the percentage of glomerular filtered phosphate which is excreted into urine expressed as the ratio of the clearance of phosphate to creatinine clearance^5,14. Therefore, PTD is associated with elevated urinary excretion of phosphate in the presence of hypophosphatemia¹¹. FePO4 has been used to assess for PTD in people living with HIV (PLHIV) on tenofovir regimens^5,15–18.

We investigated the effect of an ART regimen on proximal tubular function (PTF) as measured by FePO4 in adolescents and young adults living with HIV. We hypothesized that a TDF-regimen was associated with a decline in PTF.

Methods

We conducted a cross-sectional study involving adolescents and young adults receiving either TDF or ABC-based regimens at the University Teaching Hospitals (UTHs) in Lusaka, Zambia. SmartCare, a national HIV electronic medical database, was used to select eligible participants between May 2018 and March 2019 using a checklist to verify eligibility.

All participants were aged between 10 and 24 years who had received ART for a minimum of 12 months and had a documented normal estimated glomerular filtration rate (eGFR) at initiation of the current ART regimen. Participants with prior diagnosis of diabetes mellitus at the time of ART initiation, current use of prednisone (> 10 mg/day) or dexamethasone (> 1 mg/day) or any other corticosteroid, history of taking vitamin D supplementation within the past six months, or pregnancy at the time of enrolment into the study were excluded. Written informed consent and/or assent for participation in this study were obtained from each participant and/or parent or guardian. The study was approved by the University of Zambia Research Ethics Committee (REF. No. 010-01-18) and the Zambia National Health Research Authority. The study adhered to the Declaration of Helsinki

Results

The crude effects of covariates on FePO4

The following covariates were moderately positively correlated with PTF as measured by FePO4; age (r=0.33, p<0.0001), and BSA (r=0.32, P<0.0001) as shown in Figure 1. Other factors with weakly positive correlations with FePO4were fasting blood glucose (FBG) (r=0.16, p= 0.026), serum uric acid r=0.23, p=0.002), serum urea (r= 0.15, p=0.034), urine protein excretion (r=0.21 p=0.0043). The rest of the factors were not significantly correlated.

Figure 1. Scatterplots with linear predictions and Lowess lines for FePO4.

Age by cART (Panel 1.1); Serum Urea by cART (Panel1.2); Haycock BSA by ART (Panel 1.3); Urine β₂-MCR by cART Regimen (Panel 1.4).

The crude effects of the covariates for prediction of FePO4 (PTF) are depicted in Table 3. A TDF regimen, dipstick proteinuria, age, BSA, serum urea, urine protein excretion, urine β_2-MCR, and CRP were all associated with worsening FePO4 or PTF.

Table 3. Crude predictors of tubular dysfunction as measured byFePO4 (N = 179).

	Simple linear Regression Crude estimates of association with FePO4 (%)
Parameters	β-coefficient	T	95% CI	P-value
Age (1year increase)	0.59	4.26	0.32 - 0.87	<0.001
Sex, Male vs Female	1.25	1.04	-1.12 - 3.63	0.299
BSA (1 m2 increase)	7.65	3.41	3.23 - 12.1	<0.001
ART regimen, ABC vs TDF	3.72	3.19	1.45 - 6.02	0.002
ART duration (1 month increase)	0.004	0.13	-0.05 - 0.06	0.896
HgB (1g/L increase)	-1.09	-0.56	-4.97 - 2.78	0.579
Current CD4+ cell (1cells/μL increase)	-0.001	-0.63	-0.005 - 0.003	0.531
Viral load (1 copy/mL increase)	-0.00001	-0.60	-0.0001- 0.00003	0.549
S-Uric acid (1 mg/dL increase) S-Uric acid ≤ 250 vs ≥250	0.01 2.46	1.91 1.84	-0.0005 – 0.03 -0.19 – 5.11	0.058 0.068
Urea (1 mmol/L increase)	1.33	2.10	0.08 – 2.59	0.037
eGFR (1 ml/min/1.73m2 increase)	-0.01	-0.40	-0.06 – 0.04	0.687
α1-MCR (ng/mL)	0.001	0.12	-0.02 – 0.03	0.905
U-Protein Excretion (1g/mmol/1.73m2 increase) U-Protein Excretion < 0.1 vs ≥ 0.1	3.28 5.14	2.67 3.54	0.85 – 5.70 2.27 – 8.00	0.008 <0.001
U PCR (1 mg/mmol increase)	0.02	2.37	0.002 – 0.03	0.019
U-Dipstick, Normal vs Proteinuria	0.13	-3.46	-0.20 - -0.06	<0.001
Urine β2-MCR (µg/g)	0.01	4.48	0.01 – 0.02	<0.001
CRP (1mg/dl increase)	5.34	8.05	4.03 - 6.65	0.005

FePO4, Fractional Excretion of Phosphate; Urine β₂-MCR, Urine β₂-microglobulin Creatinine Ratio; CRP, C-Reactive Protein; Urine α₁-MCR, Urine α₁-microglobulin Creatinine Ratio; UPCR, Urine Protein Creatinine Ratio; ART=Antiretroviral Therapy; TDF, tenofovir Diproxil Fumarate; BSA, body surface are; S-phosphate, Serum phosphate; U-phosphate 2, Urine phosphate; S-Uric acid, Serum Uric acid; eGFR, estimated Glomerular Filtration Rate; U-Dipstick; Urine Dipstick; U-Protein Excretion, Urine Protein Excretion, S-Creatinine, Serum Creatinine; FBS, Fasting Blood Sugar; TmPGFR, Maximal Tubular reabsorption of phosphate per glomerular filtration rate; β-coef, Beta-coefficient; T, T-Statistic; 95% CI, 95% confidence Interval. P-value ≤ 0.050 was considered significant

Adjusted relationship of PTF as measured by FePO4

The adjusted effects of the significant or clinically important covariates associated with FePO4 at univariate analysis are shown in Table 4. After adjusting for sex, age, body, BSA, ART regimen, serum uric acid, urine β₂-MCR, CRP and UPCR, a TDF regimen was not associated with worsening FePO4 or PTF. Age (coef 0.637, 95% CI 0.612-1.113) and urine-β₂_MCR (coef 0.010, 95% CI 0.006-0.014) were associated with worsening FePO4 or PTF. The other covariates independently associated with worsening FePO4 or PTF, were UPCR (coef 0.013, 95% CI 0.001-0.025, and CRP (coef 0.155, 95% CI 0.033-0.277). Figure 2 shows the prediction of FePO4 with Urine-β₂ MCR and age. ART regimen showed a big difference in the crude analysis (Coef 3.72, 95% CI 1.45-6.02, p=0.002) but little difference in the adjusted analysis (Coef -2.3, 95% CI -6.93-2.07, p=0.287). CRP, Haycock, and urea also showed big changes between crude and adjusted analysis. When ART regimen was only adjusted for age, CRP, urea and haycock, ART regimen was only confounded by age.

Table 4. Model 1 (FePO4 Model).

FePO4(mmol/L)	Coef.	Std.Error	T-Statistic	P-Value	95% C I
Age (years)	0.637	0.241	2.65	0.009	0.162-1.113
Urine-β2_MCR(µg/g)	0.010	0.002	4.60	<0.001	0.006-0.014
CRP (mg/dL)	0.155	0.062	2.52	0.013	0.033-0.277
ART Regimen, TDF	-2.433	2.28	-1.07	0.287	-6.932-2.065
Upcr(mg/mmoL)	0 .013	0.006	2.09	0.038	0.001-0.025
S-Uric acid (mg/dL)	-0.002	0.008	-0.22	0.825	-0.015- 0.018
Haycock BSA(m2)	2.714	4.431	0.61	0.541	-6.037-11.46
Sex, Female	1.876	1.197	0.99	-0.322	-1.175 -3.551
Urea (mmol/L)	0.809	0.607	1.33	0.184	-0.389-2.007
_Cons	-11.32	4.097	-2.76	0.006	-19.40-3.23

FePO4, Fractional Excretion of Phosphate; Urine β₂-MCR, Urine β_2-microglobulin Creatinine Ratio; CRP, C-Reactive Protein; UPCR, Urine Protein Creatinine Ratio; ART=Antiretroviral Therapy; TDF, tenofovir Diproxil Fumarate; BSA, body surface area; S-phosphate 1, Serumphosphatespline 1; S-phosphate 2, Serumphosphatespline 2; S-Uric acid, Serum Uric acid; bsa, body surface area;β-coef, Beta-coefficient; Std. Err, Standard Error; 95% CI, 95% confidence Interval. P-value ≤ 0.050 was considered significant

Figure 2. Prediction of FePO4 by Urine β2-MCR (Panel 2.1); and Age (Panel 2.2).

Discussion

In this cross-sectional study among black African adolescents and young adults living with HIV on two different ART regimens, the prevalence of PTD as measured by FePO4 was 6%. We found a higher prevalence of isolated PTD in the TDF group (10%) than the ABC group (2%), but this was non-significant after adjusting for age.

The clinical implication of subclinical PTD without progressive reduction in eGFR remains obscure^5,19,20. In fact, only four participants had mild reduction of glomerular function (eGFR<90mls/mL/1.73m²), and three of these were on TDF with normal PTF implying that reduced eGFR was probably independent of PTD.

A tenofovir regimen was not associated with a worsening PTF after adjusting for confounding. This was unexpected finding as TDF exposure has been implicated with urine phosphate wasting^13,23. Although, some studies show no association between TDF and urine phosphate wasting^5,12,24,25, our study was compromised by the substantial age difference between the groups and further analysis showed that age confounded the effect of ART regimen on PTF. Therefore, it is likely that a randomized controlled trial is required to answer this question definitively. Our study suggests that a position of equipoise is present - we have no evidence that TDF is associated with worsened tubular dysfunction - and therefore a clinical trial is justified.

Of concern was the lower serum phosphate and higher urine phosphate in the TDF group than the ABC group respectively. In fact, all the four patients that had low serum phosphate were on TDF and three had PTD implying that TDF may be contributing significantly to urinary phosphate losses resulting in hypophosphatemia with possible long-term implications for bone health.

Nonetheless, available evidence indicates that hypophosphatemia associated with TDF maybe transient and normalizes during ART²¹. Pontrelli et al. among 49 adolescents showed normalization of serum phosphate levels without interruption of therapy at 2 years of follow-up after an initial significant decline in the groups receiving TDF. However, the authors in their conclusion still thought that the long-term clinical impact of a TDF regimen would be answered by a prospective study with a longer follow up period²¹.

There was no significant difference between the two groups with respect to eGFR. This finding is consistent with other studies in adolescents and young adults PLHIV that did not find significant changes in eGFR between participants on a TDF versus non-TDF regimens^{5,11,13,21,24}. Of note, our study population was younger without co-morbidities like diabetes mellitus and hypertension which are risk factors for TDF-induced renal dysfunction in adults¹² which partly explains our findings.

Urine β₂-microglobulin creatinine ratio (β_2-MCR) has been reported in some studies to be a marker of PTD in PLHIV^6,23,26. We also report an independently significant association between urine β_2-MCR and worsening PTF or FePO4. Unlike our study, there are also reports of no association of urine β_2-MCR with PTF²⁷.

The reasons for the lack of association between Urine β_2-MCR and tubular dysfunction are unclear in these studies, except that urinary pH may have played a role. Studies that did not control urine pH by treating the urine samples with acid or alkali at the time of urine collection reported negative results²⁷.

Likewise, protein excretion is enhanced in PLHIV with subclinical PTD without glomerular function impairment³.

In univariable analysis, dipstick proteinuria, protein excretion and UPCR were all associated with worsening PTF or FePO4. UPCR, the only urine protein covariate considered in multivariable analysis, was independently associated with worsening PTF orFePO4. Our findings are comparable with other studies that considered proteinuria to be a marker of PTD³. Therefore, proteinuria may be a cheaper tool to use in resource-limited settings to monitor for early PTD. CRP was independently associated with elevated FePO4, indicating the possible role of inflammation in PTD^12,28.

Our study’s strengths included a relatively larger sample size than most pediatric studies, and a comparison of participants on two different ART regimens to control for confounding. The median duration of 36 months on ART allowed us to assess the medium to long-term renal impact of TDF exposure. Furthermore, because our participants were drawn from patients who receive ART in the general HIV care programs, even though at a tertiary care level, our findings may be generalized to the general population of adolescents and young adults with HIV.

There were also limitations that affected our study findings. As this was a cross sectional study, we could not compare our findings with the pre-ART PTF because in our setting, FePO4 measurements are not done routinely. As earlier stated, the big difference in age between the two groups confounded the effects of ART on PTF. Therefore, more research is needed to investigate if TDF is associated with PTD in adolescents with similar age structure.

The majority (>90%) of patients in the TDF group were previously switched from ABC, so we don’t know the impact of this on our findings. None of our participants reported using nephrotoxic medications like non-steroidal anti-inflammatory drugs, but we did not collect information on use of such medications, so there is possibility of some residual confounding.

Conclusion

Even though subclinical PTD was more common among participants in the TDF group than the ABC group, a TDF regimen was not associated with worsening PTF as measured by FePO4. Age was a confounder on the effect of ART on PTF and therefore, further research is required. CKD was very rare with only four participants with mildly reduced eGFR. Of concern, though not significant, were the four participants on TDF with hypophosphatemia