**3. Results**

#### **3.1. Immunological data**

All FIV-seropositive cats, as determined by rapid test and then WB analysis, also scored positive for FIV DNA in whole blood samples (data not shown). The experimentally infected cats included in the study were routinely examined for CD4+ and CD8+ T lymphocyte subsets by flow cytometry and respective CD4+/CD8+ T cell ratio calculated. Inversion of the CD4+/CD8+ T cell ratio due to a selective decline in the absolute number of the CD4+ T cells was confirmed in 71.4% cats infected with FIV-Pisa M2, 76.5% of FIV-Petaluma infected cats and in all cats infected with both virus strains (Table 1). Although diminution of CD4+ T-cells and inversion CD4+/CD8+ correlated positively with duration of infection, no significant relationships were found between immunological data and renal alteration.

Feline Immunodeficiency Virus (FIV) Infection in Cats: A Possible Cause of Renal Pathological Changes 381

1 (< 140) 9/13 5/5 20/21 11/11 2 (140-249) 3/13 0/5 1/21 0/11 3 (250-439) 0/13 0/5 0/21 0/11 4 (> 440) 1/13 0/5 0/21 0/11

< 2 g/L 3/21 5/17 10/28 5/11 **> 2 g/L 18/21 12/17 18/28 6/11** 

proteinuric) ND 0/12 0/18 0/6

proteinuric) ND 2/12 9/18 4/6 **> 0.4 (proteinuric)** ND **10/12 9/18 2/6** 

GS 0/21 3/10 0/9 0/2 GNS 10/21 7/10 7/9 1/2 GNS+T 8/21 0/10 2/9 1/2

Experimentally FIV-infected cats Petaluma Pisa M2 Petaluma +

Pisa M2

Pisa-M2) and 1/2 cats (infected with both strains) had glomerulo non-selective and tubular

No clinical and selected laboratory parameters in SPF cats enclosed in the control group

Naturally FIV-infected cats

**CD4+/CD8+** < 2.0 ND 13/17 20/28 11/11

Legend: ND - not done; GS - glomerulo selective; GNS - glomerulo non-selective; GNS+T - glomerulo non-selective

Table 2 summarizes the main histological glomerular alterations observed in naturally and experimentally FIV-infected cats. Glomerular changes were detected in 18 of 21 naturally infected subjects. Mild mesangial matrix increase with occasional segmental glomerulosclerosis was observed in 9 subjects (Figure 1A), while immune mediated glomerulonephritis of mesangioproliferative type was detected in 3 animals. In eight naturally FIV-infected cats, amyloid deposition was also detected (Figure 1E). These were segmental and focal in six cases and diffuse in two. In all cases the amyloid deposits were KMnO4 sensitive. In experimentally FIV-infected animals, glomerular changes were detected in 9 of 17 Petaluma-infected cats, in 12 of 28 Pisa-M2-infected cats and in six of the 11 cats infected with both strain. In Petaluma-infected cats mesangial widening was observed in six subjects, while 3 showed a mesangioproliferative glomerulonephritis. In Pisa-M2-infected cats mesangial widening was detected in six subjects, five showed a

**Table 1.** Inversion of the CD4+/CD8+ T cell ratio, serum creatinine value, proteinuria, and urine

proteinuria (Table 1).

were found.

**IRIS staging** 

**urine protein concentration** 

< 0.2 (non

0.2-0.4 (borderline

protein/creatinine (UPC) ratio according to the IRIS classification.

(substaging)

**SDS-PAGE** 

and tubular.

**3.3. Light microscopy** 

**serum creatinine**  (stage 1 - 4)

**UPC** 

#### **3.2. Clinical data**

Total serum protein, albumin and globulins concentration values in experimentally infected cats were in the reference range, regardless infecting virus isolate and time post inoculation. None of the 56 SPF cats included in our study presented clinical signs of azotemia.

Serum creatinine concentration was measured in 37 experimentally infected cats and only one cat, infected with FIV isolate Pisa M2 had slightly increased concentration of serum creatinine (144 μmol/L), mild proteinuria (3.9 g/L), and UPC ratio of 0.43, which is classified as stage 2 of chronic renal failure (CRF) [26].

Urine protein concentration was measured in all 56 experimentally and 21 naturally infected cats. Eighteen naturally infected cats (82%) had mild to sever proteinuria, mean value 26.46±22.41 (range 3.5 – 62 g/L). In experimentally infected cats, proteinuria (> 2mg/L) ranged from 18% in cats infected with both virus strains with mean value 14.3±22.73 (2.7-60 g/L), 32% in cats infected with strain Pisa M2 (mean value 11.78±22.80, ranging 2.2 – 100) and 58% in cats infected with Petaluma strain, in which the mean value of urine proteins concentration was the lowest 4.25±2.81 (2 - 8.25 g/L) (meaning not clear to me). UPC in proteinuric cats was calculated in order to confirm the renal proteinuria. UPC values were the highest in cats infected with Pisa M2 (mean 3.27±6.34; ranged 0.53-17.63), slightly lower in cats infected with both virus strains (mean 3.0±4.39; ranged 0.55-13.01) and the lowest in cats infected with Petaluma strain (mean 2.56±3.03; ranged 0.31 – 7.02).

In addition, electrophoresis of urine proteins was performed in an attempt to establish the localization and severity of renal alteration. 10/21 naturally infected cats had glomerulo nonselective proteinuria, 8/21 glomerulo non-selective and tubular, and only 3 of 21 cats had glomerulo selective proteinuria. Glomerulo selective proteinuria was confirmed only in 3 proteinuric cats, infected with Petaluma strain. In the others experimentally infected cats proteinuria glomerulo non-selective was most frequently found: 7/10, 7/9 and 1/2 cats infected with Petaluma, Pisa-M2 and both strains respectively. Remaining 2/9 (infected with Pisa-M2) and 1/2 cats (infected with both strains) had glomerulo non-selective and tubular proteinuria (Table 1).

No clinical and selected laboratory parameters in SPF cats enclosed in the control group were found.


Legend: ND - not done; GS - glomerulo selective; GNS - glomerulo non-selective; GNS+T - glomerulo non-selective and tubular.

**Table 1.** Inversion of the CD4+/CD8+ T cell ratio, serum creatinine value, proteinuria, and urine protein/creatinine (UPC) ratio according to the IRIS classification.

#### **3.3. Light microscopy**

380 Immunodeficiency

**3. Results** 

alteration.

**3.2. Clinical data** 

as stage 2 of chronic renal failure (CRF) [26].

**3.1. Immunological data** 

All FIV-seropositive cats, as determined by rapid test and then WB analysis, also scored positive for FIV DNA in whole blood samples (data not shown). The experimentally infected cats included in the study were routinely examined for CD4+ and CD8+ T lymphocyte subsets by flow cytometry and respective CD4+/CD8+ T cell ratio calculated. Inversion of the CD4+/CD8+ T cell ratio due to a selective decline in the absolute number of the CD4+ T cells was confirmed in 71.4% cats infected with FIV-Pisa M2, 76.5% of FIV-Petaluma infected cats and in all cats infected with both virus strains (Table 1). Although diminution of CD4+ T-cells and inversion CD4+/CD8+ correlated positively with duration of infection, no significant relationships were found between immunological data and renal

Total serum protein, albumin and globulins concentration values in experimentally infected cats were in the reference range, regardless infecting virus isolate and time post inoculation.

Serum creatinine concentration was measured in 37 experimentally infected cats and only one cat, infected with FIV isolate Pisa M2 had slightly increased concentration of serum creatinine (144 μmol/L), mild proteinuria (3.9 g/L), and UPC ratio of 0.43, which is classified

Urine protein concentration was measured in all 56 experimentally and 21 naturally infected cats. Eighteen naturally infected cats (82%) had mild to sever proteinuria, mean value 26.46±22.41 (range 3.5 – 62 g/L). In experimentally infected cats, proteinuria (> 2mg/L) ranged from 18% in cats infected with both virus strains with mean value 14.3±22.73 (2.7-60 g/L), 32% in cats infected with strain Pisa M2 (mean value 11.78±22.80, ranging 2.2 – 100) and 58% in cats infected with Petaluma strain, in which the mean value of urine proteins concentration was the lowest 4.25±2.81 (2 - 8.25 g/L) (meaning not clear to me). UPC in proteinuric cats was calculated in order to confirm the renal proteinuria. UPC values were the highest in cats infected with Pisa M2 (mean 3.27±6.34; ranged 0.53-17.63), slightly lower in cats infected with both virus strains (mean 3.0±4.39; ranged 0.55-13.01) and the lowest in

In addition, electrophoresis of urine proteins was performed in an attempt to establish the localization and severity of renal alteration. 10/21 naturally infected cats had glomerulo nonselective proteinuria, 8/21 glomerulo non-selective and tubular, and only 3 of 21 cats had glomerulo selective proteinuria. Glomerulo selective proteinuria was confirmed only in 3 proteinuric cats, infected with Petaluma strain. In the others experimentally infected cats proteinuria glomerulo non-selective was most frequently found: 7/10, 7/9 and 1/2 cats infected with Petaluma, Pisa-M2 and both strains respectively. Remaining 2/9 (infected with

None of the 56 SPF cats included in our study presented clinical signs of azotemia.

cats infected with Petaluma strain (mean 2.56±3.03; ranged 0.31 – 7.02).

Table 2 summarizes the main histological glomerular alterations observed in naturally and experimentally FIV-infected cats. Glomerular changes were detected in 18 of 21 naturally infected subjects. Mild mesangial matrix increase with occasional segmental glomerulosclerosis was observed in 9 subjects (Figure 1A), while immune mediated glomerulonephritis of mesangioproliferative type was detected in 3 animals. In eight naturally FIV-infected cats, amyloid deposition was also detected (Figure 1E). These were segmental and focal in six cases and diffuse in two. In all cases the amyloid deposits were KMnO4 sensitive. In experimentally FIV-infected animals, glomerular changes were detected in 9 of 17 Petaluma-infected cats, in 12 of 28 Pisa-M2-infected cats and in six of the 11 cats infected with both strain. In Petaluma-infected cats mesangial widening was observed in six subjects, while 3 showed a mesangioproliferative glomerulonephritis. In Pisa-M2-infected cats mesangial widening was detected in six subjects, five showed a mesangioproliferative glomerulonephritis (Figure 1B), and one cats a membranoproliferative glomerulonephritis (Figure 1D). The exam of the kidneys from the eleven cats infected with both Petaluma and Pisa-M2 strains demonstrated the presence of mesangial widening in three subjects and mesangioproliferative glomerulonephritis in other three cats. Ten of the 16 SPF cats, sacrificed at 12 months post-infection showed no renal alteration (three and seven subjects infected with Petaluma and Pisa-M2, respectively), while 4 presented mesangial widening (two cats each infected with Petaluma and Pisa-M2) and two glomerulonephritis (one each infected with Petaluma and Pisa-M2). Of the 13 subjects sacrificed at 24 months post-infection, eight showed no alterations (4 each infected with Petaluma and Pisa-M2), while mesangial widening was observed in two cats (one each infected with Petaluma and Pisa-M2) and glomeruloneprhitis was detected in remaining three cats (two infected with Petaluma and one with Pisa-M2). Finally, 9 of the 27 cats sacrificed after 30 months post-infection, nine had mesangial widening (three each infected with Petaluma, Pisa-M2, and both Petaluma and Pisa-M2), while glomerulonephritis was detected in seven cats (four infected with Pisa-M2 and 3 with both Petaluma and Pisa-M2). Eleven of these cats, i.e. those sacrificed after 30 months post-infection, had no renal changes. In affected glomeruli of naturally and experimentally infected cats, dilatation of the Bowman's space was a frequent finding and, occasionally, protein droplets were present in prominent epithelial cells, particularly in subjects with an heavy proteinuria. The presence of mesangial widening and glomerulonephritis was detected both in experimentally (15/56 and 12/56, respectively) and naturally infected cats (8/21 and 3/21, respectively). Of note, naturally infected cats presented glomerular amyloid deposits that were never detected in the experimentally infected ones (P<0.001).

Feline Immunodeficiency Virus (FIV) Infection in Cats: A Possible Cause of Renal Pathological Changes 383

**Figure 1.** A. Naturally FIV-infected cat. Mesangial widening. Mild increase in mesangial matrix with a minimal increase in intraglomerular dellularity. Jones' periodic acid-silver methenamine stain. (Bar = 80 μm) B. Experimentally Pisa-M2-infected cat. Mesangioproliferative glomerulonephritis. Focal mild proliferation of mesangial cells. Hematoxylin-Eosin stain. (Bar = 80 μm) C. Experimentally Pisa-M2 infected cat. Mesangioproliferative glomerulonephritis. Segmental deposition of IgG. Strepavidin biotin peroxidise complex method, Mayer's hematoxylin counterstain. (Bar = 80 μm) D. Experimentally Pisa-M2-infected cat. Membranoproliferative glomerulonephritis. Mesangial enlargement and thickening of capillary walls. Hematoxylin-Eosin stain. (Bar = 80 μm) E. Naturally FIV-infected cat. Mild amyloid deposition (pale structurless material), occupaing the slightly expanded mesangial areas. H-E stain. (Bar = 80 μm) F. Naturally FIV-infected cat. The amyloid is immunoreactive with the antiserum to feline AA amyloid. Strepavidin biotin peroxidise complex method, Mayer's hematoxylin counterstain. (Bar = 80

μm).


**Table 2.** Main histological glomerular findings in naturally and experimentally FIV-infected cats and uninfected controls.

infected ones (P<0.001).

Mesangioproliferative

Membranoproliferative

uninfected controls.

**Glomerular** 

**Histopathological alterations** Control

cats

Naturally FIVinfected cats

Bowman's space dilatation 0/4 8/21 3/17 6/28 3/11 Mesangial widening 0/4 8/21 6/17 6/28 3/11

glomerulonephritis 0/4 3/21 3/17 5/28 3/11

glomerulonephritis 0/4 0/21 0/17 1/28 0/11 Segmental glomerulosclerosis 0/4 1/21 0/17 0/28 0/11 Glomerular amyloidosis 0/4 8/21 0/17 0/28 0/11 No alterations 4/4 3/21 8/17 16/28 5/11

**Table 2.** Main histological glomerular findings in naturally and experimentally FIV-infected cats and

Experimentally FIV-infected cats

Petaluma Pisa M2 Petaluma +

Pisa M2

mesangioproliferative glomerulonephritis (Figure 1B), and one cats a membranoproliferative glomerulonephritis (Figure 1D). The exam of the kidneys from the eleven cats infected with both Petaluma and Pisa-M2 strains demonstrated the presence of mesangial widening in three subjects and mesangioproliferative glomerulonephritis in other three cats. Ten of the 16 SPF cats, sacrificed at 12 months post-infection showed no renal alteration (three and seven subjects infected with Petaluma and Pisa-M2, respectively), while 4 presented mesangial widening (two cats each infected with Petaluma and Pisa-M2) and two glomerulonephritis (one each infected with Petaluma and Pisa-M2). Of the 13 subjects sacrificed at 24 months post-infection, eight showed no alterations (4 each infected with Petaluma and Pisa-M2), while mesangial widening was observed in two cats (one each infected with Petaluma and Pisa-M2) and glomeruloneprhitis was detected in remaining three cats (two infected with Petaluma and one with Pisa-M2). Finally, 9 of the 27 cats sacrificed after 30 months post-infection, nine had mesangial widening (three each infected with Petaluma, Pisa-M2, and both Petaluma and Pisa-M2), while glomerulonephritis was detected in seven cats (four infected with Pisa-M2 and 3 with both Petaluma and Pisa-M2). Eleven of these cats, i.e. those sacrificed after 30 months post-infection, had no renal changes. In affected glomeruli of naturally and experimentally infected cats, dilatation of the Bowman's space was a frequent finding and, occasionally, protein droplets were present in prominent epithelial cells, particularly in subjects with an heavy proteinuria. The presence of mesangial widening and glomerulonephritis was detected both in experimentally (15/56 and 12/56, respectively) and naturally infected cats (8/21 and 3/21, respectively). Of note, naturally infected cats presented glomerular amyloid deposits that were never detected in the experimentally

**Figure 1.** A. Naturally FIV-infected cat. Mesangial widening. Mild increase in mesangial matrix with a minimal increase in intraglomerular dellularity. Jones' periodic acid-silver methenamine stain. (Bar = 80 μm) B. Experimentally Pisa-M2-infected cat. Mesangioproliferative glomerulonephritis. Focal mild proliferation of mesangial cells. Hematoxylin-Eosin stain. (Bar = 80 μm) C. Experimentally Pisa-M2 infected cat. Mesangioproliferative glomerulonephritis. Segmental deposition of IgG. Strepavidin biotin peroxidise complex method, Mayer's hematoxylin counterstain. (Bar = 80 μm) D. Experimentally Pisa-M2-infected cat. Membranoproliferative glomerulonephritis. Mesangial enlargement and thickening of capillary walls. Hematoxylin-Eosin stain. (Bar = 80 μm) E. Naturally FIV-infected cat. Mild amyloid deposition (pale structurless material), occupaing the slightly expanded mesangial areas. H-E stain. (Bar = 80 μm) F. Naturally FIV-infected cat. The amyloid is immunoreactive with the antiserum to feline AA amyloid. Strepavidin biotin peroxidise complex method, Mayer's hematoxylin counterstain. (Bar = 80 μm).

Tubulointerstitial alterations were frequently detected in naturally and experimentally infected cats. In the former, degeneration of tubular epithelial cells was observed in ten cats, tubular microcysts in eight, and giant protein tubular casts in four subjects (Table 3). Only six Petaluma-infected cats showed degenerative changes of tubular epithelial cells, while no alteration were detected in the other eleven animals. In Pisa-M2-infected cats, degenerative changes were observed in eleven subjects, tubular microcysts in two and giant protein casts in only one subject, while 17 cats presented no tubular alterations. In cats infected with both strains, degenerative changes of tubular epithelial cells was detected in three cats, tubular microcysts in two and giant protein tubular casts in one subjects. Eight cats showed no alterations. Interstitial alterations were commonly observed in naturally infected cats (Table 4) and consisted of interstitial infiltration by lymphocytes and plasmacells. This was scanty periglomerular (eight subjects), diffuse without fibrosis (six subjects) and diffuse with interstitial fibrosis (three subjects). Interstitial amyloidosis was detected in seven subjects, while no interstitial alterations were detected in four naturally infected cats. Also in these cases the amyloid deposits were KMnO4 sensitive. Experimentally infected cats had seldom interstitial alterations. Scanty periglomerular infiltrates were detected in 1/12 Petalumainfected subjects sacrificed at 24 months post-infection, 3/17 cats infected with Pisa-M2 (one sacrificed at 24 months and two sacrificed ≥ 30 months post-infection) and 2/6 cats infected with both viruses and sacrificed ≥ 30 months post-infection.

Feline Immunodeficiency Virus (FIV) Infection in Cats: A Possible Cause of Renal Pathological Changes 385

Renal changes

widening Glomerulonephritis Tubular

alterations

Interstitial infiltrates

Tubular alterations were more frequently detected in naturally infected cats (18/21) than in experimentally ones (22/56). Particularly, the presence of giant protein tubular cats (4/21 *vs* 2/56; P<0.=5) and tubular microcysts (8/21 vs 4/56; P<0.05) was more frequently detected in naturally than experimentally infected subjects. Interstitial alterations were also markedly more frequently in naturally infected cats (17/21) compared to experimentally infected cats (6/56; P<0.001), particularly the presence of diffuse interstitial infiltrates and interstitial

In Table 4, the renal alterations according virus strain inoculated and time post-inoculation are presented. 14/29 cats without any histological renal alteration observed had the lowest mean value of urine protein concentration (uP) 3.95±1.37 g/L (2.5 – 7.05) and expected the lowest UPC mean value of 0.45±0.12 (0.25-0.67). The mean values of uP and UPC in 10 proteinuric cats with mesangial widening was slightly higher: 3.54±1.52 g/L (2.0 – 5.4) and 0.81±0.44 (0.53 – 1.94), respectively. Five cats with similar mesangial alteration had uP concentration lowest then 2.0 g/L. Significantly higher mean values were established in 12 cats with glomerular alteration. Mean value of uP in this group was 21.53±29.39 g/L (2.8 – 100.0) and UPC ration was 4.8±5.77 (0.90 – 17.63). All 20 cats with tubular alteration had gloerulonephritis (9/20) or mensangial widening (11/20) too. Three of them were unproteinuric. Only four of six cats with interstitial infiltrates were proteinuric, two 24 months

amyloidosis was never detected in the latter group.

pi and 4 after more then 30 months pi.

No alterations Mesangial

(n=6) 3 2 1 2 0

(n=10) 7 2 1 2 0

(n=7) 4 1 2 1 1

(n=6) 4 1 1 2 1

(n=4) 1 3 0 3 0

(n=12) 5 3 4 7 2

**Table 4.** Renal alterations detected in FIV experimentally infected cats sacrificed with different lasting.

5 3 3 3 2

inoculated

Petaluma

Pisa M2

Petaluma

Pisa M2

Petaluma

Pisa M2

Petaluma + Pisa M2 (n=11)

Lasting Virus

12 months pi

24 months pi

> 30 months pi


**Table 3.** Main histological tubular and interstitial findings in naturally and experimentally FIV-infected cats and uninfected controls.

Tubular alterations were more frequently detected in naturally infected cats (18/21) than in experimentally ones (22/56). Particularly, the presence of giant protein tubular cats (4/21 *vs* 2/56; P<0.=5) and tubular microcysts (8/21 vs 4/56; P<0.05) was more frequently detected in naturally than experimentally infected subjects. Interstitial alterations were also markedly more frequently in naturally infected cats (17/21) compared to experimentally infected cats (6/56; P<0.001), particularly the presence of diffuse interstitial infiltrates and interstitial amyloidosis was never detected in the latter group.

384 Immunodeficiency

**Histopathological** 

Degeneration of tubular

**alterations** 

**Tubular** 

**Interstitial** 

Scanty periglomeral

Diffuse interstitial infiltrates

Diffuse interstitial infiltrates

cats and uninfected controls.

Tubulointerstitial alterations were frequently detected in naturally and experimentally infected cats. In the former, degeneration of tubular epithelial cells was observed in ten cats, tubular microcysts in eight, and giant protein tubular casts in four subjects (Table 3). Only six Petaluma-infected cats showed degenerative changes of tubular epithelial cells, while no alteration were detected in the other eleven animals. In Pisa-M2-infected cats, degenerative changes were observed in eleven subjects, tubular microcysts in two and giant protein casts in only one subject, while 17 cats presented no tubular alterations. In cats infected with both strains, degenerative changes of tubular epithelial cells was detected in three cats, tubular microcysts in two and giant protein tubular casts in one subjects. Eight cats showed no alterations. Interstitial alterations were commonly observed in naturally infected cats (Table 4) and consisted of interstitial infiltration by lymphocytes and plasmacells. This was scanty periglomerular (eight subjects), diffuse without fibrosis (six subjects) and diffuse with interstitial fibrosis (three subjects). Interstitial amyloidosis was detected in seven subjects, while no interstitial alterations were detected in four naturally infected cats. Also in these cases the amyloid deposits were KMnO4 sensitive. Experimentally infected cats had seldom interstitial alterations. Scanty periglomerular infiltrates were detected in 1/12 Petalumainfected subjects sacrificed at 24 months post-infection, 3/17 cats infected with Pisa-M2 (one sacrificed at 24 months and two sacrificed ≥ 30 months post-infection) and 2/6 cats infected

with both viruses and sacrificed ≥ 30 months post-infection.

Control cats

Naturally FIVinfected cats

epithelial cells 0/4 10/21 6/17 11/28 3/11 Tubular microcysts 0/4 8/21 0/17 2/28 2/11 Giant protein tubular casts 0/4 4/21 0/17 1/28 1/11 No alterations 4/4 3/21 11/17 17/28 8/11

infiltrates 0/4 8/21 1/17 3/28 2/11

without fibrosis 0/4 6/21 0/17 0/28 0/11

with fibrosis 0/4 3/21 0/17 0/28 0/11 Interstitial amyloidosis 0/4 7/21 0/17 0/28 0/11 No alterations 4/4 4/21 16/17 25/28 9/11

**Table 3.** Main histological tubular and interstitial findings in naturally and experimentally FIV-infected

Experimentally FIV -infected cats

Petaluma Pisa M2 Petaluma +

Pisa M2

In Table 4, the renal alterations according virus strain inoculated and time post-inoculation are presented. 14/29 cats without any histological renal alteration observed had the lowest mean value of urine protein concentration (uP) 3.95±1.37 g/L (2.5 – 7.05) and expected the lowest UPC mean value of 0.45±0.12 (0.25-0.67). The mean values of uP and UPC in 10 proteinuric cats with mesangial widening was slightly higher: 3.54±1.52 g/L (2.0 – 5.4) and 0.81±0.44 (0.53 – 1.94), respectively. Five cats with similar mesangial alteration had uP concentration lowest then 2.0 g/L. Significantly higher mean values were established in 12 cats with glomerular alteration. Mean value of uP in this group was 21.53±29.39 g/L (2.8 – 100.0) and UPC ration was 4.8±5.77 (0.90 – 17.63). All 20 cats with tubular alteration had gloerulonephritis (9/20) or mensangial widening (11/20) too. Three of them were unproteinuric. Only four of six cats with interstitial infiltrates were proteinuric, two 24 months pi and 4 after more then 30 months pi.


**Table 4.** Renal alterations detected in FIV experimentally infected cats sacrificed with different lasting.

#### **3.4. Immunohistochemistry**

The results of immunohistochemical investigations are summarized in table 5. By IF and IHC positive specimens showed segmental, predominantly granular mesangial deposits of IgG, IgM and C3, while rarely scattered granular deposits were detected along the capillary loops (Figure 1C). IgA staining was never observed. Cellular infiltrates were characterized by the presence of IgG secreting plasma cells and scattered IgM. Large proteinaceous casts were positive for IgG and weakly for IgA. Amyloid deposits were always positive for the mouse monoclonal anti-human AA and the rabbit polyclonal against the feline AA amyloid (Figure 1F), while were always negative for the rabbit polyclonal anti-AL amyloid.

Feline Immunodeficiency Virus (FIV) Infection in Cats: A Possible Cause of Renal Pathological Changes 387

investigations described kidney abnormalities in 12 of 15 cats [16] and 10 of 14 cats [17] with naturally acquired FIV infection. Six of the twelve subjects of the first study presented lesions that caused a marked increase in serum BUN and creatinine concentration and heavy glomerular non-selective proteinuria; the other nine cats with renal abnormalities, the urine protein content was higher than normal range (>0,2g/l) [16]. Results obtained in the present study showed similar findings. 18 of 21 (82%) of naturally infected cats had mild to sever proteinuria (mean value of 26.46±22.41 (ranging from 3.5 – 62 g/L). 10/21 cats had glomerulo

The investigations carried out on experimentally FIV-infected SPF cats demonstrated renal alterations partially similar to those detected in naturally FIV-infected ones. Particularly, mesangial widening with or without segmental glomerulosclerosis and immune mediated glomerulonephritis were observed in these subjects, no matter that they were infected with different FIV isolates, maintained in isolation units, and sacrificed at different times post-

Mesangial widening with segmental to diffuse glomerulosclerosis [16], nephrosclerosis [29] and thickened Bowman's membrane [28] have been previously described in naturally FIVinfected cats. These alterations represent glomerular reactions common to many apparently unrelated, clinical entities that are currently believed to result from intraglomerular hemodynamic alterations [31]. In FIV-infected cats hemodynamic alterations might be mediated by sustained production of lymphokines and/or factors of mesangial proliferation with activity on glomerular capillary permeability as a consequence of the chronic systemic viral infection. Although controversial, increasing evidence supports a direct effect of the virus on renal cells either as a result of exposure to viral proteins or direct renal parenchyma infection. The use of a HIV-1 transgenic mouse model demonstrated a direct etiologic link between HIV- 1 expression in kidney and the development of segmental glomerulosclerosis in HIV associated nephropathy (HIVAN) with unique viral-host interactions, which depends on inherent features of the virus and, at the same time, host response [32]. In FIV infection the direct role of the virus in the pathogenesis of renal alterations is postulated by the presence of p24 viral antigen in tubular epithelial cells as well as scattered interstitial inflammatory and glomerular cells and by detection of FIV *gag* DNA and RNA sequences in

Even if FIV-infected cats often present hypergammaglobulinemia, which is believed to be triggered by chronic polyclonal B cell activation [34], which, in turn, can lead to the production of immune complexes [34,35] and auto-antibodies [36], immune complex glomerulonephritis are infrequent. In a previous study on 15 naturally FIV-infected cats only one subjects showed IgG deposits in mesangial areas [16). In this study IgG deposits were detected in 3/21 naturally and in 11/56 experimentally FIV-infected cats, associated with segmental and focal mesangioproliferative glomerulonephritis in 13 cases and only with a membranoproliferative glomerulonephritis. Even if, as mentioned, immune mediated glomerulonephritis seem uncommon in FIV-infected cats, previous studies demonstrated that the mean concentration of circulating IgG immune complex (CIC) in FIV-infected cats were significantly higher than in control cats, while IgM levels increased only slightly. The

non-selective proteinuria, 8/21 of them combined with tubular proteinuria.

infection.

these subjects [17,33].


**Table 5.** Main immunohistochemical findings in naturally and experimentally FIV-infected cats and uninfected controls.
