Focal segmental glomerulosclerosis (FSGS) is characterized by sclerosis in some but not all glomeruli and only in parts of individual glomeruli. The incidence of FSGS ranges from 0.2 to 1.8/100,000 population per year (1), which continues to increase in diverse populations (2–5). Primary FSGS refers to an idiopathic form of disease where the mechanisms of pathogenesis are poorly understood. However, it is generally accepted that podocyte loss is a cause of segmental lesion formation, suggesting that podocyte injury is a key event in the development of FSGS (6, 7).

Standard first-line therapy for patients with primary FSGS with nephrotic-range proteinuria involves corticosteroids, often also employed with blockade of the renin angiotensin system (RAS) (8, 9). Complete remission is defined as proteinuria ≤0.3 g/24 h, or protein to creatinine ratio of <30 mg/mmol, stable serum creatinine and serum albumin>35 g/L, whilst partial remission is defined as a > 50% reduction in peak proteinuria and/or to sub-nephrotic levels (0.3–3.5 g/24 h or 30–350 mg/mmol total protein to creatinine ratio) (9–11). Unfortunately, response to first-line therapy is variable and unpredictable, with 40–60% of patients failing to achieve remission or having relapsing proteinuria (defined as proteinuria >3.5 g/d after complete remission was achieved) (8, 12), both of which are largely attributed to steroid resistance (13–18). There is therefore significant clinical value in developing approaches to indicate the response to first-line therapy in primary FSGS, particularly to avoid the severe multi-organ side effects of corticosteroids (19–21) in patients who are not likely to respond to steroid therapy (22).

Murine and human podocytes express corticosteroid (23–25) and angiotensin receptors (26) suggesting that standard first-line therapy for primary FSGS may have direct action on podocytes that are independent of their systemic effects. In vivo and in vitro studies have provided strong evidence that steroids and RAS blockade have direct therapeutic effects on podocytes, including enhanced podocyte survival, resulting in the preservation of podocyte number (24, 27–29). Findings from animal studies indicate that the degree of podocyte depletion is associated with the degree of resulting pathology (7). Taken together, these findings suggest that the degree of podocyte depletion at the time of primary FSGS diagnosis may be associated with the subsequent response to therapy.

Using diagnostic biopsies from patients with primary FSGS, this study aimed to determine if podocyte number, density and/or size and indices of glomerulosclerosis can indicate the clinical response to first-line therapy in patients with primary FSGS. The findings indicate that podocyte number per glomerulus as well as the degree of glomerulosclerosis at the time of diagnosis may aid in deciding therapy regimens.

This is the first study to analyze podometrics and indices of glomerulosclerosis in diagnostic biopsies of patients with primary FSGS who were subsequently shown at 6 months to be either responders or non-responders to first-line therapy. Our results show that total podocyte number per glomerulus at the time of biopsy was significantly higher in responders than non-responders at 6 months, whereas glomerulosclerotic index and the percentage of glomeruli with global sclerosis were significantly lower in responders. Moreover, threshold values at the time of biopsy of approximately 216 podocytes per glomerulus, 20% of glomeruli with global sclerosis, 50% of glomeruli with FSGS lesions and a GSI score of 1 were able to indicate the response to first line therapy at 6 months.

The mechanisms of action and target cells of corticosteroids are still not completely understood, although recent studies suggest the efficacy of glucocorticoid therapy is achieved, at least in part, through direct effects on podocytes (53). The present findings demonstrate that patients with primary FSGS who responded to first-line therapy had more podocytes per glomerulus at the time of biopsy than patients who did not respond. Studies have also reported increased podocyte survival and preservation of podocyte number following corticosteroid therapy (24, 54).

Although steroid treatment is considered first-line therapy in primary FSGS, it is avoided in patients in whom side effects of steroids are of particular concern (22). For these patients, more conservative therapy may be considered such as blood pressure control achieved with RAS blockade (ACEi/ARBs) and dietary modifications (8), as observed in approximately 30% of patients in both groups in the present study. Moreover, steroid treatment is often prescribed in combination with ACEi/ARBs, which was the case in 38% of responders and 50% of non-responders in the present study. RAS blockade lowers systemic blood pressure which in turn reduces intraglomerular capillary pressure, reducing mechanical distension (stress) of the capillary tuft and thereby protecting podocytes (55). However, podocytes express angiotensin II type 1 receptors and this increases following mechanical stress in vitro (56). Mechanical stress on podocytes also increased podocyte angiotensin II production and apoptosis by 2.5 fold, which was ameliorated by RAS blockade (56). Taken together, these findings suggest that ACEi/ARBs have direct effects on podocyte survival, independent from systemic blood pressure control.

Normal adult human glomeruli contain approximately 500–600 podocytes each (57, 58). Interestingly, several studies have identified that a podocyte number of approximately 200 or less can indicate poorer outcomes. Lemley et al. (59) found that patients with IgA nephropathy and with fewer than 200 podocytes per glomerulus had increased frequency of GFR < 90 mL/min/1.73 m², a higher percentage of global glomerulosclerosis and high shunt magnitude. Similarly, Andeen et al. (60) found patients with early stage diabetic nephropathy had 268 ± 73 (mean ± standard deviation) podocytes per glomerulus, whilst those with advanced disease had 144 ± 52 podocytes per glomerulus. Although these studies did not evaluate therapeutics specifically, the findings support the concept that patients with podocytopathies with fewer than around 200 podocytes per glomerulus are of increased clinical concern and may require more intricate therapeutic strategies than standard first-line therapy. Similarly, ten of the nineteen patients in the present study had 216 or fewer podocytes per glomerulus - this included five of the six non-responders and four of the thirteen responders, suggesting that patients with fewer than approximately 200 podocytes per glomerulus may be more unlikely to respond to first-line therapy.

In contrast to podocyte number per glomerulus, podocyte density is an index of relative podocyte depletion which has repeatedly been shown to be a key event in the onset and progression of glomerulosclerosis (61, 62). We therefore expected that differences in both podocyte number and podocyte density would be found between responders and non-responders to first-line therapy. However, we found no difference in podocyte density between these two groups.

Although the present study found that podocyte density may not be useful in identifying patients who are likely to respond to first-line therapy, podocyte density undoubtedly remains a very valuable tool for assessing glomerular disease. A podocyte density of 100 podocytes per 10⁶ μm³ glomerular volume is considered a critical threshold that results in dysfunctional glomeruli, protein leakage, glomerulosclerosis and decreased renal function (63). Further decreases in podocyte density require exponential increases in podocyte hypertrophic compensatory adaptations. However, this is difficult to accomplish given that podocytes have a limited capacity to increase in size. Failure of podocyte hypertrophy to match the enlarged glomerulus drives progression of glomerular disease (64).

Median podocyte density for both groups in the present study was less than 100 per 10⁶ μm³ at the time of biopsy. Interestingly, all non-responders had a podocyte density less than this value, as did 62% of responders. For both groups of patients, podocyte volume was significantly and negatively correlated with podocyte number and podocyte density, evidence of podocyte hypertrophy. Although not statistically significant, it is of interest that podocyte cytoplasmic volume was 60% higher in non-responders than responders (p = 0.07), while total podocyte volume was 48% higher in non-responders (p = 0.08). This suggests that podocytes in non-responders had undergone greater hypertrophy than podocytes in responders, indicative of increased podocyte hypertrophic stress. Non-responders also had a higher level of glomerulosclerosis. Taken together, these data support the podocyte depletion hypothesis (6, 64).

In addition to podocyte number being able to distinguish treatment responders from non-responders in the present study, three indices of glomerulosclerosis also distinguished between these patients – the percentage of glomeruli with global sclerosis, the percentage of glomeruli with FSGS lesions, and GSI. While these findings are perhaps not surprising, to our knowledge this is the first study to report that indices of glomerulosclerosis have predictive clinical value in patients with primary FSGS. Interestingly, in a study of ANCA-associated glomerulonephritis, Brix et al. (65) developed and validated a clinicopathologic score to indicate renal outcomes. They found that the percentage of normal glomeruli (without scarring, crescents or necrosis) was the strongest indicator of death-censored end stage renal disease. Brix et al. (65) also identified predictive threshold values for these three parameters. With the recent development of deep learning algorithms and new morphometrics to interrogate renal histopathology (66), one can envisage rapid progress in the development of new strategies to indicate kidney outcomes from diagnostic biopsies and functional data and thereby more accurately indicate and refine therapy.

The relationships between urinary/serum data at biopsy and podocyte number per glomerulus and podocyte density were analyzed to determine if non-invasive urinalysis could be used to determine the severity of podocyte depletion at diagnosis. No significant correlations were observed, although the relationship between serum creatinine and podocyte number approached statistical significance. It was expected that increased proteinuria and serum creatinine, and decreased eGFR would be associated with greater podocyte depletion. This was the case in a study of patients with IgA nephropathy where podocyte density was significantly correlated with serum creatinine and eGFR (67).

A limitation of this single center study is the relatively small sample size. Archival kidney biopsy tissue was obtained from patients who required a biopsy to confirm diagnosis of primary FSGS over a 12-year period (2009 to 2020). While 84 patients were initially recruited, 65 patients were excluded, leaving 13 responders and 6 non-responders. While novel and clinically relevant findings were obtained, a larger sample size may have found that podocyte density was higher in responders than non-responders, as was found for podocyte number. The small study size also meant that differences between groups could not be fully appreciated. For instance, non-responders were older, but the age difference between groups was not significant. Treatment regimens also differed, with not all study patients having documented ACEI/ARB therapy which is now standard of care alongside any immunosuppressive therapy (8, 9). Due to the retrospective nature of the study, data collection was limited to what was recorded in patient notes whereby 24-h urine protein results were not available for 16% of patients at time of biopsy and 74% of patients at 6 months. However, spot urine protein levels have been shown to be comparable to 24 h urine protein calculations for the clinical outcomes of end stage renal failure and death (68). Given the retrospective nature of the study and the small sample size, further studies incorporating stratification for confounders such as patient age and treatment regimen are required to fully understand the utility of podometrics and glomerulosclerosis indices for determining therapeutic options.

In conclusion, these findings suggest that primary FSGS patients with higher podocyte number per glomerulus and less glomerulosclerosis at the time of diagnostic biopsy respond to first-line therapy at 6 months. A podocyte number of less than approximately 200 per glomerulus, a GSI greater than 1, and a percentage global sclerosis greater than 20% may all be associated with a lack of response to first-line therapy. Podometric and glomerulosclerosis analyses of diagnostic biopsies may be of clinical value in determining optimum therapeutic options for patients with primary FSGS, however, larger, prospective studies would be needed to confirm this.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving humans were approved by the Monash University Human Research Ethics Committee (14344). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ or their legal guardians/next of kin.

NZ: Data curation, Formal analysis, Investigation, Writing – original draft. KH: Methodology, Writing – review & editing. DN-P: Methodology, Supervision, Writing – review & editing. PK: Investigation, Methodology, Supervision, Writing – review & editing. JL: Data curation, Investigation, Methodology, Writing – review & editing. SG: Methodology, Writing – review & editing. VP: Conceptualization, Writing – review & editing. JB: Conceptualization, Project administration, Supervision, Writing – review & editing. LC-M: Conceptualization, Data curation, Formal analysis, Project administration, Supervision, Writing – original draft, Writing – review & editing.