Nephroangiosclerosis not related to hypertension: A matter to resolve in the era of precision medicine

Nephroangiosclerosis (NAS) associated with hypertension continues to be one of the most causes of end stage renal diseases in Europe, but it is still poorly studied. The prevalence of NAS shows a large variability due to the difference among different countries regarding clinical presentations and the indication to perform renal biopsy. The study aimed to investigate the prevalence in biopsy-proven NAS patients and the association with hypertension and/or glomerulonephritis (GN). We included all patients referred for native kidney biopsy between 2003–2021 at Policlinic Umberto I of Rome. From 837 patients who underwent renal biopsy NAS was diagnosed in 80 (10.5%) patients. Serum creatinine was significantly higher in NAS [2.07 mg/dl (IQR 1.13–5.2) vs 1.1 mg/dl (IQR 0.8–2.1), p < 0.001] compared to patients without NAS. Hypertension was present in 45% of patients with NAS. Proteinuria was significantly higher in patients with mild-moderate NAS compared to patients with severe NAS [2.6 g/die (IQR 1–5) vs 1.5 g/die (IQR 0.86–2.3), p < 0.05]. We did not find any significant differences, including histological features, between NAS patients with hypertension and NAS patients without hypertension (p > 0.05). IgA nephropathy, focal segmental glomerulosclerosis and membranous nephropathy were the most frequent GN associated. In conclusion no specific histological features are reported in NAS with and without hypertension. More information on the phenotype, clinical presentation and markers are needed to improve histological and clinical diagnostics.


INTRODUCTION
Nephroangiosclerosis (NAS), hypertensive nephrosclerosis, and benign nephrosclerosis are commonly used to describe the same clinical condition characterized by changes in the kidneys usually attributed to the effects of hypertension [1].However, the histopathological changes are not specific to hypertensive kidney damage.In course of chronic kidney disease (CKD) of any cause, including patients without history of hypertension, in the elderly or in diabetic patients, the same changes can be found [2].Thus, NAS in clinical practice is a diagnosis of exclusion and the real prevalence has been overestimated and varies among different countries.According to the ERA-EDTA Registry Annual Report [3], hypertensive nephrosclerosis is responsible for end stage renal disease (ESRD) in 16% of patients starting dialysis while NAS, biopsy proven, ranges from 2.7% to 20% in native kidney biopsy registry [4][5][6][7][8].
In a specimen of renal biopsy medial thickening and intimal fibrosis of medium and small-sized arteries up to the interlobular arteries, arteriolar thickening and hyalinosis are observed.A global glomerulosclerosis or secondary segmental sclerosis can occur, particularly in a perihilar location and tubulointerstitial fibrosis are often observed [9].For this reason, by definition, the diagnosis of NAS can only rest on histology but in clinical practice, the diagnosis is based on clinical manifestations as hypertensive retinopathy, left ventricular hypertrophy, mild proteinuria, and progressive CKD with long-term essential hypertension [10].Regarding the presence of hypertension in NAS, over the past 30 years, nephrologist and hypertensiologist wondered if kidney was culprit or victim of hypertension [11].
Based on this background, the aims of the present study were to describe the prevalence of NAS biopsy-proven and clinical and laboratory presentation; (2) to evaluate the association of NAS with hypertension; (3) to evaluate the association of NAS with other glomerulonephritis; (4) to evaluate the relationship between NAS and renal resistive index.

METHODS
Renal native biopsies from 837 consecutive patients examined in 18 years (2003-2021) at the Policlinic Umberto I of Rome, were collected.Specimens from pediatric patients, kidney transplants and cancers were excluded from the study.
The retrospective study was conducted in accordance with the protocol, good clinical practice principles and the Declaration of Helsinki statements.The informed consents were collected at the time of biopsy and the study was approved by the local Ethics Committee in 2021.
Clinical and laboratory data were collected, recorded, and inserted into a database at the time of biopsy.For each patient history, complete physical examination, proteinuria, urinalysis, serum creatinine level (sCr), blood urea nitrogen (BUN) was recorded.The assessment of total urinary protein excretion has been performed using 24 h urine collection.
Clinical diagnosis of NAS was not suspected before to kidney biopsy, but history of hypertension and antihypertensive drugs were recorded prior to performing the procedure.
Hypertension, measured with the subject in the sitting position, was defined as attended office systolic blood pressure (SBP) values at least 140 mmHg and/or diastolic blood pressure (DBP) values at least 90 mmHg in accordance with the criteria of the guidelines of the European Society of Hypertension and the European Society of Cardiology [12].SBP and DBP values were 140/90 mm Hg as the maximum blood pressure allowed to perform renal biopsy as reported by several studies [13].
Every participant underwent renal ultrasonographic assessment immediately before kidney biopsy, within 1 hour of the procedure.
Glomerular filtration rate was estimated using the Chronic Kidney Disease Epidemiology [14].
Renal Doppler Ultrasound was performed by two single expert investigators throughout the recruitment, blinded to the patients' clinical data (Aplio Ultrasound System SSA-790 with convex 3.5 MHz probe, Toshiba, Tokyo, Japan) to detect microcirculation or renal artery stenosis.Renal Doppler evaluation of the arcuate arteries in the region of the corticomedullary junction and the interlobar arteries along the border of medullary pyramids was performed by placing the probe at three different positions (mesorenal, superior and inferior pole) over both kidneys, guided by color flow.The gain was set so that background echoes were barely visible.Doppler gate width was kept small, and the angle of insonation was corrected.An anterior approach was used to detect renal artery origin, and an oblique, lateral approach was used for the intermediate tract and intrarenal vessels.RRI was calculated as: (peak systolic frequency shiftminimum diastolic frequency shift)/peak systolic frequency shift.The average of three measurements for each Doppler parameter of arcuate and interlobar arteries in both kidneys was calculated.RRI > 0.7 was considered abnormal [15].Previously, for other studies performed by the same operators, the following coefficients were calculated: Weighted kappa, used to evaluate the intra-rater reliability by the same observer, was 0.975 for RRI and the intra-patient coefficient of variation for RRI was 1.2%.

Renal biopsy and histology
Renal tissue was obtained by percutaneous needle biopsy.Tissue cores were received within 10 min of biopsy and divided in three portions for immunofluorescence and light and electron microscopy.Multiple paraffin sections were stained with periodic acid-Schiff (PAS), hematoxylin and eosin and PAS-silver methenamine.All biopsy samples had at least 10 glomeruli.
Immunofluorescence was performed on 5 μm cryostat sections using polyclonal fluorescein isothiocyanate-conjugated antibodies to IgG, IgM, IgA, C3, C1q, kappa, lambda and fibrinogen (Dako, Carpinteria, CA, USA).The severity of glomerular lesions was graded semiquantitatively on a scale of 0 to 3 according to the percentage of glomerular involvement: 0 = absence of lesions, 1 = involvement of 1-30% of specimen/total number of glomeruli, 2 = involvement of 31-60% of specimen/total number of glomeruli, 3 = involvement of 61-100% of specimen/total number of glomeruli.The following glomerular lesions were evaluated: karyorrexis/fibrinoid necrosis, leukocyte exudation, cellular and fibrous crescents, segmental/global sclerosis.Tubular lesions evaluated were tubulitis and tubular atrophy; interstitial lesions evaluated were inflammatory infiltration and fibrosis.The arterial and arteriolar changes evaluated were arteriosclerosis and arteriolar hyalinosis.For arteriosclerosis we examined the percentage of arteries (median caliber 140 μm) showing intimal fibrosis; for arteriolosclerosis we examined the percentage of arterioles (median caliber 50 μm) with hyaline thickening.
For vascular lesions, if the changes are focal, the most severe lesion present gives the final grade (0 = absent; 1 + = increased wall thickness but to a degree that is less than the diameter of the lumen; 2 + = wall thickness that is equal or slightly greater to the diameter of the lumen; 3 + = wall thickness that far exceeds the diameter of the lumen with extreme luminal narrowing or occlusion).
Two pathologists, without prior knowledge of any information concerning each patient, performed these morphological evaluations throughout the recruitment.

Statistical analysis
The coefficient of kurtosis was used to evaluate normal distribution of data.All the results are expressed as median and interquartile range (IQR).Group comparisons were made by Kruskal-Wallis test.Spearman's rank correlation coefficient was used to test for an association between numerical variables.The chi-square test or Fisher's exact test, as appropriate, were used to compare categorical variables.p-values < 0.05 were considered significant.SPSS version 26.0 software was used for statistical analysis.

RESULTS
We retrospective analyzed data from 837 patients who underwent renal biopsy from 2003 to 2021.We excluded data from 74 patients with transplanted kidney, so the final analysis was performed on 763 patients.
Table 1 summarizes clinical and demographical characteristics of patients.
The histological diagnosis of NAS was present in 80 (10.5%) patients.
Table 4 summarize the comparative analysis between patients with mild/moderate NAS and patients with severe NAS.
Systemic arterial hypertension was found in 36 (45%) NAS patients and disease duration was 7 (5-13) years.None of patients had diagnosis of renal artery stenosis at the time of enrollment.We did not find any significant differences, including histological features, between NAS in patients with systemic arterial hypertension and NAS patients without systemic arterial (p > 0.05).We stratified patients with NAS according to the number of antihypertensive medications.Twenty-one patients with NAS were treated with ≥ 3 antihypertensive medications.The number of antihypertensive medications was higher in patients with NAS and associated systemic arterial hypertension (n = 36) compared to patients with NAS without associated systemic arterial hypertension (n = 44) [15 (41.7%) vs 6 (13.6%), p < 0.01].
The analysis performed in 490 patients due to missing data showed RRI ≥ 0.70 (54.4% vs 33.7%, p < 0.01) and systemic arterial hypertension (45% vs 26.8%, p < 0.001) more frequently in patients with NAS compared to patients without NAS.
Although the analysis was performed only in 57 patients due to missing data, patients with severe NAS had more frequently RRI ≥ 0.70 than patients with mild-moderate NAS (70% vs 45.9%, p < 0.05).

DISCUSSION
NAS is characterized by non-immune-mediated vascular lesions, essentially with renal arterial lesions, interstitial fibrosis, tubular atrophy, and glomerulosclerosis that evolves towards CKD [4].
In the present study histological diagnosis of NAS was present in 80 (10.5%) patients undergoing renal biopsy.In clinical practice the prevalence of NAS shows a large variability due to the difference of ethnicity among different countries and the indication to perform renal biopsy [3][4][5][6][7][8].
Patients with NAS showed a worsening of renal function, mainly in patients with severe grade of NAS than patients without NAS, while proteinuria did not show differences between groups.Podocyte damage plays an important role on proteinuria onset and only few studies have examined podocyte depletion in NAS patients.
Wang et al demonstrates a reduction in podocyte number and intrarenal gene expression of podocyte-associated molecules in patients with NAS-associated hypertension.They found an association between glomerular podocyte density and intrarenal expression of podocyte-associated molecules with GFR and the degree of renal fibrosis, confirming that podocyte loss is important in the pathogenesis of NAS associated hypertension.However, although mild proteinuria is associated with NAS, Wang et al did not demonstrated an association between proteinuria levels and intrarenal gene expression of podocin [16].In our study different proteinuria levels were found between mild-moderate NAS compared to patients with severe NAS.Increased proteinuria levels in mild-moderate NAS could be mainly due to a greater number of GN associated in this group.
However, data on podocyte damage and proteinuria in NAS are limited and not fully clarified.
Clinical phenotypes in NAS biopsy-proven have been proposed.A long-standing hypertension, mild to moderate CKD, mild proteinuria, no hematuria and in absence of diabetes, had a positive predictive value for NAS in the 97% of African American patients but it was only 48% in Italian patients [17,18].In these different populations there are several factors that can contribute to the development of NAS.A second and environmental hit have been postulated in African Americans, among which variants in the apolipoprotein L1 gene (APOL1) often cause secondarily elevated blood pressure, while in white population NAS is linked to more often due hypertension and cardiovascular risk factors   [ 19,20].Among other causes seem to play a role the reninangiotensin-aldosterone system (RAAS) metabolic derangements, oxidative stress, atherosclerosis, obesity and endothelial dysfunction [4].Anyway, the mechanisms linking hypertension and NAS are still unclear since it's not been established which is the primary trigger [11].
The mechanisms that have been postulated suggest that chronic hypertension leads in narrowing of preglomerular arteries and arterioles, with decreased in glomerular blood flow.Alternatively, the elevated blood pressure leads to glomerular hypertension and hyperfiltration causing glomerulosclerosis with GFR reduction.However, these mechanisms are not exclusive and may coexist simultaneously in the kidney [21].
Hypertension is the hallmark in patients with kidney disease mainly in ESRD.In the present study, all patients were referred for kidney related problems, therefore we can suppose that the different prevalence of hypertension in NAS was secondary to a significant eGFR reduction (median value of eGFR was 57.6 ml/min in not NAS vs 28.3 ml/min in NAS).
In the group of NAS, only 45% of the patients had systemic arterial hypertension.We did not find significant differences between NAS patients with systemic arterial hypertension and NAS patients without systemic arterial hypertension.
It is well known that high blood pressure is correlated to CKD, but the capacity of mild-to-moderate hypertension to produce renal damage has been questioned.Guyton et al demonstrated more than 30 years ago, the crucial role of the renal sodium excretion capacity in blood pressure regulation [22].Although primary renal diseases can cause hypertension, and malignant hypertension, characterized by specific lesions as fibrinoid necrosis and myointimal hyperplasia, is correlated with the development of CKD and ESRD, the real risk of kidney injury due to mild hypertension is still a matter of debate [23].
The histologic changes in NAS have been described also in the diabetic nephropathy, in the aging and in advanced stages of nephropathies [19].
In our study diabetes was present in only 6 (7.5%) of patients with NAS and the median age of NAS patients was 48 (36-63) years.Some authors suggest that when essential hypertension evolves towards ESRD, other conditions should be considered among which atherosclerotic renal vascular disease, malignant or accelerated hypertension, occult primary renal disease [23,24].
In 43 (53.8%)patients NAS was associated with glomerulonephritis (GN) and the most frequent were IgA nephropathy, focal segmental glomerulosclerosis and membranous nephropathy.The only difference was that patients with NAS and associated GN had significantly increased proteinuria compared to patients with isolated NAS.Hallan et al. proposed a diagnostic algorithm to diagnose NAS based on proteinuria < 0.75 g die, systolic blood pressure > 155 mmHg and age > 75 years.
However, there were false-positive cases with treatable disease as primary GN or interstitial nephritis suggesting wider indications to perform kidney biopsy [8].
Finally, patients with NAS vs not-NAS and patients with severe NAS vs mild-moderate NAS had more frequently RRI ≥ 0.70.Renal resistive index is a sonographic parameter that reflects changes in blood flow of the intrarenal arcuate or interlobar arteries.High RRI values are associated to hemodynamic parameters that assess arterial stiffness such as systolic and diastolic blood pressure, pulse pressure and pulse wave velocity [25].Renal resistive index has been studied in hypertensive patients [26,27] but elevated RRI are also present in patients with diabetes, CKD and in renal biopsy histological findings such as glomerulosclerosis, arteriolosclerosis, tubule-interstitial fibrosis [28].
In a previously study, we found association between arteriolosclerosis biopsy-proven and higher RRI, regardless of the underlying primary GN and the concurrent presence of arterial hypertension and DM [29].Thus, RRI could be a marker of vascular histological changes of NAS regardless the hypertension history.
The diagnosis of NAS is based both on assessment of histology and clinical assessment and frequently lacks precision.Although NAS correlated to hypertension continues to be one of the most causes of ESRD in Europe, it is still poorly studied.
The study has several limitations related to its retrospective design.First, the sample size is relatively small because this is a single-center study.Second, all secondary causes of hypertension that might contribute to NAS in some patients were not completely excluded.
In the era of precision medicine, we cannot be satisfied of non-specific definition of disease, that allows a diagnosis by exclusion.Kidney biopsy has no specific findings for NAS with and without hypertension.More information on the phenotype, presentation, markers are needed to improve clinical diagnostics and treatment.

SUMMARY
What is known about the topic • Nephroangiosclerosis (NAS) associated with hypertension is one of the most causes of end stage renal diseases in Europe.
• NAS in clinical practice is a diagnosis of exclusion and the real prevalence has been overestimated.

Table . 1
. Clinical and demographical characteristics of 763 patients.IQR Interquartile range, M Male, F Female, eGFR Estimated glomerular filtration rate, BP Blood pressure.

Table . 4
. Comparative analysis between patients with severe nephroangiosclerosis (NAS) (n = 35) and patients with mild and moderate NAS (n = 45).Subanalysis performed in 57 patients due to missing data.NAS Nephroangiosclerosis, M Male, F Female, IQR Interquartile range, eGFR Estimated glomerular filtration rate, RRI Renal resistive index. *